front cover · 2015-06-19 · 59. chemical characterization of ia antigens of the mouse major...
TRANSCRIPT
FRONT COVER
The Nobel Prize in Physiology and Medicine for 1981 was awarded on December 10, 1981 to Dr. Roger Sperry. Professor Sperry is shown receiving his Prize from King Carl Gustav XVI.
We are proud.
A REPORT FOR THE YEAR 1981-82
ON THE RESEARCH AND OTHER ACTIVITIES
OF THE
DIVISION OF BIOLOGY
AT THE
CALIFORNIA INSTITUTE OF TECHNOLOGY
PASADENA, CALIFORNIA
ii
STAPP OP BIOLOGY 1982
Constance R. Katz: Compilation, typing and layout.
Elizabeth T. Hanson: Copy editing.
The helpful assistance of Christina Balber, Isabella Lubomirski and Lody Kempees is greatly appreciated
RESEARCH REPORTS
Much of the research work summarized here has not yet been reported in print, in many instances because it is not yet complete. For that reason this report is not intended as a publication and should not be cited as such. Individual projects should be referred to only if specific permission to do so is obtained from the investigator responsible for the material. References are made here to published papers bearing on the projects reported. Publications by members of the Division, covering the period Jwte 1981-April 1982, are listed separately, at the end of the research reports of each group.
Introdu~tion. . . . . . . . . . Staff of Instruction and Research
MOLECULAR BIOLOGY AND BIOCHEMISTRY
TABLE OF CONTENTS
Research Reports
Giuseppe Attardi -Summary ••••••••••••••••••••••••••••••• 1. Transcription initiation sites and rRNA gene transcription in human mitochondrial DNA . . . . . 2. Sequence analysis and precise mapping of the 3' ends of the human mitochondrial ribosomal RN As 3. Search for the mitochondrial RNA processing enzyme(s). • • • • • • • • • • • 4. An in vitro transcription/translation system . . . . . . . . . . . . . . . . . 5. Determination of the steady-state levels and metabolic stabilities of tRNA from
HeLa cell mitochondria • • • • • • • • • • • • • • • • • • • • • • • • 6. Analysis of proteins binding at the origin of replication of HeLa cell mtDN A • 7. Identification of the translation products specified by the
unidentified reading frames of human mtDNA •••••••••••••• 8. Human dihydrofolic acid reductase cDNA analysis . . . . . . . . . . . . 9. DHFR-specific sequences in chromosome fractions from V A2B-6A3 . . . . .
10. Organization of the DHFR gene and the amplified unit in a MTX-resistant human cell line . 11. Loss of double minute chromosomes and appearance of homogeneously staining regions in
a human MTX R cell line, V A2B-6A3 • • . • • • • • • • • • • • • • • • • • • • • 12. Chromosomal localization of the human dihydrofolate reductase gene in human cells 13. Isolation of dihydrofolate reductase deficient human cells • • • • • • • • • • • 14. Organization of the ribosomal RNA genes isolated from a human genomic library.
Eric H. Davidson - Summary • • • • • • • • • • • • • • • • • • • • • • • 15. A sea urchin gene represented in both maternal RNA and embryo nuclear RNA . 16. Sequence conservation in and around a sea urchin gene . . . . 17. Sequence polymorphism in regions surrounding structural genes. 18. Gene organization in sea urchin mitochondrial DNA . .... . 19. Mitochondrial transcripts in sea urchin embryo RNA . . . . . 20. Homologues of mitochondrial genes in sea urchin nuclear DNA . 21. Stage-specific expression of sea urchin embryo RNA sequences. 22. Interspersed polyadenylated RN As of sea urchin eggs and embryos 23. Structure and fate of sea urchin maternal RNA • 24. DNA transformation of sea urchin eggs . . . . . . . . . . 25. Culture of sea urchin embryos to sexual maturity. . . . . . 26. Organization and expression of actin genes in the sea urchin . 27. A study of the sea urchin "bindin" gene . . . . . . . . . . 28. Antibodies to proteins of the vitelline layer of sea urchin eggs 29. Interspersed middle frequency repeats in human and gorilla DNA . 30. Quantitative estimates of poly(A)+ mRNA in early mouse embryos 31. A cloned genomic library of a marine species of the phylum Bryozoa 32. Genome size and DNA complexity of Plasmodium falciparum
William J. Dreyer - Summary • • • • • • • • • • • • • • 33. Analysis of gp70-like molecules on murine tumors . . . . . 34. Detection on human cells of molecules resembling retroviral gp70 35. Genes related to retrovirus envelope genes in the human genome . 36. Structural studies of a human leukemia antigen . . . . . . . . 37. Melanoma surface antigen p97 is related to transferrin . . . . . 38. Protein chemical and recombinant DNA facility . . . . . . . . 39. Speculations on the role of mobile genes and cell-surface molecules in embryogenesis .
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Leroy E. Hood - Summary. • • • • • • • • • • • • • • • • • • • • , • • • • • • • • • • 37 40. Chromosomal arrangement of antibody heavy chain variable region genes • . . . • • • . . . . 38 41. Sequence organization of the T15 Ytt gene family . . . . . • . • . . . . . . . . • • • . • 39 42. Evolutionary analysis of a small immunoglobulin heavy chain variable region (V H) multigene family 39 43. Sequence organization of the J558 gene family in BALB/c mice . . . . . . . . • . . • . . . 40 44. Molecular genetics of anti~treptococcal antibodies. • . . . . . . . . . . . • . • . • . . . 41 45. Structure and evolution of human immunoglobulin gamma constant region genes . . . • . . . . 41 46. Molecular analysis of human B-cell differentiation using B-cell leukemias and immunodeficiencies 42 47. Rearrangement of cloned immunoglobulin genes introduced into B cell lines • . . . . . . 42 48. The molecular nature of the gene(s) encoding the T-cell receptor . . . . . . . . . . • . 43 49. Molecular dissection of the mouse major histocompatibility complex . . . . . . . . • • 43 50. Assignment of cosmid clones encoding mouse transplantation antigens to defined regions of
the mouse major histocompatibility complex. . . . . . • . 44 51. Studies of genes encoding H-2 molecules of the ''P" haplotype • • . . . . • . . . . 45 52. Structure of genes encoding mouse transplantation antigens • . . . • • • . . . • . 45 53. DNA sequence analysis of the Tia genes of the BALB/c mouse . . . . . . • • • • • 46 54. Identification of the coding functions of the cloned genes of the H-2 and Tla regions of
the mouse by DNA-mediated gene transfer . . . . • . . • • . . . . . . . . . . • 46 55. Studies on the gene structure, expression and function of mouse class I transplantation antigens 47 56. Generation of recombinant histocompatibility antigen genes and
analysis of their expression in mouse L cells . . . . .- • . . . . . . . . 47 57. Expression of histocompatibility genes in mouse teratocarcinoma cells . . 48 58. Biochemical analyses of mouse L cells transformed with class I genes of the
mouse major histocompatibility complex . • . . . • . • • • . . • . . 49 59. Chemical characterization of Ia antigens of the mouse major histocompatibility complex 49 60. Development of protien microsequencing methodology 49 61. Structural analysis of the acetylcholine receptor . 50 62. Generalized recombination in E. coli . • . . . 50 63. Miniplasmids, microplasmids, replicon modules. 51 64. Genes for reproductive hormones of Aplysia . . 51
Norman H. Horowitz - Summary • • • • • • • 53 65. Ornithine synthesis by an ornithine-deficient triple mutant of Neurospora 53 66. The function of cellular siderophores . . . . . 54 67. Search for siderophore receptors in Neurospora • . . . . • . • . • 54 68. Utilization of triacetylfusigen for iron transport . . . • • . . • . • 55 69. Isolation of an autonomously replicating plasmid in Neurospora crassa. 55
Elliot M. Meyerowitz - Summary • • • • . • • • • • • • • • • • • 56 70. Effect of a-ecdysone on RNA metabolism in Drosophila salivary glands 57 71. Transcript mapping of the 68C RNAs . . . . • . . . . . 58 72. Comparative sequence analysis of Drosophila glue proteins 58 73. Sequence analysis of 68C puff DNA. • . . . • . . . . . 59 7 4. Two ways in which the 68C puff is not controlled. . . . . 59 75. Molecular limits of the 68C glue puff . . . . • . • . • . 60 76. Analysis of the 68C cluster in Drosophila species other than D. melanogaster. 60 77. Mutagenesis of the 68C region . . . • . . . • . . . • . • . . . . . . 60 78. Characterization of mutations affecting development of the Drosophila eye 61 79. Preliminary characterization of the genome of Arabidopsis thaliana. 61
Herschel K. Mitchell - Summary • • • • • • • • • • • • • • • • 62 80. Developmental abnormalities in Drosophila induced by heat shock 62 81. The morphogenesis of cell hairs on Drosophila wings . . . . . . • 62 82. Gradients of differentiation in wild-type and bithorax mutants of Drosophila. 63 83. Effects of heat shock on messenger RNA synthesis, stability, and
translation in differentiating Drosophila wings . . . . . . 63 84. Changes in actin gene expression during wing development. . . . 64
James H. Strauss Jr. - Summary • • • . • • • • • • • • • • • 65 85. The 3'-noncoding regions of alphavirus RN As contain repeating sequences 65 86. Sequence studies of several alphavirus genomic RN As in the region containing
the start of the subgenomic RN A. • . . . . . . • . . . • . . . • 65 87. Comparative studies of the 51-terminal sequences of several alphavirus
genomic RN As and a family of their defective interfering RN As • 66 88. Construction of Sindbis virus defective interfering RN As in vitro . 66 89. Evolution of alphaviruses . . . . • . . . . . . . 66 90. Sequence analysis of Ross River virus 268 RNA 67 91. Intracellular transport of Sindbis virus glycoproteins 67 92. Analysis of the glycosylation of several alphaviruses 68 93. Studies on a small glycoprotein produced by Sindbis virus 68
94. Sequencing of the region of the Sindbis-genome encoding the nonstructural proteins .. 95. Study of the nonstructural proteins of Sindbis virus . 96. Flavivirus proteins . . . . . . . . . . . .
Barbera J. Wold - Summary • • • • • • • • 97. A selection system for LDL receptor function 98. Homologous recombination in animal cells. . 99. Isolation and characterization of cell lines resistant to high levels of mevinolin.
CELLULAR BIOLOGY AND BIOPHYSICS
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Howard C. Berg - Summary • • • • • • • • Adaptation in E. coli chemotaxis . . . . . . Signal processing times in E. coli chemotaxis The chemotactic impulse response . . . . . Chemiosmotic coupling to the flagellar motor and membrane ATPase of Streptococcus Dynamics of the flagellar motor of Streptococcus Effect of eosin on motility of Streptococcus . Mechanism of gliding motility • • • • • • • The phototactic response of Chlamydomonas. Chiasma interference in eukaryotic organisms The avoidance response in Phycomyces . . .
Charles J. Brokaw - summary • • • • • • • Bending patterns of Chlamydomonas flagella. Activation of non-motile flagella. . . . . . Movement of sperm flagella with and without a terminal filament Sulfate inhibition of flagellar motility Monoclonal antibodies to alpha tubulin •
Jolm J. Hopfield - Summary • • • • • • Emergent properties of neural networks , The dynamics of CO binding to heme proteins Electron transfer processes . . . . . . . .
Elias LaF.arides - summary • • • • • • • • Structural analysis of desmin and vimentin genes . Isolation of neurofilament protein and glial fibrillary acidic protein cDNAs Cyclic AMP-modulated phosphorylation of intermediate filament proteins in cultured avian myogenic cells . . . . . . . . . . . . . . . . . . . . . A possible role for synemin revealed by immunoelectron microscopy . . . . Biochemical characterization of the intermedaite filament associated protein, synemin Isolation of a new high molecular weight protein associated with desmin and vimentin filaments from avian embryonic skeletal muscle . . . . . . . . . . . Changes in the composition of intermediate filaments during muscle development Planar anisotropy in the avian erythrocyte plasma membrane . . . Widespread occurrence of avian spectrin in non-erythroid cells . . . Characterization of skeletal muscle filamin . . . . . . . . . . . The methylation of heat shock proteins at lysyl and arginyl residues. The effect of sodium arsenite on tropomyosin phosphorylation
Jean-Paul Revel - SUmmary • • • • • • • • • • • • • • • Tissue specificity of the gap junction protein • • • • • • • Studies to identify the gene coding for the gap junction protein. Genetic analysis of gap junctions in cultured mammalian cells The isolation of gap junctions from rat heart. . . . . . . Modulation of gap junction permeability. . . . . . . . . Orientation of the lens junctional protein in the membrane A neutron diffraction study of lens junctions. . . . Cell junctions in the development of feather germs .
CELLULAR NEUROBIOLOGY
Jeremy p. Brockes - summary • • • • • • • • • • • • • • • • • • 138. Antibodies to the voltage-sensitive sodium channel . . . . . . . . . 139. Characterization of Trembler mouse Schwann cells in vivo and in vitro 140. Latent infection of nerve cells by HSV-1 141. Glial growth factor •••••••••••••••••••••••
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A characterization of monoclonal antibodies generated against rat dorsal root cells . Studies on blastemal cells from regenerating limbs of Urodeles.
A. James Hudspeth - Summary • • • • • • • • • • • • • • • In vivo adaptation in hair cells and primary neurons of the bullfrog sacculus Motion of hair-cell stereocilia in the auditory receptor organ of the alligator lizard . Characteristics of voltage-, ion-, and time-dependent ionic conductances in isolated hair cells
Mary B. Kennedy - Summary. • • • . • . . . • • • • • • • • • Substrate specificity and possible "autophosphorylation" of rat brain calmodulin-dependent synapsin I kinase • • • • • • • • • • • • • Purification of synapsin I kinase • . • • • • • • • • • • • • • • Preparation and selection of hybridomas which produce monoclonal antibodies to partially purified synapsin I kinase
Henry A. Lester - Summary • • • Agonist concentration-jumps at nicotonic receptors. Characterization of single ionic channels opened by nicotinic agonists. The properties of the Bis-Q activated channel and the nature of desensitization A study of the nicotinic acetylcholine receptor using a photoisomerizable competitive antagonist . Studies of tissue-cultured cardiac muscle . . . . . . . . . . . . . . Photolabile proton donors and pH control of gap junctions in Chironomus Intracellular pH and gap junctional permeability in early embryos. Photosensitive calcium chelates . . . . . . . . . . . . . . . The application of a photoactivatable cAMP analogue to study the mechanism of afterdischarge in bag cell neurons . . . . • . . . Photoactivated cyclic nucleotides probe the kinetics of calcium channel regulation in heart Torpedo synaptosomes . . . . . . . . Physiology of nerve and muscle cultures. Soft X-ray microscopy . . . . . .
Felix Strumwasser - Summary • • • Calcium determination in bag cells • Agents that affect the neuronal circadian rhythm in the eye . Enhanced protein phosphorylation with exogenous protein kinase in extracts of Aplysia eyes during day versus night . . • . . . . . Altered patterns of protein phosphorylation in extracts of Aplysia eyes as a function of photoperiod. Mapping neuron~ activity in the eye of Aplysia wiwith high resolution ( H)2-deoxyglucose autoradiography. • • • • • • • • • • • • • • • Physiology of cultured Aplysia photoreceptors • • • • • • • • • • • • • • • • • • An economical real-time neuronal spike sorter . . . . . . . . . . . . . . • . . . High potassium stimulation of 35s-methionine incorporation into atrial gland peptide B Atrial gland peptide B immunohistochemistry . . • . . . • . . . . . . . • . . .
NEUROBIOLOGY AND BEHAVIORAL BIOLOGY
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Jolm M. Allman - Summary • • • • • • • • • • • • • • • • • • • The organization of the cortical visual areas in a strepsirhine primate. Ontogeny of monoaminergic receptors . . . . . . . Involvement of cyclic AMP in visual cortical plasticity . • . . . . . Energy metabolism in the visual cortex . . . . • . . . • • • . . . Quick changes in ocular dominance . . . . . • • . . . • . • . • . Antagonistic direction-specific mechanisms in Area MT in the owl monkey. Regrowth of central catecholaminergic fiber$ in cat visual cortex following localized lesions with 6-hydroxydopamine . . . . . • • . . . Morphology of catecholaminergic terminals in cat visual cortex . . . . . Distribution of $-adrenoreceptors in cat visual cortex . . . . • . . . • The "critical period" for plasticity in dark-reared cats; dependence on catecholamines. Segregation of geniculocortical afferent terminals in layer IV of cat visual cortex Illusions of depth produced by moving random dots • • • • • • • • • • • • • • Single unit electrophysiology in the CNS of two visually predatory arachnids . • . The structure and function of vocalizations in free ranging owl monkey . . . . . Physiological properties of norepinephrine-containing cells in cat locus coeruleus . Restoration of neuronal plasticity in cat visual cortex by stimulation of the locus coeruleus
Masakazu Konishi - Summary • • • • • • • • • • • • • • • • • • • • • • • • Neuronal control of bird song production . . . . . . . • • • . . • . . . . • . Intracellular staining and microanatomy of single neurons in song system brain slices
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Acoustic parameters underlying neuronal responses to song in a vocal control nucleus of white-crowned sparrows • • • • • • • • • • • • The auditory periphery: Extraction of binaural cues for sound localization.
Marianne I!. Olds - summary. • • • • • • • • • • • • • • • • • • • • Study of the effects [>roduced by the neurotoxin 6-hydroxydo[>amine injected neonatally in the rat Effects of [>ermanent de[>letion of do[>amine in the brain of the rat • • • • • • • • Behavioral and biochemical effects of deafferenting the hippocampus neonatally of its noradrenergic input . . . . • . . . . . . . . . • • . • . • . . Anatomical basis and function of learned enhanced neural responsiveness in the auditory system of the rat . . . . . . • . . • • • • • . • .
R. W. Sperry -SUmmary. • • • • • • • • • • • • • • • • • • • • Hemispheric differences in ability to recognize figure and background. Background influenee on perception of size and location in left and right hemispheres • Right/left processing of perspective cues for visual distance in commissurotomy subjects Visual field abnormalities in commissurotomy subjects • . . • • • • . . . . . • . . Naming of stimuli felt with the left hand following forebrain commissurotomy • • • • • Serial reversal learning: Two hemispheres are better than one and the left is better than the right Lexical decision and semantic facilitation in the split brain • . • • • • • . . Disconnection syndrome as a model for laterality effects in the normal brain. • Left hemisphere superiority for perception of sequentially-presented stimuli . . Right hemisphere superiority for processing mental images of rectangular solids Hemispheric specialization for oriented lines . . • . • • • . . . • . • . • Hemispheric differences in facial discrimination . . . . . • . . . • . • . • Sequential processing in the two hemispheres of split-brain monkeys • . • • • Interhemispheric communication in partially split-brain monkeys for perceptual processes Interhemispheric communication in partially split-brain monkeys during learning • . . •
David C. Van Essen - SUmmary. • • • • • • • • • • • • • • • • • • • • • • • • • Functional specialization for motion analysis in the middle temporal area of the macaque Analysis of motion in three-dimensional space in the middle temporal area of the macaque. Cortical and subcortical connections of Area MT in·the macaque • • • • • • • • • • • • Spatial organiz~tion of directionally-selective neurons in the middle temporal area of macaque. Functional properties of single cells in visual areas V2, VP and VA of ventral extrastriate cortex in the macaque . . • • • • • . . . • . . . . . • . Transformations in the visual representation in the retino-geniculo-striate pathway • The pattern of ocular dominance stripes in macaque striate cortex • Two stages of synaptic reorganization in the rabbit soleus muscle •• Mechanisms involved in the control of geniculate cell size in the cat
NEUROGENETlCS
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Seymour Benzer - summary • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Monoclonal antibodies against the Drosophila nervous system • • • • . . • . . . • . • . . . . . Identification of polypeptides recognized by monoclonal antibodies directed against Drosophila tissues. Monoclonal antibodies specific to nuclei. • • • • • • • • . • • • . Generation of monoclonal antibodies against Drosophila retina • • • . Monoclonal antibodies reveal antigenic profiles of Drosophila cell lihes Antibodies distinguish cell ty[>es in [>rimary culture of Droso[>hila CNS Lens-s[>ecific antibody binds to known lens-specific [>Olype[>tides • Whole mount staining procedure • • • • . . . . . . . . Cloning the Shaker gene • • • • • • • • • • • • • • • Single channel studies in Drosophila mutants. • • • . • • Giant fiber activation of direct flight muscles in Drosophila Antenna! physiology of Drosophila . . . . • . • . . . . Antenna! biochemistry . • • . • . . . . • . • . . . . cAMP phosphodiesterase in normal and dunce flies . . . . Dosage sensitivity and regulation of cyclic AMP phos[>hodiesterase 11 for the dunce memory mutant gene of Droso[>hila • • • • • • • • • • • • • • • • SU[>[>ression of the female sterility [>henotY[>e associated with the dunce memory mutant of Droso[>hila • • • • • • • • • • • • • • • • • • Hormonal regulation of cyclic nucleotide [>hOS[>hodiesterase activity in clonal Droso[>hila cell lines
Ronald J. Konopka - summary • • The chronotransposon hypothesis . Cloning the [>er locus • • • • • • Isolation of new clock mutants. . Temperature dependence of period in five clock mutants
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241. Mosaic studies of circadian and courtship song oscillations. . . • . . . • • . . 242. Genetic studies of sensory neuron projection patterns in Drosophila melanogaster.
DEVELOPMENTAL GENETICS
l!dward B. Lewis - Summary • • • • • • • • • • • • • • • • • • • • • • • • 243. Localization of bithorax complex (BX-C) and antennapedia complex (ANT-C) gene
activities along the body axis of Drosophila . . • . • . • . . • . . 244. A search for a new cis-regulatory region within the bithorax complex . 245. Mitochondrial DNA polymorphism in Drosophila • • • • • • • • • •
BIOLOGY/CHEMISTRY GRADUATE STUDENTS
246. A cluster of Drosophila cuticle genes • • • • • • • • • • • • • • • • • • • 247. Characterization of genes encoding muscle proteins in Drosophila melanogaster 248. Molecular biology of the acetylcholine receptor from Torpedo • 249. Developmentally regulated expression of Drosophila actin genes 250. Do Drosophila actin genes require intrans for expression? . • .
Graduates • . . . . . • • . • . . . Financial Support • • • • • • • • • • Author Index (by page number). • • • • Financial Support Index (by page number)
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INTRODUCTION
Rothenberg
Wold
3
During the past year, our attention has focused on completion of the new Braun Laboratories in Memory of Carl F and Winifred H Braun. The exterior and the surrounding landscaping, shown in final form in the photograph on page 6, were completed in May. Lee Hood's research group began preparations for their big move later in the summer, after completion of additional work to set up laboratory facilities inside the building.
Tiie Faculty
Dr. Ellen Rothenberg, arrived in June to begin work as Assistant Professor of Biology. Ellen did her undergraduate work in biochemistry at Harvard, and then went to work with. David Baltimore at M.I. T. for her Ph.D. work. Her developing interest in immunology led her to the Sloan-Kettering Cancer Center, where she was a Jane Coffin Childs postdoctoral fellow, and then to the Salk Institute. She is interested in cellular immunology, both for its own sake and as a model developmental system. Focusing on the immune system of mice, she is studying the changes in gene expression that take place in T lymphocytes during their functional maturation and the mechanisms by which hormones and cell-cell contacts drive this process. This involves examination of the synthesis of specific polypeptides and their messenger RNAs in different subpopulations of cells isolated from the thymus. Ellen will be joined later this year by our two other new faculty, who will move into the Braun Laboratories, Professors John Abelson and Melvin Simon.
Earlier this year, we welcomed Dr. Barbara Wold, who returned to. our Division as Assistant Professor of Biology. Barbara did her undergraduate work in zoology, at Arizona State University, and then came to Caltech to do her Ph.D. work in developmental biology with Professor Eric Davidson. Since receiving her Ph.D. in 1978, she has been a postdoctoral fellow at Columbia University. Barbara has set up her laboratories at the east end of the first floor of the Kerckhoff Laboratories. Barbara•s research, which focuses on the use of modern molecular biological techniques to gain understanding of the function of cell surface receptor proteins, is described on pages 70-72 of this report.
The ftrrival of a new faculty member who is a Caltech Ph.D. is particularly appropriate this year, because Professor Norman Horowitz, who received his Ph.D. from our Division in 1939, retired from our faculty at the end of June. Norman began his work here studying the development of marine embryos under the direction of Professor Albert Tyler. Subsequently, he came under the influence of Professor George Beadle, and began an association that resulted in an appointment to our faculty in 1947, the development of Neurospora as a definitive model system for biochemical genetics, and ultimately to the 11one gene -- one protein" hypothesis which is now difficult to imagine as revolutionary. Subsequently, Norman1s developing interest in the problem of the origin of life in prebiotic environments led him to involvement with the search for life on Mars, and he went on a part-time basis to the Jet Propulsion Laboratory to be head of its Biosciences Section from 1965 to 1970. After this project was completed, he served as Executive Officer for the Biology Division from 1971-1976, and then for three years as our Division Chairman. His most recent research, described on pages 53-56, continues to reflect his interest in adaptations of organisms to life under harsh environmental conditions, such as the Martian environment.
Peter Lowy, Senior Research Associate in Biology, also retired at the end of June. Peter came to Caltech in 1949 to work with Professor Henry Borsook. Following Professor Borsook's retirement in 1968, Peter worked with several members of our Division, principally Professor Herschel Mitchell, and also served our Division by filling the position of Radiation Safety Officer and taking responsibility for many other duties related to safety within our Division.
Both Norman and Peter will be staying in Pasadena, and we will hope to see them frequently.
Horowitz Lowy
4
Honors and Awlll'ds
As our cover proclaims, this year was an exceptional one, with the award of the 1981 Nobel Prize in Physiology and Medicine to Professor Roger Sperry, thus ending many years of speculation by his friends and colleagues who felt that he would eventually receive this highly deserved award. Roger received half of the 1981 prize in recognition of his contributions to understanding the function of the human brain-specifically his work on the functional specialization of the two hemispheres of the brain and the new insights which his work provided into the implications of the specialization and separateness of the two hemispheres. When the prize was announced on October 9, 1981, Roger was found to be away on a camping and fishing expedition in Baja, California-leaving his colleagues to deal with the hullabaloo associated with the prize announcement. Roger and Norma went to Stockholm in December to accept the prize at the ceremonies on December 10, an experience that Roger heartily recommends to all of us! His friends ·and colleagues in the Division of Biology and in the Institute celebrated with a party on April 6, 1982; some scenes from that occasion are shown on the next page, as well as other mementos of Roger's illustrious achievements.
Our Division Chairman, Professor Leroy Hood, was elected to membership in the National Academy of Science and also the American Academy of Arts and Sciences. Professor Hood also was selected as the Stanhope Bayne-Jones Memorial Lecturer, Johns Hopkins Medical School; Carter-Wallace Lecturer, Princeton University; and Marrs McLean Lecturer, Baylor College of Medicine.
Professor Seymour Benzer gave The John M. Prather Lectures, at Harvard University, and The Croonian Lecture to the Royal Society, London.
Associate Professor Jeremy P. Brockes was awarded a McKnight Foundation Neuroscience Development Award for advanced research.
• Professor Eric H. Davidson was appointed the Norman Chandler Professor of Cell Biology, and was elected Fellow of the
American Association for the Advancement of Science.
Professor Edward B. Lewis was awarded an honorary Ph.D. Degree by the University of Umea, Sweden.
Assistant Professor Elliot M. Meyerowitz was awarded an Alfred P. Sloan Research Fellowship; Sigma Xi Grant-in-Aid of Research associated with the Procter Prize (which was previously awarded to George w. Beadle, our first Division Chairman).
Professor Elias Lazarides' graduate student Bruce Granger won the 1982 Milton and Francis Glauser Doctoral Prize for the greatest degree of originality in his Ph.D. thesis research.
Professor Mark Konishi's graduate student Mark Gurney won the 1981 Donald Lindsley Prize for the most outstanding Ph.D. Thesis in Behavioral Neuroscience submitted during 1980-1981.
Associate Professor David Van Essen's graduate student John Maunsell has received an Intra-Science Research Foundation Graduate Student Award for 1982.
Hood Davidson
Scenes from reeeption at the Athenaeum
Roger end Swedish students
Roger at Oberlin College graduation year 1935
Wife, Norma, and Queen Astrid of Sweden
Daughter, Janeth Sperry, next to Ammonite discovered by Roger--t>ossibly largest
in the world
6
The Albert Tyler Memorial Lecture -- 1981
On November 17, 1981, the ninth Albert Tyler Memorial Lecture was given by Dr. Karl Illmensee, of the University of Geneva. This lecture, nExperimental Genetics of the Mammalian Embryo," described Dr. Illmensee's pioneering studies using nuclear transplantation and transformation methods to construct mouse embryos containing new combinations of genetic information, and to provide new understanding of the developmental potentialities of the genomes of different cells in the mammalian embryo.
mmensee
Bratm Laboratories
BIOLOGY DIVISION STAFF
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STAFF OF INSTRUCTION AND RESEARCH DIVISION OF BIOLOGY
Leroy E. Hood, Chairman Charles J. Brokaw, Associate Chairman A. James Hudspeth, Executive Officer James H. Strauss, Executive Officer
Professors Emeriti
Professors
9
Biology Biochemistry
. Genetics Biology
Physiology
• Biology . James G. Boswell Professor of Neuroscience
Biology Biology
Norman Chandler Professor of Cell Biology Biology
Biology and Applied Science Ethel Wilson Bowles and Robert Bowles Professor of Biology
Roscoe G. Dickinson Professor of Chemistry and Biology Biology Biology
Bing Professor of Behavioral Biology Thomas Hunt Morgan Professor of Biology
Biology Biology
• Albert Billings Ruddock Professor of Biology
Associate Professors
Assistant Professors
Sherman Fairchild Distinguished Scholars
Hixon Professor of Psychobiology Biology
Biology Biology Biology Biology Biology Biology
Biology Biology Biology Biology
Biology Biology Biology
Distinguished Garnegie Senior Research Associate
Roy J. Britten, Ph.D. • Biology
10
Charles R. Hamilton, Ph.D. Barbara R. Hough-Evans, Ph.D. Takuji Kasamatsu, M.D., Ph.D. Peter H. Lowy, Doctorandum . Marianne E. Olds, Ph.D.
Carol H. Sibley, Ph.D.
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Henry v. Huang, Ph.D. Michael w. Hunkapiller, Ph.D. Lawrence M. Kauvar, Ph.D. Jeanne M. Nerbonne, Ph.D. Roger M. Perlmutter, M.D., Ph.D. Nancy S. Petersen, Ph.D . . Carol Readhead, Ph.D. M. Viswanath Reddy, D.Sc. John w. Roberts, Ph.D. Janet M. Roman, Ph.D. Ignacio Sandoval, M.D., Ph.D. Ellen G. Strauss, Ph.D. Mark A. Tanouye, Ph.D. Terry L. Thomas, Ph.D. S. Barbara Yancey, Ph.D.
Senior Research Associates
Visiting Professor
Biology Biology Biology Biology Biology
Biology
Visiting Associates
Pitzer College Karolinska Institute, Stockholm
Huntington Research Institute, Pasadena DNAX Research Institute, Palo Alto . The Australian National University
Arizona State University Universidad Central de Venezuela
Faculte des Sciences de Poitiers Stevens Institute of Technology
• University of Montreal University of Alberta
University of Southern California . Jet Propulsion Laboratory
Universidad Complutense de Madrid . Universita di Pavia
• Wakayama Medical College . University of Tour;;, France
Applied Molecular Genetics, Inc. Weizmann Institute, Rehovot
Veterans Administration Medical Center .Centre National de la Recherche Scientifique, Paris
. Yale University University of Chicago
University of Southern California School of Medicine • Stanford University
California State University, ·Los Angeles . University of California, Los Angeles
Senior Research Fellows
Del Webb Research Fellows
Lee D. Chabala, Ph.D. Thomas Holton, Ph.D.
Christopher R. Kintner, Ph.D. Terry T. Takahashi, Ph.D.
Biology Biology Biology Biology Biology Biology Biology Biology Biology Biology Biology Biology Biology Biology Biology
Hans Lennart Adler, Ph.D. John R. Bell, Ph.D. Ingrid Blikstad, Ph.D. Andreas Burkhalter, Ph.D. Carlos V. Cabrera, Ph.D. Lars Carlsson, Ph.D. Anne Chomyn, Ph.D. Camilo A. L. S. Colaco, Ph.D. Cheryl M. Corsaro, Ph.D. Claus-Jens W. Doersen, Ph.D. Richard H. Douglas, Ph.D. Lawrence C. Fritz, Ph.D. Shinobu c. Fujita, Ph.D. David L. Gard, Ph.D. Robert S. Goodenow, Ph.D. Johanna A. Griffin, Ph.D. Karen Goldman Herman, Ph.D.
Carlos F. Arias-Ortiz, B.S., M.S. Mark K. Bennett, B.S. Steven M. Block, B.A. Beverley J, Bond, A.B. George J. Carman, A.B. Stephen T. Crews, B.A. Alice M. Cronin-Golomb, B.A. Madeline A. Crosby, B.S. Thomas E. Crowley, B.S. Kurt Eakle, B.S. Ruth A. Eatock, M.Sc. Jay w. Ellison, B.S. Ngozi E. Erondu, B.S., M.B. Douglas A. Fisher, A.B. Karl J. Fryxell, B.A., B.S. George L. Gaines III, B.S. Boning Gao, B.S. Mark D. Garfinkel, B.A. Richard H. Gomer, B.A. Herman Gordon, B.A. Bruce L. Granger, B.A. Steven H. Green, B.S.
Gosney Research Fellows
Yassemi Capetanaki, Ph.D. Ian W. Duncan, Ph.D.
Constantin N. Flytzanis, Dr.rer.nat John W. Grula, Ph.D.
Janine Perlman, Ph.D. Charles M. Rice III, Ph.D.
Research Fellows
Akira Ishihara, Ph.D. Howard T. Jacobs, Ph.D. Steven A. Johnson, Ph.D. Shahid M. M. Khan, Ph.D. Joan A. Kobori, Ph.D. Ellen B. Kraig, Ph.D. Leslie S. Leutwiler, Ph.D. Paolo Mariottini, Ph.D. EveLynn McGuinness, Ph.D. Andrew P. McMahon, Ph.D. Andrew Moiseff, Ph.D. Charlotte K. Omoto, Ph.D. Lee K. Opresko, Ph.D. Anders Orn, Ph.D. Maureen G. Price, Ph.D. Elizabeth A. Repasky, Ph.D. Samuel J. Rose III, Ph.D.
Graduate Students
Tim Hunkapiller, B.S. Kent R. Jennings, B.Sc. Lawrence C. Katz, B.A. Stuart K. Kim, B.A. Michael King, B.A. Mitchell Kronenberg, B.A. Mauri E. Krouse, B.S. Baruch Kuppermann, A.B. James L. Lee, B.S. Greg Erwin Lemke, S.B. David E. Levy, B.A. Richard S. Lewis, B.S. Donna L. Livant, B.A. Susana Lopez-Charreton, B.S. Jeffrey N. Masters, B.S. William W. Mattox, B.S. John H. R. Maunsell, B.S. Jeffrey T. Mayne, S.B. James S. Mccasland, B.S., B.A. Paul W. Meyer, B.S. Katharine S. Mixter, B.A. David A. Myers, B.A.
Members of the Prof,...ional Staff
Gisela w. Charlang, Ph.D. Suzanna J. Horvath, Ph.D. Francis M. Miezin, MSEE
Shigeru Sakonju, Ph.D. Margit Schardin, Ph.D. Robert E. Sheridan Jr., Ph.D. Rosemary J. Shott, Ph.D. Donald J, Silvert, Ph.D. Michael Steinmetz, Ph.D. Iwona T. Stroynowski, Ph.D. Katherine A. Stygall, Ph.D. David B. Teplow, Ph.D. Tadmiri R. Venkatesh, Ph.D. Kazushige Watabe, M.D. Martin M. Weinstock, Ph.D. Jiyoung K. Yang, Ph.D. Martha C. Zuniga, Ph.D. Stephen L. Zipursky, Ph.D.
Jay J. Myers, B.A., M.A. John J. Ngai, B.A. Bruce J. Nicholson, B.Sc. Dominic Orr, B.Sc. Jing-hsiung James Ou, B.S. Vann Parker, B.S. James W. Posakony, B.S. Robert E. Pruitt, B.S. Antonio A. Reyes, B.S., M.S. Arthur Roach, B.Sc. Loveriza A. 8armiento, B.S. Jeffrey E. Segall, B.A. Beverly Taylor Sher, B.A. Sandra L. Shotwell, A.B. David W. Sivertsen, B.S. Randall F. Smith, B.S. Michael P. Snyder, B.A. Yi Henry Sun, B.S. Chung Wang, B.S., M.S. Astar Winoto, B.A. Joanne M. Yeakley, B.S. Lei Yu, S.S., M.S.
11
12
Maria Alonso, B.S. Helen Alvarez Cynthia Akutagawa Eugene Akutagawa, B.S. Carolyn Anderson, B.A. David J. Baker Rollin H. Baker' B. A., M.S. Carlzen Balagot A. Anthony Balber Fargo Balliett, B.S. Bennett J. Berson, B.S. Amy M. Canada Jeffrey D. Carpenter Paul K. Cartier Ill, A.B. Alessandra Cellini, Ph.D. Susanna Chan Su-Ming Chiang, B.S., M.S. Maria A. Clancy M. Patricia Conley, B.S. Michael Connolly, B.S. Adriana Cortenbach Loring G. Craymer III, Ph.D. Trau Cuong Maria A. DeBruyn Arthur W. DeJohn Michael Douglas, M.S. Arger L. Drew Jean E. Edens Eveline Eichenberger Vincent R. Farnsworth, M.A. Peggy Feyen Doris T. Finch, B.A. Eef Goedemans Maria R. Gomez
Manuel Acevedo-Ruiz John J. Beahan Alison Blake R. J. Brandenburg Kimberly D. Carr Steven Chin Reese E. Faucette Lisa L. F!itz Tracy T. Furutani
Research Staff
Leila Gonzalez Margaret M. Griffith Less B. Grim, B.A. Luci Hansen, A. A. Wanetta Harrington, B.A. Nancy I. Harris Richard L. Hudspeth, B.S., M.S. Richard A. Jacobs, B.S., M.S. Jeannette Johnstone Bertha E. Jones Gertrude Jordan Bennetta T. Keeley Chin Sook Kim, M.S. Rosina K. T. Kinzel Patrick F. Koen Susan Shu-Ai Tsai Lai, B.S. Fred R. Larsen Patrick S. Leahy, B.S. Sharon W. Lee, B.A., M.S. Thomas E. Lee, B.S. Edith M. Lenches, B.S. Ilga Lielausis Eva H. Lujan Lois E. MacBird Mary J. Macchi Josephine Macenka, B.S. Susan L. Mailheau, B.A., B.S. Janet M. McNicholas Amanda E. Milgram, A.B. Laurie S. Minamide, B.A. Karyl Minard, B.S. Barbara Moore, B.S. Sandra M. Nakada, B.S. Bradford Ng, B.S.
Student Assistants
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Kethleen R. Sheedy Eric Sinn Steven R. Swanson John C. Terrell David P. Watkins Jeanne M. Weaver Yi-Wang Wong Fonda Wu
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Lody Kempees, Supervisor Sandra L. Carta Ruth M. Erickson
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13
MOLECULAR BIOLOGY AND BIOCHEMISTRY
Giuseppe Attardi
Roy J. Britten
Eric H. Davidson
William J. Dreyer
Leroy E. Hood
Norman H. Horowitz
Elliot M. Meyerowitz
Herschel K. Mitchell
James H. Strauss Jr.
Barbara J. Wold
Professor: Giuseppe A ttardi Visiting Associates: Nestor E. Gonzalez-Cadavid, Julio
Montoya, Monica Mottes Research Fellows: Anne Chomyn, Claus-Jens W. Doersen,
Paolo Mariottini, Jiyoung K. Yang Visiting Scientist: Alessandra Cellini Graduate Students: George L. Gaines Ill, Michael P.
King, Jeffrey N. Masters, Barry J. Maurer* Research staff: Doris T. Finch, Benneta R. Kelley, Susan
Shu-Ai Tsai Lai Laboratory Staff: Arger L. Drew, Maria R. Gomez,
Rosina K. T. Kinzel, Wanda L. Owens
*Division of Chemistry and Chemical Engineering, California Institute of Technology.
SUpport: The work described in the following research reports has been supported by:
Biomedical Research Support Grant (NIH) Cenci Bolognetti Foundation, Rome National Institutes of Health, USPHS
Summary: Our laboratory has been pursuing for some
time an in-depth analysis of the informational content and
of the gene organization, expression and control in human
mitochondrial DNA (mtDNA). The understanding of the
basic features of this system has made rapid progress in
the past three years with the determination of the
complete sequence of human mitochondrial DNA in Dr. F.
Sanger's laboratory in Cambridge and the parallel work
carried out in our laboratory on the detailed organization
of the mitochondrial DNA transcripts and on their
structural and metabolic properties. This work has
revealed the extraordinary degree of compactness and
economy of this genome and its simplified and, at the
same time, highly evolved mode of expression. The
analysis of mitochondrial DNA transcription has led us to
propose a model (tRNA punctuation model) whereby the
tRNAs, rRNAs and mRNAs encoded in the heavy strand
are transcribed in the form of polycistronic molecules in
which tRNA sequences separate with nearly absolute
regularity the rRNA and mRNA sequences, being in most
cases butt-jointed to them. These polycistronic molecules
are destined to be processed to mature species by precise
endonucleolytic cleavages, occurring in general
immediately before and after a tRNA sequence: an
enzyme (or enzymes) that recognizes the cloverleaf
structure of tRNAs, like the RNAse P of bacteria, is
presumably involved in this process. The mechanism by
which a differential control of expression of the rRNA,
tRNA and protein coding genes is achieved in this unique
system has been the focus of much interest in our
laboratory in the past year. Particular attention has been
17
given to the identification of the initiation sites for
transcription, to the elucidation of the mode by which the
differential rate of rRNA and mRNA synthesis is produced
and to the analysis of the mechanism of RNA processing.
Evidence has been obtained for the occurrence of two
initiation sites of heavy strand transcription, one located
very near to the 5' end of the 12S rRNA gene and the
other 20 to 40 nucleotides upstream of the tRNA Phe gene
that flanks the 12S rRNA gene on the 5' side. The
existence of two initiation sites for heavy strand
transcription can be correlated with the previously
obtained evidence pointing to two different heavy strand
transcription events, one leading to the synthesis of a
polycistronic molecule corresponding to almost the entire
heavy strand, and the other restricted to the rDNA region
and responsible for the synthesis of the bulk of rRNA.
The latter transcription of the rDNA region is pre
maturely terminated at the 31 end of the 168 rRNA, which
can be folded in a stem-loop structure resembling in a
rudimentary form the hairpin-oligo(U) signal postulated
for bacterial termination-attenuation. Which of the two
heavy strand promoters controls rRNA synthesis and how
the use of the two promoters is regulated in order to
modulate the relative rates of rRNA and mRNA synthesis
is at present being investigated. An in vitro transcription
system has been developed that will prove to be useful for
dissecting the process of initiation of transcription and its
control. In another line of investigation, an in vitro assay
has been developed for the identification and isolation of
the mitochondrial RNA processing enzyme(s) operating in
the formation of the mature rRNA, tRNA and mRNA
species from the polycistronic precursor molecules.
Another problem concerning the human mitochondrial
genetic system that has been the object of inquiry in our
laboratory in the past year has been the nature of the
polypeptides encoded in the eight unidentified reading
frames (URFs) of human mitochondrial DNA, which make
up about 40% of its information content. In collaboration
with Dr. Russell Doolittle of the University of California
at San Diego, we have started a project aimed at
identifying these polypeptides by using antibodies
prepared against synthetic peptides corresponding to
appropriate regions of the reading frames. The first
results obtained using this approach applied to cytochrome
c oxidase subunit II have been fully successful, justifying
the expectation that it may be possible in the near future
to identify several of the proteins encoded in the URFs.
18
This should open the way to the isolation of these proteins
and to their further characterization.
A considerable part of the activity of the laboratory
has continued to be devoted in the past year to the
analysis of the dihydrofolic acid reductase (DHFR) gene
amplification in human cells in culture. Significant
progress has also been made in this area. Thus, screening
of phage A libraries of DNA from different chromosome
fractions of a methotrexate-resistant human cell line and
from the parental cell line, has led to the isolation of
presumably all the genomic fragments constituting the
DHFR gene; these are at present being analyzed in order
to determine the organization of the gene. Analysis of
DNA from double minute chromosomes has provided
evidence for rearrangements of some of the DHFR genes.
Furthermore, a systematic analysis of the evolution of the
chromosomal constitution of one of the methotrexate
resistant cell lines has revealed a complex relationship
between the double minute chromosomes and the
"homogeneously staining regions11 located in identifiable
chromosomes.
1. TRANSCRIPTION INITIATION SITES AND rRNA GENE TRANSCRIPTION IN HUMAN MITOCHONDRIAL DNA
Investigators: Julio Montoya, Giuseppe Attardi
The sequence analysis of the human mitochondrial
RNAs and their alignment with the DNA sequence have
revealed that the H-strand sequences coding for the
rRNAs, poJy(A)-containing RNAs and tRNAs are
immediately contiguous to each=°ther, extending con
tinuously from coordinate 2/100 to coordinate 95/100
(relative to the origin of replication taken as 0/100). This
arrangement is consistent with a model of transcription of
the H strand in the form of a single molecule that is
processed by precise endonucleolytic cleavages before and
after each tRNA sequence to yield the mature products
(Montoya et al., 1981; Ojala et al., 1981; Attardi et al.,
1982).
To localize the initiation sites of transcription of the
heavy and light strands of HeLa cell mitochondrial DNA,
two approaches have been followed: (1) "capping" in vitro
the mitochondrial RNA with (a-32P)-GTP and vaccinia
virus guanylyltransferase and mapping the "capped" ends
by DNA transfer hybridization and Sl nuclease protection
experiments (in collaboration with T. Christianson, D.
Levens and M. Robinowitz at the Department of Medicine,
Biochemistry and Biology, University of Chicago), and
(2) Sl nuclease protection experiments utilizing nascent
RNA molecules isolated from transcription complexes.
Using these approaches, a main initiation site for the
heavy strand transcription and one for the light strand
transcription have been identified near the origin of HeLa
cell mtDNA replication.
The rate of synthesis of the rRNA species is 20 to 60
times higher than that of most of the mRNAs (Attardi
et al., 1982). It has been proposed that a premature
termination of transcription at the 3' end of the 168 rRNA
cistron would produce a larger molar yield of the rRNA
species relative to that of the mRNAs and of the majority
of the tRNAs. To study in more detail the mode of
transcription of the rDNA region of the HeLa cell
mtDNA, the individual RNA species encoded in this region
have been analyzed by RNA sequencing, measurements of
kinetics of labeling and high resolution mapping experi
ments. The results indicate the existence of a dual
pathway of transcription of the rDNA region, which
probably plays a major role in the regulation of the
relative rates of rRNA and mRNA synthesis. One
pathway is a part of the process of continuous tran
scription of nearly the entire H strand. In particular,
processing of nascent transcripts and poly(A) addition at
the 31-terminal nucleotide of the 168 rRNA or very close
to it results in the formation of a polyadenylated RNA
species (RNA 4) that corresponds precisely in mapping
position to the whole rDNA region. This polyadenylated
RNA 4 is probably further processed to give rise to a
small fraction, polyadenylated, of 16S rRNA (RNA 10).
The other pathway of transcription of the rDNA region
produces transcripts that are terminated at or very near
the 3' end of the 168 rRNA; the 3'-end terminal region of
this RNA can be folded into a stem-loop structure that
resembles, in a rudimentary form, the hairpin-oligo(U)
signal postulated for bacterial termination-attenuation.
These terminated transcripts are the immediate pre
cursors of the bulk of the mature 16S rRNA and 12S
rRNA.
References: Attardi, G., Cantatore, P ., Chomyn, A., Crews, 8.,
Gelfand, R., Merkel, C., Montoya, J. and Ojala, D. (1982) In: Mitochondrial Genes. P. Slominski, P. Borst and G. Attardi (Eds.), pp. 51-71. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Montoya, J., Ojala, D. and Attardi, G. (1981) Nature 290, 465-470.
Ojala, D., Montoya, J. and Attardi, G. (1981) Nature 290, 470-474.
2. SEQUENCE ANALYSIS AND PRECISE MAPPING OF THE 3' ENDS OF THE HUMAN MITOCHONDRIAL RIBOSOMAL RNAs
Investigators: Julio Montoya, Donald T. Dubin*, Kathleen D. Timko*, Giuseppe Attardi
The 3'-terminal segments of the two HeLa cell
mitochondrial rRNA species have been sequenced by
subjecting 3'-end labeled samples of highly purified
components to RNAse T1 or RNAse A digestion, finger
printing the products and further characterizing them by
secondary enzymatic digestion. The results, when
correlated with the mtDNA sequence, clearly indicate
that the 31-end nucleotide of the transcribed moiety of
almost 90% of the 12S rRNA molecules corresponds in the
DNA sequence to a nucleotide immediately contiguous to
the following tRNA Val gene, while the transcribed moiety
of the remainder of the 12S rRNA molecules is shorter by
one, or perhaps two or three, nucleotides. Both the major
and the minor 128 rRNA species have been found to be in
their majority oligoadenylated at their 3' ends, exhibiting
mostly stretches of 1 to 5 As. A similar analysis of the
16S rRNA has shown the occurrence of two major types of
transcribed moiety comprising about 90% of the total, and
several minor ones. Some ambiguities in the precise
localization in the mtDNA sequence of the 3'-terminal
residues of the 168 rRNA transcripts have been resolved
by high resolution mapping experiments utilizing the s 1 protection technique and analysis of the products on
sequencing gels. The results clearly show that the 31
termini of the two major types of transcribed moiety
correspond in the DNA sequence to the nucleotide
immediately adjacent to the tRNA Leu gene or to the
preceding one; furthermore, the 3' ends of the minor types
of transcribed moiety form with those of the major types
a cluster of nucleotides, mostly contiguous, in the mtDNA
sequence. All species of transcribed moiety have been
found to be oligoadenylated. The present results point to
a certain imprecision in the process leading to the
formation of the 3' ends of 16S rRNA and, together with
other data, support a model whereby termination of
transcrition, rather than processing, is responsible for
generating these 31 ends. The observation of oligo
adenylation in the HeLa cell mitochondrial rRNAs
confirms and extends similar findings in the homologous
hamster RNAs, and suggests a general role of adenylation
in the processing or termination of mtDNA transcripts.
*Department of Microbiology, CMDNJ-Rutgers Medical School, Piscataway, New Jersey.
3. SEARCH FOR THE MITOCHONDRIAL RNA PROCESSING ENZYME(S)
Investigator: Claus-Jens W. Doersen
19
The sequence analysis of human mitochondrial RNAs
has shown, by comparison with the DNA sequence
(Anderson et al., 1981), that the sequences of the heavy
strand of mitochondrial DNA coding for the rRNAs and
poly(A)-containing RNAs are in nearly every case
immediately contiguous at both ends to a tRNA coding
sequence (Crews and Attardi, 1980; Montoya et al., 1981;
Ojala et al., 1981). This unique genetic arrangement,
together with other data from this laboratory, supports a
model whereby the heavy strand of DNA is transcribed as
a single polycistronic RN A which is processed by precise
endonucleolytic cleavages, punctuated by the tRNA
sequences, to yield the mature rRNAs, poly(A)-containing
RN As, and tRN As.
An investigation aimed at identifying the enzyme(s)
involved in the processing of mitochondrial RNAs has
begun with the construction of a pBR322-derived plasmid
containing an insert of mitochondrial DNA with tRNA
coding sequences. This plasmid can be transcribed in vitro
utilizing a phage-specific promoter to yield an RNA
species containing primarily mitochondrial DNA
sequences. Experiments are currently in progress to test
the appropriateness of in vitro synthesized RNA as a
substrate for in vitro RNA processing activity in
fractionated mitochondrial preparations.
References: Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M.
H. L., Coulson, A. R., Drouin, J., Eperson, L c., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Stader, R. and Young, I. G. (1981) Nature 290, 457-465.
Crews, S. and Attardi, G. (1980) Cell 19, 775-784. Montoya, J., Ojala, D. and Attardi, G. (1981) Nature 290,
465-470. Ojala, D., Montoya, J. and Attardi, G. (1981) Nature 290,
470-474.
4. AN IN VITRO TRANSCRIPTION/TRANSLATION SYSTEM
Investigators: George L. Gaines, Gary G. Gibbs*
We are actively developing a functional in vitro
transcription and translation system with isolated mito
chondria. Many of the questions involving gene expression
in the mitochondria require these systems from both
feasibility and manipulation aspects. Currently we are
studying the parameters for maximum labeling of RNA
species using radioactive precursors. ATP concentration
20
has profound effects upon the efficiency of the
transcription system. Further investigations include
parallel studies on in vivo transcription/translation,
effects of drugs upon the in vitro systems, and potential
mitochondrial-nuclear interactions.
*Undergraduate, California Institute of Technology.
5. DETERMINATION OP THE STEADY-STATE LEVEUI AND METABOLIC STABILITIES OP tRNA PROM HeLa CELL MITOCHONDRIA
Investigator: Michael P. King
In an effort to understand better the transcription of
the HeLa cell mitochondrial genome, work has begun to
quantitate the relative amounts of tRNAs and to investi
gate their metabolic properties. Initially, mitochondrial
RNAs from in vivo, long-term labeled cells are isolated
from EDTA-washed mitochondria by sedimentation in a
sucrose density gradient. The 4S peak is then run on an
acrylamide-urea gel; bands detected by ethidium bromide
are cut, eluted and further purified by ion-exchange
chromatography. These purified tRNAs are hybridized to
separated strands of mtDNA to determine the relative
steady-state levels of light and heavy strand transcripts.
The metabolic properties of the tRN As will be determined
by measuring their kinetics of accumulation using labeled
RNA precursors. Future studies will use specific frag
ments of mtDNA containing individual tRNA genes or a
cluster of tRNA genes. Of particular interest are the
three tRNAs whose genes surround the 12S and 16S rRNA
genes because of the different pathways of transcription
in this area (see Abstract No. 1).
6. ANALYSIS OP PROTEINS BINDING AT THE ORIGIN OP REPLICATION OP HeLa CELL mtDNA
Investigator: Michael P. King
A previous investigation has shown the association of a
protein complex or membrane fragment with HeLa cell
mtDNA at or near its origin of replication (Albring et al.,
1977). Crosslinking mtDNA in situ with psoralen deriva
tives indicates the presence of a protected region near the
origin of replication in vivo (De Francesco and Attardi,
1981). This evidence, indicating that the mtDNA is
attached in vivo to the inner mitochondrial membrane, is
of particular interest because the region bound contains
both the origin of replication and the proposed promoters
of heavy and light strand transcription (see Abstract
No. 1). Preliminary work is focusing on the isolation and
characterization of the protein(s) specifically associated
with the mtDNA in this complex.
References: Albring, M., Griffith, J. and Attardi, G. (1977) Proc. Nat.
Acad. Sci. USA 74, 1348-1352. De Francesco, L. and Attardi, G. (1981) Nucleic Acids
Res. 9, 6017-6030.
7. IDENTIFICATION OF THE TRANSLATION PRODUCTS SPECIFIED BY THE UNIDENTIFIED READING FRAMES OF HUMAN mtDNA
Investigators: Paolo Mariottini, Anne Chomyn, Giuseppe Attardi
The mitochondrially synthesized polypeptides number
up to 26, as determined by bidimensional SDS-gel electro
phoresis of proteins radioactively labeled with 35smethionine in the presence of emetine (Ching, 1980).
Three of the bands in a unidirectional separation of these
proteins have been identified as being the three largest
subunits of cytochrome c oxidase (Hare et al., 1980).
Genes for cytochrome b and for ATPase subunit 6 have
been recognized in the mtDNA sequence (Attardi et al.,
1982), but the corresponding polypeptides have not yet
been identified in gel autoradiograms. The mtDNA
includes eight additional reading frames coding for
proteins different from those mentioned above (Anderson
et al., 1981). These genes have not yet been identified as
to the polypeptides that they specify.
In collaboration with Donna Strong and Russell
Doolittle of the University of California at San Diego, we
have begun a project aimed at identifying the proteins
encoded in the unidentified reading frames of human
mtDNA. The immediate aim is to assign bands or spots on
a gel to one or another reading frame. The approach is to
use purified antibodies directed against chemically
synthesized short peptides, corresponding to sections of a
particular reading frame, in immunological reactions with
mitochondrial proteins. We have obtained positive results
in a test case using antibodies directed against two
different peptides derived from the coding sequence for
cytochrome c oxidase subunit II.
References: Anderson, S., Bankier, T., Barrell, B. G., de Bruijn, M. H.
L., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H., Stader, R. and Young, I. G. (1981) Nature 290, 457-465.
Attardi, G., Chomyn, A., Montoya, J. and Ojala, D. (1982) Cytogenetics and Cell Genetics, in press.
Ching, E. (1980) Ph.D. Thesis, California Institute of Technology.
Hare, J. F., Ching, E. and Attardi, G. (1980) Biochemistry 19, 2023-2030.
8. HUMAN DIHYDROFOLIC ACID REDUCTASE cDNA ANALYSIS
Investigator: Jeffrey N. Masters
Dihydrofolic acid reductase (DHFR) cDNA clones have
been isolated (Morandi et al., 1982) and used to determine
the human DHFR coding sequence, to map this region on
the 3800 nt mRNA, and to determine the relationship of
the three mRNAs of 3800, 1000 and 750 nt to each other.
Restriction endonuclease mapping and DN.A transfer
experiments utilizing overlapping plasmids covering about
3500 nt, including plasmids derived from trimethoprim
resistant E. coli X2882 transformants that express the
human DHFR, have localized the DHFR coding region in
the 5'-end proximal segment of the 3800 nt mRNA,
leaving a 3' tail of about 3000 nt.
DNA sequence analysis of the 713 bp insert of pHD84,
a plasmid conferring the greatest trimethoprim resistance
to E. coli X2282, shows a reading frame starting at 22 nt
from a poly(G-C) tail, extending for 558 nt to a TAA
termination site and for 95 nt more to the other poly(G-C)
tail. This reading frame is shown to code for the human
DHFR by both the 89% homology of its predicted amino
acid sequence to that of the mouse DHFR (Stone et al.,
1979) and the 87% homology of its nucleotide sequence to
that of a partial mouse DHFR cDNA sequence (Nunberg
et al., 1980), A nucleotide sequence homology of 73% is
found in the 22 nt following, on the 3' side, the TAA; the
homology then falls to 21 % for the next 61 nt, indicating a
lack of selective pressure on this 3' region.
DNA sequence analysis also shows the presence in
pHD84 of a poly(A) stretch just prior to the poly(G-C)
tail. This result has led to speculation that this plasmid
contains the cDNA of the 750 nt mRNA. Analysis of
other plasmids has confirmed this hypothesis and shown
that the cDNA of the 1000 nt mRNA has also been cloned.
References: Morandi, C., Masters, J. N., Mottes, M. and Attardi, G.
(1982) J. Mo!. Biol. 156, 583-607. Nunberg, J. H., Kaufman, R. J., Chang, A. C. Y ., Cohen,
S. N. and Schimke, R. T. (1980) Cell 19, 355-364. Stone, D., Paterson, S. J., Raper, J. H. and Phillips, A. w.
(1979) J. Biol. Chem. 254, 480-488.
9. DHFR-SPECIFIC SEQUENCES IN CHROMOSOME FRACTIONS FROM V "21HA3
Investigator: Anne Chomyn
V A2B-6A3 is a human cell line resistant to high
concentrations of metotrexate (1.8 x 10-4 M) (Masters
21
et al., 1982), which has been carried both on plates and in
suspension culture since its isolation two and a half years
ago. Six months after their adaptation to the
maintenance concentration of methotrexate, the cells in
suspension contained both double minutes and a 11homogeneously staining region" in an identifiable
chromosome. Both types of chromosome abnormalities
contain amplified genes for dihydrofolate reductase
(DHFR) (see Biology 1981, No. 12).
I have developed a rapid chromosome fractionation
method that yields high molecular weight DNA. Southern
blot hybridization of restriction enzyme digested DNA
from normal chromosomes and from double minutes with a
DHFR cDNA probe has shown the presence of DHFR
specific sequences in both fractions. However, the double
minute DNA appears to contain, relative to the normal
chromosome DNA, much less of the Eco RI fragment that
has been identified in genome blots as representing the
5' end of the coding sequence. This result suggests that at
least some of the double minutes contain DHFR genes
with rearrangements involving the 5' end of the gene.
Reference: Masters, J., Keeley, B., Gay, H. and Attardi, G. (1982)
Molec. Cell. Biol. 2, 498-507.
10. ORGANIZATION OF THE DHFR GENE AND THE AMPLIFIED UNIT IN A MTX-RESISTANT HUMAN CELL LINE
Investigator: Jiyoung Kim Yang
All the various MTX-resistant human cell lines, which
were isolated in this laboratory (Masters et al., 1982),
exhibit the presence of small acentric chromosomal
elements, designated as double minute chromosomes. The
number of these double minute chromosomes in resistant
variants does not show any obvious correlation with the
level of DHFR activity nor with the instability of the
drug-resistant phenotype. However, 6A3, a variant
resistant to 1.8 x 10-4 M methotrexate derived from the
human line VA2-B, showed a close relationship between
loss of double minutes and the decrease in DHFR activity
after removal of MTX in the meditim (Masters et al.,
1982). This cell line has a DHFR activity which is 150
times higher than that of the parental cell line. At least
25% of the increased activity appears to be stable after
removal of MTX and persists when the double minutes
have substantially disappeared. These results indicate
that the amplified DHFR genes are associated partly with
22
double minute chromosomes and partly with the normal
chromosomes.
Normal chromosomes and double minute chromosomes
were prepared by differential centrifugation from 6A3
cells, which were grown in suspension and arrested in
metaphase by vinblastin sulfate. Southern blot hybridiza
tion of Eco RI-digested total 6A3 DNA and normal
chromosome DNA with a DHFR cDNA clone, pHD84
(Morandi et al., 1982), 32P-labeled by nick-translation,
revealed the same positive fragments as those found in
the DNA from the parental cell line. A similar analysis of
minute DNA showed the existence of the same fragments
but diminution of the 5' fragment (see Abstract
No. 9).
DNAs from normal chromosomes and minute
chromosomes of 6A3 were utilized to construct libraries,
using phage A Charon 4A as a vector. The libraries were
screened by in situ plaque hybridization. Approximately
0.2% of the recombinant phages from the normal
chromosome DNA library and about 0.5% of those from
the minute DNA library gave positive signals with nick
translated pHD84. After analyzing approximately 100
positive phages, we obtained several overlapping gene
fragments corresponding to the 3'-end portion of the
coding region but were not able to find fragments
corresponding to the 51-end portion. Attempts to isolate
these fragments by differential hybridization, using
pHD84, which contains the entire protein coding sequence,
and pHD43, which lacks the coding sequences on the 5'
end side, enabled us to obtain eight different phages
containing overlapping inserts, including some fragments
corresponding to the 5'-end portion of the gene. Analysis
of the inserts of these phages showed that they account
for the entire DHFR gene. Detailed studies of these
fragments are being carried out.
In addition, three different phages containing positive
fragments of unusual sizes were isolated. These frag
ments have also been identified recently in the DNAs
from the parental cell line VA2-B, from HeLa cells, and
from 6A3 cells by genomic blot hybridization and have not
been found to be amplified in the DNA from the last
source. One of these fragments is being analyzed at the
present time.
References: Masters, J., Keeley, B., Gay, H. and Attardi, G. (1982)
Molec. Cell. Biol. 2, 498-507. Morandi, c., Masters, J. N., Mottes, M. and Attardi, G.
(1982) J. Mol Biol. 156, 583-607.
11. LOSS OF DOUBLE MINUTE CHROMOSOMES AND APPEARANCE OF HOMOGENEO!:ILY STAINING REGIONS IN A HUMAN MTX CELL LINE, V ~-A3
Investigator: Barry J. Maurer
A karyotype analysis of several methotrexate-resistant
(MTXR) human cell variants isolated in this laboratory
from VA2-B cell lines (Masters et al., 1982) has revealed
the presence of double minute chromosomes (DMs), shown
to contain at least some of the amplified genes for
dihydrofolate reductase (DHFR) by in situ hybridization
(Biology 1981, No. 12). One variant, VA2B-6A3, exhibits,
in addition to DMs, a chromosome bearing a homoge
neously staining region (HSR). Upon extended growth in
methotrexate, VA2
B-6A3 loses almost all of its DMs and
acquires two additional HSRs in distinctive chromosomes.
An extensive analysis of this phenomenon is being carried
out on time points extending over a 16-month period.
Karyotype analysis is being performed to study the
kinetics of DM loss and HSR acquisition. Growth rate
studies are being conducted to explore whether a selective
advantage is conferred upon the cell by DHFR genes
located on HSRs as compared to those residing in DMs. In
situ hybridization experiments using a 1251-dCTP-labeled
human DHFR cDNA probe are being conducted to
determine the DHFR gene location at various stages
during the development of MTX resistance. Preliminary
results indicate that the acquisition of the two later
appearing HSRs is sequential and "catastrophic," with no
developmental intermediaries.
Reference: Masters, J., Keeley, B., Gay, H. and Attardi, G. (1982)
Molec. Cell. Biol. 2, 498-507.
12. CHROMOSOMAL LOCALIZATION OF THE HUMAN DIHYDROFOLATE REDUCTASE GENE IN HUMAN CELLS
Investigators: Barry J. Maurer, Jeffrey N. Masters
Somatic cell fusion techniques can be used to assign
genes to their particular chromosomal location (Ruddle,
1981). 1n collaboration with Frank Ruddle (Yale), we are
examining the DNA from mouse-human and Chinese
hamster-human cell hybrids by restriction enzyme and
Southern blot analysis using a human dihydrofolate
reductase (DHFR) cDNA probe, to determine the
chromosomal location of the normal DHFR gene in human
cells. This information may give insights into the
mechanism of DHFR gene amplification in methotrexate
resistant human cell lines.
Reference: Ruddle, F. H. (1981) Nature 294, 115-120.
13. ISOLATION OF DlliYDROFOLATE REDUCTASE DEFICIENT HUMAN CELLS
Investigator: Anne Chomyn
We have begun experiments aimed at the isolation of a
HeLa cell derivative that is deficient in dihydrofolate
reductase (DHFR) activity. The procedure we are using is
based on the 3H-deoxyuridine suicide technique devised by
Urlaub and Chasin (1980). The first step is the selection
of cells that have lost one functional copy of the DHFR
gene. For this purpose, cells are mutagenized and
repeatedly exposed to 3H-deoxyuridine in the presence of
a low concentration of methotrexate. The survivors are
mutagenized once more, and cells completely deficient in
DHFR are selected for surviving 3H-deoxyuridine treat
ment.
The DHFR-deficient cells will be used as the recipient
cells in chromosome mediated transformation
experiments.
Reference: Urlaub, G. and Chasin, L. (1980) Proc. Nat. Acad. Sci.
USA 77, 4216-4220.
14. ORGANIZATION OF THE RIBOSOMAL RNA GENFJ! ISOLATED FROM A HUMAN GENOMIC LIBRARY
Investigator: Monica Mattes
We reported last year the isolation, from a human
genomic library constructed in this laboratory, of clones
containing most of the 18S rRNA coding region plus a
portion (.r12.5 kb) of the large external spacer that,
together with the 18S and 28S rRNA coding regions and
the internal transcribed spacer, constitutes the rDNA
unit. Further screening of the genomic library with
radiolabeled cDNA transcripts of 288 rRNA has led to the
isolation of other clones containing the remainder of the
188 rRNA coding region plus the internal transcribed
spacer and most of the 28S rRNA coding region; one of
the clones contains, besides the above specified segment,
the remainder of the 28S rRNA gene plus about 7.5 kb of
the adjacent portion of the nontranscribed spacer.
23
Altogether, the isolated cloned segments of the rDNA unit
encompass about 30 kb.
Partially purified 45S r RN A precursor from He La cells
has been utilized to map its coding sequence in the
external spacer segment flanking the 18S rRNA gene by
the Sl nuclease protection technique. A major protected
segment extending to about 3500 nt from the 5' end of the
188 rRNA gene and a minor protected segment extending
to about 3900 nt from the same 5' end have been found.
The data are compatible with the idea that the 5' end of
the longer protected segment may be an initiation site for
transcription, while the 5' end of the shorter protected
segment may be either another initiation site or a
processing site.
PUBLICATIONS
Attardi, G., Cantatore, P., Chomyn, A., Crews, 8., Gelfand, R., Merkel, C., Montoya, J. and Ojala, D. (1982) A comprehensive view of mitochondrial gene expression in human cells. In: Mitochondrial Genes. P. Slominski, P. Borst and G. Attardi (Eds.), pp. 51-71. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Attardi, G., Chomyn, A., Montoya, J. and Ojala, D. (1982) Identification and mapping of human mitochondrial genes. Cytogenetics and Cell Genetics, in press.
Attardi, G. and Montoya, J. (1982) Analysis of human mitochondrial RNA. Methods in Enzymology, Biomembranes: Membrane Biogenesis, Assembly and Recycling, in press.
Ching, E. and Attardi, G. (1982) High resolution electrophoretic fractionation and partial characterization of the mitochondrial translation products from HeLa cells. Biochemistry, in press.
De Francesco, L. and Attardi, G. (1981) In situ photochemical crosslinking of HeLa cell mitochondrial DNA by a psoralen derivative reveals a protected region near the origin of replication. Nucleic Acids Res. 9, 6017-6030.
Dubin, D. T., Montoya, J., Timko, K. D. and Attardi, G. (1982) Sequence analysis and precise mapping of the 3'ends of HeLa cell mitochondrial ribosomal RNAs. J. Mol. Biol., in press.
Gelfand, R. and Attardi, G. (1981) Synthesis and turnover of mitochondrial RNA in HeLa cells: the mature ribosomal and messenger RNA species are metabolically unstable. Molec. Cell. Biol. 1, 497-511.
Masters, J. and Attardi, G. (1982) Amino acid sequence of the human dihydrofolic acid reductase derived from the cDNA nucleotide sequence. Nature, submitted for publication.
Masters, J., Keeley, B., Gay, H. and Attardi, G. (1982) Variable content of double minute chromosomes not correlated with degree of phenotype instability in methotrexate-resistant human cell lines. Molec. Cell. Biol. 2, 498-507.
Morandi, c. and Attardi, G. (1981) Isolation and characterization of dihydrofolic acid reductase from methotrexate-sensitive and -resistant human cell lines. J. Biol. Chem. 256, 10169-10175.
24
Morandi, c., Masters, J. N., Mottes, M. and Attardi, G. (1982) Multiple forms of human dihydrofolate reductase messenger RNA: cloning and expression in E. coli of their DNA coding sequence. J. Mo!. Biol. 156, 583-607.
Ojala, D., Crews, S., Montoya, J., Gelfand, R. and Attardi, G. (1981) A small polyadenylated RN A (78 RN A), containing a putative ribosome attachment site, maps near the origin of human mitochondrial DNA replication. J. Mo!. Biol. 150, 303-314.
Professor: Eric H. Davidson Distinguished C8rnegie Senior Research Associate: Roy J.
Britten* Senior Research Associate: Barbara R. Hough-Evans Visiting Associates: Lajos Pike**, Thomas J. M. Schopf Senior Research Fellows: John W. Roberts, Terry L.
Thomas Gosney Research Fellows: Constantin N. Flytzanis, John
W. Grula Research Fellows: Carlos V. Cabrera, Howard T. Jacobs,
Steven A. Johnson, Andrew P. McMahon, Samuel J. Rose III, Rosemary J. Shott
Graduate Students: Boning Gao, James L. Lee, James W. Posakony
Research Staff: Maria Alonso, Alison E. Blake, Richard L. Hudspeth, Patrick S. Leahy, Jane Rigg, Gilda Watts, Ronald C. Wek
Laboratory Staff: Manuel Acevedo, Carlzen Balagot, Fargo Balliett, Reese Faucette, Luci Hansen
*Concurrently a member of the staff of the Carnegie Institution of Washington. **Veterans Administration Medical Center, Sepulveda, California.
SUpport: The work described in the following research reports has been supported by:
American Cancer Society Biomedical Research Support Grant (NIH) California Foundation for Biochemical Research Norman Chandler Professorship in Cell Biology Deutsches Krebsforschungszentrum European Molecular Biology Organization Fogarty International Research Fellowship E. S. Gosney Fund National Institutes of Health, USPHS National Science Foundation Science Research Council Fellowship, England University of Chicago Veterans Administration
Summary: Interest in our laboratory has for several years
centered on studies of the organization and expression of
the genome in higher animals. We use sea urchin eggs and
developing embryos as a model system in much of our
work, and have recently added to the system by setting up
a series of vessels and aquaria in which embryos are
carried through metamorphosis to maturity. In examining
Wiseman, A. and Attardi, G. (1982) Cytoplasmically determined human cell mutants defective in mitochondrial ribosome assembly. Malec. Gen. Genetics, in press.
the organization of sequences of the sea urchin genome,
we are making use of recombinant DNA techniques for
analysis of single genes and gene families, using libraries
of genomic DNA from several sea urchin species. Thus we
are able to measure the conservation of certain sequences
through evolution and between sea urchin populations, as
well as the distribution of single copy and repeated
sequences in their vicinity in the genome. Our recent
discovery of genomic DNA sequences related to mito
chondrial DN As resulted from one aspect of· this research.
Maternal RNA (RNA sequences stored in sea urchin
eggs) and RNA extracted from embryos are used for study
of the expression of sea urchin genes during embryo
genesis. Maternal transcripts are found to include very
long RN A molecules in which gene transcripts are
interspersed with transcripts of other single copy and
repeated sequences. We have identified and selected a
number of cloned sequences of genes whose expression is
developmentally regulated-that is, whose transcripts
show large changes in prevalence between stages of
embryonic development. For more detailed analysis of
gene expression, we are working to develop a method of
transforming sea urchin genomes by introducing cloned
DNA sequences into eggs.
The sequence organization and expression of the sea
urchin actin gene family are being actively investigated,
and we are using a variety of approaches to obtain clones
of genes coding for other specific sea urchin proteins such
as the bindin protein of sperm and the vitelline layer
proteins of eggs. Other particular projects are concerned
with sequence organization and gene expression in
eukaryotes ranging from protozoa to man. In all, our aim
is to better understand the mechanisms of gene control,
and the relationships between DNA sequences, in higher
animals.
15. A SEA URCHIN GENE REPRESENTED IN BOTH MATERNAL RNA AND EMBRYO NUCLEAR RNA
Investigators: Terry L. Thomas, Eric H. Davidson
The sequence organization and transcriptional
expression of a 17 kilobase (kb) region of the sea urchin
genome have been described. This region includes a
transcription unit, designated "gene 88, 11 that is repre
sented by a typical low prevalence maternal poly(A) RNA.
The predominant maternal gene 88 transcript is about
9.5 kb in length, and analysis of the sequence organization
of the cloned genomic region included in this transcript
shows that it contains a high density of low and
moderately prevalent repeat sequences, as well as ~2 kb
of single copy sequence at the 3' end. Genome blots
carried out with DNA from different individual sea
urchins show that several allelic forms of this region
exist. The transcribed single copy sequence is only
slightly less polymorphic than is the average single copy
sequence of the Strongylocentrotus purpuratus genome.
Gene 88 single copy sequences appear to be represented in
at least three smaller transcripts in the polysomal RNA of
16-cell embryos. The nuclear RNA of later embryos also
contains gene 88 transcripts. RNA gel blots indicate that
polyadenylated gene 88 transcripts are also 9.5 kb in
length in blastu!a and gastrula nuclear RNAs. These
transcripts are probably colinear with the major maternal
RN A transcript.
16. SEQUENCE CONSERVATION IN AND AROUND A SEA URCHIN GENE
Investigators: Steven A. JolulSon, Roy J. Britten
The gene designated "gene 88" or Sp88 has been chosen
for investigation of the sequence conservation, in and
around a developmentally regulated gene, between two
related species of sea urchins, Strongylocentrotus
droebachiensis (Sd) and s. purpuratus (Sp). The Sp88-
containing Sp clone p16 has been characterized with
regard to restriction sites, the location of interspersed
repeat and single copy sequences, and the location of the
major 9.5 kb egg transC!ript (Thomas et al., 1982). To
study this gene region in Sd, a Sd library was screened
with a single copy probe, pRHl.4, which represents the 3'
terminal of 1.5 kb of the Sp88 transcript. Of the clones
selected, one representative of each allele was mapped
with various 6-cut restriction enzymes. In 7 kb of gene
region-overlap between the Sp and Sd clones, 80% of the
restriction sites are identical. Based on an average single
25
copy sequence divergence between Sp and Sd of 7%, less
than 50% of the 6-cut restriction sites should be con
served. This suggests that this region has been quite well
conserved since the two species diverged. To study repeat
conservation, Southern blots containing digestions of Sd
and Sp 1188" DNAs were reacted with an Sd total genome
tracer. These data indicate that the location and
apparent repetition frequency of interspersed repeats in
the Sp88 region is highly conserved between Sp and Sd.
Most recently, a more detailed comparison of the major
single copy region, pRH!.4 and its Sd counterpart dRH!.4,
by 4-cut restriction mapping has revealed a small insert of
about 150 nt in the Sd gene. The insert and its
surrounding sequence environment are currently under
study.
Reference: Thomas, T. L., Britten, R. J. and Davidson, E. H. (1982)
Devel. Biol., submitted for publicatfon.
17. SEQUENCE POLYMORPHISM IN REGIONS SURROUNDING STRUCTURAL GENES
Investigators: Joint W. Roberts, Joint W. Gru!a, Steven A. JolulSon, Roy J. Britten
Thermal stability measurements have demonstrated an
average 4% single copy DNA sequence polymorphism in
the sea urchin Strongylocentrotus purpuratus, with a range
of sequence polymorphism from less than 1 % to more than
10% (Britten et al., 1978). The blot hybridization method
has been used to estimate the sequence polymorphism
surrounding several S. purpuratus structural genes and in
single copy regions adjacent to expressed repeats. The
sequences surrounding different genes exhibited a range of
polymorphism from less than 1 % to about 4%; the repeat
adjacent regions exhibited a somewhat higher level.
These results confirm the thermal stability measurements
and, further, demonstrate different degrees of poly
morphism in different regions of the genome.
Individual genome blot hybridization experiments with
cDNA clone SpG6 suggested patterns in the sequence
polymorphism. For any restriction endonuclease used,
there appeared to be one or two predominant fragment
lengths (alleles), along with a series of less frequent
variants. This observation was confirmed by blot
hybridization experiments using balanced mixtures of
genomic DNA from 20-30 individuals from two different
populations. The two populations share the same pre
dominant and less frequent alleles, although quantitative
differences in the frequencies of some variants are
26
observed, suggesting some degree of isolation between the
animals in the two populations. Similar, although more
extensive, interpopulation differences are seen with the
repeat adjacent probes.
Reference: Britten, R. J., Cetta, A. and Davidson, E. H. (1978) Cell
15, 649-660.
18. GENE ORGANIZATION lN SEA URCHIN MITOCHONDRIAL DNA
Investigators: John W. Grula, John W. Roberts, James w. Posakony, Roy J. Britten
Two Strongylocentrotus purpuratus cDNA clones
(SpG30 and SpP389) encoding prevalent embryonic tran
scripts were shown to be mitochondrial by the following
criteria: hybridization with purified mitochondrial DNA;
reaction with a band comigrating with supercoiled mtDNA
in a Southern transfer of undigested S. franciscanus DNA;
and selection of a set of circularly permuted clones from
a S. franciscanus genomic library, of size and restriction
map identical with that of mtDNA. The cDNAs were
identified as genes encoding cytochrome oxidase subunit I
and 168 mitochondrial rRNA, respectively, by virtue of
specific cross-reaction with fragments of human mtDNA
in blot hybridizations, and by partial nucleotide
sequencing. Localization of the cDNA sequences in S.
franciscanus mtDNA clones, by blot hybridization,
indicated that these genes are separated by less than 1 kb,
in contrast to their organization in mammalian mtDNAs.
Additional blot hybridization analyses using human
mtDNA and subcloned segments from S. franciscanus
mtDNA confirm a difference in the organization of the
168 rRNA, 128 rRNA, and cytochrome oxidase subunit I
genes in the two mitochondrial genomes. Gene rearrange
ments, therefore, appear to have occurred in the
mitochondrial genome subsequent to the divergence of the
lines leading to the echinoderms and vertebrates.
19. MITOCHONDRIAL TRANSCRIPTS lN SEA URCHIN EMBRYO RNA
Investigators: Carlos v. cabrere, Constantin N. Flytzanis, Eric H. Davidson
Many of the most prevalent clones in sea urchin
embryo cDNA libraries were found to be mitochondrial
when screened with mitochondrial DNA. The cDNA
clones representing mitochondrial 16S rRNA and cyto
chrome oxidase mRNA (see Abstract No. 18) were used to
measure the prevalence and stability of these transcripts
in gastrula stage embryos. The 168 rRNA is the single
most prevalent embryo poly(A) RNA. The relative
prevalence of the two mRNAs is largely determined by
their turnover rates.
20. HOMOLOGUES OF MITOCHONDRIAL GENES lN SEA URCHIN NUCLEAR DNA
Investigators: Howard T. Jacobs, James W. Posakony, John W. Grula, John W. Roberts, Carlos V. Cabrera, Eric H. Davidson, Roy J. Britten
Two sea urchin embryo cDNA clones, representing
mitochondrial genes for cytochrome oxidase subunit I and
168 rRNA, were found to react with restriction fragments
of genomic DNA whose sizes and demonstrable
polymorphism were incompatible with a mitochondrial
origin. In screening a genomic library, the same set of
clones was independently selected with each of these
probes. Analysis of the structure of these cloned DN As
showed that they carried only a limited region of
homology with mtDNA, contained typical dispersed
genomic repeats and were not the result of an
adventitious ligation during cloning. Sequences
homologous with the cDNAs were localized in the cloned
nuclear element by blot hybridization. This demonstrated
that only the 3' end of the 168 rRNA gene was
represented, flanked by two homologues of the cyto
chrome oxidase subunit I gene, only one of which included
the extreme 3' end of the gene. Thermal stability analysis
and nucleotide sequencing showed differential sequence
conservation between different regions of the
mitochondrial genes and their nuclear homologues. We
interpret the results as documenting that a transposition
of sequences into the nuclear genome has occurred, and
that subsequently this domain has evolved essentially like
noncoding DNA, with a process of gross rearrangements
(duplications, deletions and insertions) superimposed on a
low rate of point mutation.
21. STAGE-SPECIFIC EXPRESSION OF SEA URCHIN EMBRYO RNA SEQUENCES
Investigators: Com;tantin N. Flytzanis, Glenn Nakamura•, Eric H. Davidson
During the early development of sea urchin embryos,
only a minor class of poly(Ai+ RN A sequences displays
sharp stage-specific changes. To study such stage-
specific transcripts, cDNA libraries made from different
embryonic stages have been screened with labeled cDNAs
+ transcribed from polysomal poly(A) RNAs of 16-cell,
blastula,
selected
gastrula and pluteus
clones that show
stage embryos. A set of
dramatic developmental
changes has been further characterized using RNA and
genome blot analysis. On this basis, one cDN A clone,
SpP25, was selected for further studies. RNA transcripts
complementary to the clone SpP25 are rare in both the
mature egg and adult tissues tested, while a dramatic
increase (more than lOOx) in transcript concentration at
gastrula and pluteus stage of the embryo has been
calculated. Only one transcript size (1450 nucleotides)
was detected at gastrula and pluteus stage. Although this
transcript is not present in total intestine RN A, a longer
transcript (2000 nucleotides) of a lower concentration was
detected. To investigate the possibility that this tran
script represents a nuclear non-processed RNA, a genomic
library was screened with the clone SpP25 and a set of
genomic clones was characterized with restriction enzyme
analysis. Two non-overlapping genomic clones have been
found that carry a homologous sequence of about 5
kilobases long wherein the cDNA clone SpP25 maps. The
question of whether these two clones represent two
distinct genes or the two chromosomal alleles of the same
gene is under current investigation. It is hoped that the
study of nuclear transcripts of embryo and adult tissues
will elucidate the mechanisms at the transcriptional or
post-transcriptional level that control the expression of
this gene, especially in the embryonic stages.
*Undergraduate, California Institute of Technology.
22. INTERSPERSED POLYADHNYLATED RNAs OF SEA URCHIN EGGS AND EMBRYOS
Investigators: James W. Posakony, Ronald c. Wek, Eric H. Davidson
Sea urchin egg and embryo polyadenylated RNAs
bearing specific repetitive sequences were analyzed by
cDNA cloning, DNA and RNA gel blot hybridization, and
DNA sequencing. It was found that the two complements
of a given repeat are carried on different sets of
polyadenylated transcripts, which are generally quite long
(>3 kilobases, with an estimated number average length of
5-6 kilobases). Within these transcripts, specific short
repetitive sequence elements are found interspersed
either with single-copy sequences or with other repeat
sequences. It was demonstrated by sequencing that one
such repeat-containing region is not translatable. The
27
sets of polyadenylated transcripts deriving from several
individual repeat families undergo substantial quantitative
and probably qualitative modulation during early sea
urchin development. Analysis of specific transcripts with
single-copy probes from repeat-containing cDN A clones
indicates that the embryo genome is transcribed to
produce at least some of the same interspersed RNAs as
are stored in the oocyte during oogenesis. Finally, the
transcripts bearing specific repeat sequences in the
polyadenylated egg RNA of two related sea urchin species
were found to be qualitatively dissimilar.
23. STRUCTURE AND FATE OF SEA URCHIN MATERNAL RNA
Investigators: Howard T. Jacobs, Eric H. Davidson
A considerable proportion of sea urchin maternal RNA
comprises transcripts much longer than conventional
mRN A, which include interspersed repeat elements and
single-copy regions. In order to investigate the structure
of these transcripts, and their relationship to message, we
are exploiting the fact that some embryo-derived single
copy cDNAs react predominantly with abundant
transcripts of this class in egg RNA. One such is the
cDNA clone designated SpPI54, which is represented in
egg RNA as 7.5 kb and 3.8 kb transcripts. The nucleotide
sequence of portions of the cDN A has been determined by
the primer extension method, using subclones in the
single-stranded DNA phage M13. This shows that there is
no extended region of open reading frame in the cDN A,
from which we infer that it is either a long 3' untranslated
region, or unprocessed mRNA precursor, or a transcript
unrelated to message. The detailed structure of the
maternal transcript is being studied using overlapping
clones from a randomly-primed cDNA library to egg
poly(At RNA, and comparing these with the gene
(available as a set of clones isolated from a genomic
library) and with the homologous clones from embryonic
cDNA libraries. If, as seems likely, the maternal species
represents a message precursor, the fact that there is only
a very low rate of de novo synthesis of SpP154 transcripts
during early development means that it will be possible to
determine if this species functions as such, by measuring
the decay rates of its message and non-message sequences
in embryo RNA.
28
24. DNA TRANSFORMATION OF SEA URCHIN EGGS
Investigators: Andrew P. McMahon, Constantin N. Flytzanis, Ronald C. Wek, Patrick S. Leahy, Eric H. Davidson
Developmental gene expression in sea urchin eggs and
embryos is being investigated by DNA transformation of
sea urchin eggs. Three procedures for the introduction of
foreign DNA are under study: (I) liposome fusion; (2)
calcium phosphate precipitation; and (3) microinjection of
unfertilized and fertilized eggs. Of these the latter has
proved the most promising, with approximately 35% of
injected unfertilized eggs surviving to feeding larvae using
the culture methods described in the following report (see
Abstract No. 25).
Appropriate vectors for the transforming DNA, and
methods for conveniently assaying the efficiency of
transformation, are being explored. One of the vectors
under current investigation contains a eukaryotic
promoter from the Herpes thymidine kinase gene,
together with the coding sequence for a prokaryotic
phosphotransferase, which inactivates the antibiotic
G-418. G-418 has been shown to perturb normal
embryonic development, resulting in plutei unable to feed,
and thus provides a potential selection system for trans
formants.
Construction of other vectors using terminal inverted
repeats and coding sequences for the transposase of known
transposable elements, which code for the protein
responsible for their own insertion into the sea urchin
genome, is under way.
Jn order to assay the transformation efficiency, a
screening system has been developed in which single
larvae are lysed directly onto nitrocellulose filters. Nick
translated DNA probes have been shown to detect single
copy sequences in an advanced larva (20-30,000 cells).
Other approaches using highly radioactive single-stranded
probes and a bioassay in which antibiotic resistance is
conferred on transformants are also being tested.
25. CULTURE OF SEA URCHIN EMBRYOS TO SEXUAL MATURITY
Investigators: Constantin N. Flytzanis, Andrew P. McMahon, Patrick S. Leahy, Eric H. Davidson
The development of a DNA transformation system for
sea urchin eggs is of basic importance for a functional
study of the meaning of specific nucleic acid sequence
features in the context of early embryonic development.
An essential experimental component of the
transformation system is a method for efficiently raising
eggs through larval development and metamorphosis to
sexual maturity. By adapting and improving the small
scale procedures described initially by Hinegardner and
Rocha Tuzzi (1981), we have developed methods for
raising more than 75% of fertilized eggs through meta
morphosis and to the juvenile adult stage. Larvae are
cultured at 16°C in seawater and are fed on a unicellular
algae (Rhodomonas sp.) which is very easy to raise in the
laboratory. Our current larval culture systems are
capable of handling up to 2000 individuals in as many
separate groups as desired. From fertilization to meta
morphosis is about six weeks. Within two weeks of
metamorphosis, the juveniles achieve sufficient morpho
logical development to begin grazing. We have found that
algae-coated rocks from one of our field stations provide
an ideal substratum for raising the juveniles in relatively
high concentrations. They attain sexual competence at
about six months after metamorphosis.
Reference: Hinegardner, R. T. and Rocha Tuzzi, M. M. (1981) In:
Marine Invertebrates. National Academy Press, Washington, pp. 291-302.
26. ORGANIZATION AND EXPRESSION OF ACTIN GENES IN THE SEA URCHIN
Investigators: Samuel J. Rose, Rosemary J. Shott, James L. Lee, Terry L. Thomas, Eric IL Davidson
Actin mRNA in the sea urchin Strongylocentrotus
purpuratus is encoded by a small multigene family
comprised of at least 11 nonallelic genes. Seven of these
genes are linked to at least one other actin gene. These
genes can be grouped into three major subfamilies based
on the sequences of 3' untranslated regions present on
actin mRNAs (Scheller et al., 1981). Gel blot analysis of
RNAs from different developmental stages and adult
tissues, using actin subfamily specific probes, shows that
the expression of these subfamilies is developmentally
regulated. Two major questions to be resolved are (1)
precisely which genes are expressed at each
developmental stage, and (2) whether actin genes that are
coordinately expressed are linked in the sea urchin
genome. To address the first question, 3' untranslated
regions from members of each actin gene subfamily are
being cloned into an M13 vector and sequenced by the
dideoxy-chain extension method. We are also sequencing
these same regions in actin cDNA clones isolated from
stage-specific cDNA libraries. A comparison of the
genomic 3' noncoding sequences with cDNA 31 un
translated sequences should . unambiguously identify the
actin gene(s) represented at each developmental stage.
Using the subfamily-specific probes, we are also
determining the absolute number of subfamily transcripts
at different stages, and the rates at which these tran
scripts are synthesized and degraded.
The long-range genomic relationship between the
expressed actin genes is being approached by isolating
35-45 kb genomic regions containing actin genes using
cosmid cloning techniques. Linkage of different actin
subfamilies will be examined by probing these cosmids
with subfamily-specific probes. The issue of coordinate
expression of linked genes is of fundamental importance in
considering the evolution and function of small gene
families.
Reference: Scheller, R. H., McAllister, L.B., Crain, w. R., Durica, D.
S., Posakony, J. W ., Thomas, T. L., Britten, R. J. and Davidson, E. H. (1981) Molec. & Cell. Biol. 1, 609-628.
27. A STUDY OF THE SEA URCHIN "BINDIN" GENE
Investigators: Boning Gao, Eric H. Davidson
During sea urchin fertilization, the interaction of egg
jelly coat fucose sulfate polymer with the sperm plasma
membrane induces the sperm acrosome reaction, which
consists of the exocytosis of the acrosome granule.
"Bindin" (the sperm-borne binding protein) is one of the
proteins in the acrosome granule. There is evidence to
support the hypothesis that bindin is the adhesive protein
binding sperm to egg (Glabe and Vacquier, 1978). It is a
species-specific protein. Bindin protein has been purified
and partially sequenced (Glabe and Vacquier, 1978). The
molecular weight is 30,500 daltons.
Cytoplasmic poly(A) RNA was prepared from sea
urchin testes. In vitro translations were carried out using
this RNA. We are doing immunoprecipitation reactions
with anti-bindin antibody (the kind gift of V. D. Vacquier)
to find out if bindin protein was made.
Also several 11-deoxynucleotide DNA fragments were
synthesized corresponding to the amino acid sequence.
We plan to use these oligonucleotides in screening
libraries of cloned sea urchin DNA for the bindin gene.
Reference: Glabe, C. G. and Vacquier, V. D. (1978) Proc. Nat. Acad.
Sci. USA 75, 881-885.
28. ANTIBODIES TO PROTEINS OF THE VITELLINE LA YER OF SEA URCHIN EGGS
Investigators: Henry Niman*, Barbara R. Hough-Evans, Eric H. Davidson
29
The vitelline layer of the sea urchin egg consists of a
number of functionally related proteins and glycoproteins.
Monoclonal antibodies against the vitelline layer proteins
have been prepared. About 30 monoclonal antibodies were
obtained which react positively with total VL protein.
Currently we are attempting to determine the individual
protein to which each monoclonal antibody reacts,
preparatory to cloning the genes for these proteins.
*Scripps Clinic and Research Institute, La Jolla, California.
29. INTERSPERSED MIDDLE FREQUENCY REPEATS IN HUMAN AND GORILLA DNA
Investigators: Jolm W. Roberts, Richard L. Hudspeth, Roy J. Britten
The distribution of Alu family and middle frequency
(greater than about 100 copy) repeats in the human
genome was investigated using randomly chosen clones
from a human recombinant DNA library. Of approxi
mately 50 clones (average insert size 16 kb) tested by a
low criterion screening procedure, 94% contained one or
more Alu family repeats. Restriction mapping and blot
hybridization of a subset of 16 Alu repeat-containing
clones suggests a random pattern of location for Alu
repeats with an average spacing between locations of
4.3 kb; this corresponds to a repetition frequency of
700,000 Alu family repeats per genome. Middle frequency
repeats were detected in this subset of recombinants by
blot hybridization to total human genomic DNA labeled to
high specific activity. On the basis of the number of
middle frequency repeats detected, we estimate an
average spacing between middle frequency repeats of
about 17 kb, and a total of about 170,000 middle
frequency repeats in the human gemome. The number of
different families of middle frequency repeats in the
human genome is not known. One recombinant which has
been analyzed in detail contains two middle frequency
repeats belonging to families with different repetition
frequencies.
A similar analysis of the gorilla genome has been
initiated. Of approximately 65 randomly chosen clones
from a gorilla recombinant DNA library (insert size
approximately 15 kb), 87% react with a total human
genome tracer, but fewer than 70% react with a cloned
30
Alu family repeat. Thus, as expected, the middle
frequency repeats in human and gorilla DNA appear to be
similar in sequence and distribution, but there seem to be
fewer copies of Alu family repeats in the gorilla genome
than in the human.
30. QUANTITATIVE ESTIMATES OF POLY(A)+ mRNA IN EARLY MOUSE EMBRYOS
Investigators: Lajos Piko, Kerry B. Clegg*
The poly(A) content of preimplantation mouse embryos
undergoes a striking fluctuation: after a transient
increase in the one-cell embryo, the poly(A) content drops
by about 70% by the late two-cell stage and increases
approximately fivefold between the two-cell and early
blastocyst stages (Pik6 and Clegg, 1982). In order to
interpret these changes in terms of numbers of poly
adenylated mRNA molecules, we analyzed the size
distribution of total poly(A) (sequences resistant to
RNase A and T1) by polyacrylamide gel electrophoresis
followed by hybridization of the eluted fractions with 3H
poly(U). The size distribution of poly(A) was similar at all
stages between the one-cell embryo and early blastocyst,
suggesting that the fluctuations in poly(A) content are not
attributable to a lengthening or shortening of the poly(A)
tracts but are due primarily to changes in the number of
these tracts. From the data on poly(A) content and the
number-average length of poly(A) (varying from 61 to 77
nucleotides), one obtains the following number of poly{A)
tracts, taken to indicate the numbers of poly{A)+ mRNA,
per embryo at different stages of development: one-cell
embryo, 2.4 x 10 7; late two-cell, 0. 7 x 107; eight-cell,
1.3x107; early blastocyst (32 cells), 3.4x 107• These
findings and other evidence suggest that the bulk of the
maternal poly(Ai+ mRNA is eliminated at the two-cell
stage and that there is a progressive build-up in the
mRNA population, due to new synthesis by the embryo,
from the two-cell stage onwards.
Reference: Piko, L. and Clegg, K. B. (1982) Devel. Biol. 89, 362-378.
*Veterans Administration Medical Center, Sepulveda, California.
31. A CLONED GENOMIC LIBRARY OF A MARINE SPECIES OF THE PHYLUM BRYOZOA
Investigators: Thomas J. M. Sehopf, Terry L. Thomas, Eric H. Davidson
DNA was prepared in standard fashion (Graham, 1978)
from a single genetic individual of Watersipora cucullata;
this is an encrusting colonial bryozoan species which is
extremely common on docks and pilings at the Kerckhoff
Marine Laboratory. The vector, AJl, was constructed by
J. Mullins (Division of Chemistry and Chemical
Engineering, Caltech) by the insertion of two polylinker
sites into the genome of AL41 {Loenen and Brammar,
1980). Vector arms were prepared by a Barn HJ digest of
ligated AJl, followed by sucrose density gradient
centrifugation. Bryozoan DNA was cleaved by partial
Mbo I digestion, and size selected bryozoan DNA was
ligated to 1-Jl and packaged (Mullins et al., 1981).
Controls showed no packaging of E. coli DNA. Packaging
efficiency was 106 plaques/µg bryozoan DNA; 4.6 x 104
plaques were obtained on bacterial strain K802. Size
analysis of Eco RI and Barn HI digests of DNA prepared
from individual plaques of the library after amplification
revealed 8 of 15 phage with recombinant inserts. The
genome size of Watersipora is unknown, but if data apply
from the only marine bryozoan whose genome size is
known {0.4 pg, Potter, 1979), then the procedures outlined
above yielded approximately one genome equivalent of
bryozoan DNA in AJl. The library will be used to screen
for various multi-gene families.
References: Graham, D. E. (1978) Anal. Biochem. 85, 609-613. Loenen, W. A. M. and Brammar, W. J. (1980) Gene 10,
249-259. Mullins, J. I., Casey, J. W., Nicolson, M. O., Burck, K. B.
and Davidson, N. (1981) J. Virol. 38, 688-703. Potter, R. (1979) In: Advances in Bryozoology. G. P.
Larwood and M. B. Abbott (Eds.), pp. 11-32. Academic Press, London.
32. GENOME SIZE AND DNA COMPLEXITY OF PLASMODIDM FALCIPARUM
Investigators: Barbara R. Hougll-Evans, JUdith Howard*
Plasmodium falciparum is the parasite which causes
the most virulent form of human malaria. P. falciparum
DNA was prepared from cells cultured in vitro in human
red blood cells. The kinetics of reassociation were
measured along with a sample of tritium-labeled E. coli
DNA. It was found that the P. falciparum genome is 90
times as large as the E. coli genome, and that it contains
a repetitive component amounting to about 1096 of the
DNA.
*Scripps Clinic and Research Institute, La Jolla, California.
PUBLICATIONS
Anderson, D. M., Richter, J. D., Chamberlin, M. E., Price, D. H., Britten, R. J., Smith, L. D. and Davidson, E. H. (1982) Sequence organization of the poly(A) RNA synthesized and accumulated in lampbrush chromosome stage Xenopus laevis oocytes. J. Mol. Biol. 155 281-309.
Cabrera, c. V., Jacobs, H. T., Posakony, J. W., Grula, J. W., Britten, R. J. and Davidson, E. H. (1982) Transcripts of three mitochondrial genes in the RNA of sea urchin eggs and embryos. Science, submitted for publication.
Cabrera, c. V., Ellison, J. W., Lee, J. L., Britten, R. J. and Davidson, E. H. (1982) Regulation of cytoplasmic mRNA prevalence in sea urchin embryos: rates of appearance and turnover for specific sequences. Manuscript in preparation.
Clegg, K. B. and Piko, L. (1982) RNA synthesis and cytoplasmic polyadenylation in the one-cell mouse embryo. Nature 295, 342-345.
Davidson, E. H. (1982) Evolutionary change in genomic regulatory organization: speculations on the origins of novel biological structure. In: Evolution and Development. Dahlem Konferenzen. J. T. Bonner (Ed.), pp. 65-84. Spring_er-Verlag, Berlin.
Davidson, E. H., Hough-Evans, B. R. and Britten, R. J. (1982) Molecular biology of the sea urchin embryo. Science, in press.
Davidson, E. H. and Posakony, J. W. (1982) Repetitive sequence transcripts in development. Nature, in press.
Davidson, E. H., Thomas, T. L., Scheller, R. H. and Britten, R. J. (1982) The sea urchin actin genes, and a speculation on the evolutionary significance of small gene families. In: Genome Evolution. G. A. Dover and R. B. Flavell (Eds.), pp. 177-191. Academic Press, London.
Dawid, I., Britten, R. J., Davidson, E. H., Dover, G. A., Gallwitz, D. F., Garcia-Bellido, A., Kafatos, F. C., Kauffman, S. A., Moritz, K., Ohno, S., Schmidtke, J. and Schutz, G. (1982) Genomic change and morphological evolution. Group Report. In: Evolution and Development. Dahlem Konferenzen. J. T. Bonner (Ed.), pp. 19-39. Springer-Verlag, Berlin.
Flytzanis, C. N., Brandhorst, B. R., Britten, R. J. and Davidson, E. H. (1982) Developmental patterns of cytoplasmic transcript prevalence in sea urchin embryos. Devel. Biol. 91, 27-35.
Grula, J. W., Hall, T. J., Hunt, J. A., Giugni, T. D., Davidson, E. H. and Britten, R. J. (1982) Sea urchin DNA sequence polymorphism and reduced interspecies differences of the less polymorphic DNA sequences. Evolution, in press.
Professor: William J. Dreyer Senior Research Fellow: Janet M. Roman Research Fellow: David B. Teplow Graduate Student: David E. Levy Research Staff: Carolyn Anderson, Gayle-Linda Westrate
Support: The work described in the following research reports has been supported by:
Biomedical Research Support Grant (NIH) National Institutes of Health, USPHS National Science Foundation
31
Hough-Evans, B. R. and Howard, J. (1982) Genome size and DNA complexity of Plasmodium falciparum. Biochem. Biophys. Acta, submitted for publication.
Jacobs, H. T., Thomas, T. L., Posakony, J. w., HoughEvans, B. R., Britten, R. J. and Davidson, E. H. (1982) Mechanisms of eukaryotic gene regulation. In: Perspectives in Differentiation and Hypertrophy. W. Anderson (Ed.), Elsevier, New York, in press.
Jacobs, H. T., Posakony, J. W., Xin, J.-H., Grula, J. w., Britten, R. J. and Davidson, E. H. (1982) Mitochondrial genes shared with the nuclear genome in the sea urchin. Manuscript in preparation.
Lasky, L. A., Lev, Z., Thomas, T. L., Xin, J.-H., Lee, A. s., Britten, R. J. and Davidson, E. H. (1981) The expression of abundant and rare mRNA sequences during sea urchin development. In: Progress in Developmental Biology. H. w. Sauer (Ed.), pp. 75-86. Gustav Fischer Verlag, Stuttgart (Fortschritte der Zoologie 26).
Mauron, A., Kedes, L., Hough-Evans, B. R. and Davidson, E. H. (1982) Accumulation of individual histone mRNAs during embryogenesis of the sea urchin Strongylocentrotus purpuratus. Devel. Biol., in press.
Moore, G. P., Pearson, W.R., Davidson, E. H. and Britten, R. J. (1981) Long and short repeats of sea urchin DNA and their evolution. Chromosoma 84, 19-32.
Piko, L. and Clegg, K. B. (1982) Quantitative changes in total RNA, total poiy(A), and ribosomes in early mouse embryos. Devel. Biol. 89, 362-378.
Posakony, J. W., Scheller, R. H., Anderson, D. M., Britten, R. J. and Davidson, E. H. (1981) Repetitive sequences of the sea urchin genome. Nucleotide sequences of cloned repeat elements. J. Mo!. Biol. 149, 41-67.
Scheller, R. H., Anderson, D. M., Posakony, J. w., McAllister, L. B., Britten, R. J. and Davidson, E. H. (1981) Repetitive sequences of the sea urchin genome. Subfamily structure and evolutionary conservation. J. Mo!. Biol. 149, 15-39.
Scheller, R. H., McAllister, L.B., Crain, W.R., Durica, D. s., Posakony, J. W., Thomas, T. L., Britten, R. J. and Davidson, E. H. (1981) Organization and expression of multiple actin genes in the sea urchin. Molec. & Cell. Biol. 1, 609-628.
Thomas, T. L., Britten, R. J. and Davidson, E. H. (1982) An interspersed region of the sea urchin genome represented in both maternal poly(A) RNA and embryo nuclear RNA. Devel. Biol., submitted for publication.
Thomas, T. L., Posakony, J. W., Anderson, D. M., Britten, R. J. and Davidson, E. H. (1981) Molecular structure of maternal RNA. Chromosoma 84, 319-335.
Xin, J.-H., Brandhorst, B. P., Britten, R. J. and Davidson, E. H. (1982) Cloned embryo mRNAs not detectably expressed in adult sea urchin coelomocytes. Devel. Biol. 89, 527-531.
Summary: The focus of this laboratory is on cell-surface
protein molecules that function as mediators of cellular
communication and on the genes that code for such
molecules. Certain cell-surface proteins under study are
receptors for diffusible molecules. Other proteins, which
are members of highly polymorphic families of molecules,
may play a role in cell-cell interactions during
32
embryogenesis. Little is known about these molecules
since it has been difficult or impossible for anyone to
isolate such scarce cell-surface proteins in amounts
suitable for conventional analysis. We have approached
this problem by: (1) development of highly sensitive
instrumentation capable of analyzing very small quantities
of proteins; (2) analysis of molecules from tumor cell
surfaces, an approach that provides a cloned population of
cells that express substantial amounts of the specific
proteins of interest free of related molecules that are
expressed on other cell types; and (3) taking advantage of
gene cloning techniques to isolate the relevant genes.
In previous years we invested considerable effort in
the design and construction of microchemical instrumen
tation. That program has been successful and the desired
instruments are now in routine use (see Abstract No. 38).
As an example, the high sensitivity of the new sequencing
instrument now enables us to obtain useful amino-terminal
sequence information on very small quantities of proteins
isolated after analytical scale separation by SD5-gel
electrophoresis (see Hewick et aL, 1981 and Abstract
No. 37).
Just as homogeneous antibodies are most readily
obtained from cloned tumor cell lines or from hybridomas,
we have used tumors as a source of cell-surface receptors.
As an example, one of our collaborative efforts (see
Abstract No. 37) has been the study of a "tumor antigen"
(an iron receptor) expressed on most human melanomas.
In this case, a monoclonal antibody provided an effective
method for the isolation of this molecule. The amino-acid
sequence that we obtained allowed us to determine the
function of the receptor and also to synthesize a DNA
probe that is now being used to screen a cDNA library.
Several other cell-surface receptors that are
differentially expressed on specific normal and tumor cell
types are also under study in collaborative projects. We
not only wish to characterize such molecules but also hope
that the DNA sequences in and near the relevant
structural genes will provide insights into the differential
control of gene expression in specific classes of normal
and cancerous cells.
A closely related but more adventuresome aspect of
our study of cell-surface receptors is aimed at studying
the hypothetical "area code" molecules that are believed
to play a key role in cell-cell interaction during develop
ment (Dreyer et al., 1967; Hood et al., 1977). Such
molecules are also expected to be most easily studied
when expressed on cloned tumor cell lines. Of particular
interest is a family of molecules that is so diverse that
each new sarcoma cell line studied to date expresses a
molecule that appears to be serologically unique. This
type of antigenic diversity is very reminiscent of the V
regions of antibodies that provide the molecular basis for
the expression of "unique" antigens {idiotypes) on B-cell
tumor lines. We are interested in learning more about the
"unique" antigens of tumors and the presumptive multi ...
gene family(s) that codes for them. This project is
discussed in Abstract Nos. 34, 35 and 39. As described in
Abstract No. 35, we have also begun analyses of a family
of human genes that are related to a gene family in the
mouse that codes for one class of cell-surface "tumor
antigens."
Our aim in each of these closely related studies is to
increase our understanding of cell-surface proteins, the
genes that code for them, and the genetic mechanisms
that program their expression on specific cell lineages.
References: Dreyer, W. J., Gray, W.R. and Hood, L. (1967) Cold Spring
Harbor Sy mp. Quant. Biol. 32, 353-367. Hewick, R. M., Hunkapiller, M. w., Hood, L. E. and
Dreyer, w. J. (1981) J. Biol. Chem. 256, 7990-7997. Hood, L., Huang, H. V. and Dreyer, W. J. (1977) J.
Supramolec. Struct. 7, 531-559.
33. ANALYSIS 01' gp70-LIKE MOLECULES ON MORINE TUMORS
Investigators: Janet M. Roman, William J. Dreyer
When skin cells are transformed in experimental
animals, essentially every tumor expresses a cell--surface
molecule(s) that differs from those expressed on other
tumors derived in the same way. Even those tumors that
arise in different sites on the same animal express
different molecules (unique tumor antigens). We wish to
isolate these molecules and the genes that code for them.
These studies are based on the hypothesis that molecules
that function as unique "tumor" antigens are actually
molecules involved in the function of the normal cell from
which the tumor developed and that the genes that code
for these molecules are inherited in the germline of those
animals. This is known to be true for plasmacytomas,
where unique tumor antigens are the variable regions of
antibody molecules. The large family of molecules that
are expressed on other types of tumors may also play a
key role in normal cellular function.
Using syngeneic antisera that recognize unique tumor
antigens on murine fibrosarcomas, we have detected, by
immunoprecipitation studies, 70-90 K dalton molecules
that cross react with the major envelope protein (gp70) of
murine retroviruses (Roman et al., 1981). We have no
evidence that these are unique tumor antigens. However,
they are the only molecules precipitated by these
antisera, and they have the properties and the complexity
expected of such molecules. Each tumor line bears at
least two gp70-like molecules, each with extensive iso
electric point heterogeneity, and the patterns of these
molecules on two-dimensional (2D) SDS gels are stable,
heritable characteristics of each individual tumor line,
whether passaged in vivo or in vitro. Migration of these
molecules on 2D gels relative to an internal standard
confirms that distinctly different patterns are displayed
by the molecules from different tumors.
Reference: Roman, J. M., Hirsch, J., Readhead, C., Levy, D.,
DeOgny, L. and Dreyer, W. J. (1981) Transplant. Proc. 13, 1782-1786.
34. DETECTION ON HUMAN CELLS OF MOLECULES RESEMBLING RETROVIRAL gp70
Investigator: Janet M. Roman
The intriguing series of molecules found on the
fibrosarcomas discussed in Abstract No. 33 cross react
with the retroviral envelope protein gp70, and some of
these may in fact be carried by genes related to
retroviruses. Although we feel that these molecules may
play roles other than in virus formation, perhaps during
normal development and differentiation, the pursuit of
this question in the mouse, whose genome contains many
copies of retrovirus genes, leaves room for many counter
explanations. We therefore turned to humans, whose
genome has not been shown to contain any complete
retroviral sequences, and whose cells have not been shown
to produce any endogenous retroviruses, despite a very
extensive search.
Cell lines of human fibrosarcomas were grown and
labeled with 1251. Cells were lysed and proteins precipi
tated with baboon endogenous virus-specific antibodies
prepared in goats, and the precipitated molecules run on
two-dimensional (2D) gels. The primary molecules
migrated on these 2D gels in a glycoprotein type of
pattern that very closely resembled the patterns of gp70-
like molecules from murine cells, although the molecular
weight was slightly lower. Again, wide isoelectric point
heterogeneity was seen, just as for the murine gp70s.
These human molecules did not cross react with a murine
gp70.
35. GENES RELATED TO RETROVIRUS ENVEWPE GENES IN THE HUMAN GENOME
Investigators: Janet M. Roman, Carolyn Anderson, William J. Dreyer
33
The detection of retrovirus envelope-like genes in
humans, whose genome has not been shown to carry genes
capable of being expressed as retroviruses, would raise the
possibility that these genes may play a role in normal
cellular processes.
Retrovirus envelope proteins are involved in cell
recognition by the virus and they determine the virus host
range. Consequently, these proteins display a great deal
of polymorphism, even within a single species such as the
mouse, where they have been carefully analyzed. If one
considers the hypothesis that such molecules function in
normal cell-cell interactions and that they have been
"parasitized" by viruses, the expectation is that the
proposed normal envelope-like molecules are also quite
polymorphic. We therefore chose to look for the
structural genes for such molecules using a retrovirus
probe from baboons, a species evolutionarily related to
humans, and to do our initial screening tests under very
low stringency conditions.
We have screened a lambda library of human DNA
using a 3.1 kb probe of baboon endogenous retroviral DNA
that contains a 3' portion of the pol (polymerase) gene, the
entire gp70 (envelope) gene, the p15E gene and the U3
region of the 3' LTR. (The cloned baboon endogenous virus
was obtained from M. Cohen.) By using a probe containing
sequences flanking the envelope gene, we hoped to
optimize our chances of detecting the envelope gene, as
these flanking sequences are highly conserved in retro
viruses that haye been studied. Our initial screening has
yielded a number of clones. We have selected and
rescreened several of these, and are presently analyzing
them. We will_ use the envelope-like regions of these
clones to rescreen the human library at higher
stringencies. Recently, three other laboratories have also
found evidence for retrovirus-related genes in humans
(Martin et al., 1981; Noda et al., 1982; M. Cohen et al.,
private communication).
Our own focus continues to be on envelope-like genes
and we will seek to answer the following questions. (1) Do
humans, like mice, have a very large family of envelope
like genes (see Martin et al., 1981; Phillips et al., 1982)?
(2) Are any of these genes actively transcribed and
translated? (3) ls expression of these genes regulated by
34
tissue-specific developmental controls, again as seems to
be true in mice (see references in Hara et al., 1981)?
(4) Is the glycoprotein that we detected on the surface of
some human sarcomas related to retroviral envelope
proteins (see Abstract No. 34)? (5) Does the DNA
sequence in and near these genes provide clues as to the
origin and/or expression of these genes? (6) Do these
genes play any role in normal human development and/or
in cancer, or are they useless passenger DNA?
References: Hara, I., Izui, S., McConahey, P. J., Elder, J. H., Jensen,
F. c. and Dixon, F. J. (1981) Proc. Nat. Acad. Sci. USA 78, 4397-4401.
Martin, M. A., Bryan, T., Rasheed, s. and Khan, A. S. (1981) Proc. Nat. Acad. Sci. USA 78, 4892-4896.
Noda, M., Kurihara, M. and Takano, T. (1982) Nucleic Acids Res. 10, 2865-2878.
Phillips, S. J., Birkenmeier, E. H., Callahan, R. and Eicher, E. M. (1982) Nature 297, 241-243.
36. STRUCTURAL STUDIES OF A HUMAN LEUKEMIA ANTIGEN
Investigators: David B. Teplow, Marcus Braun•, William J. Dreyer
Cells of patients with acute lymphoblastic leukemia
(ALL) often display a common cell-surface antigen termed
CALLA. CALLA is a glycoprotein of 100,000 molecular
weight. CALLA-specific monoclonal antibodies have been
used to immunoprecipitate CALLAs from a variety of
radioiodinated leukemia cell lines and to compare them by
two-dimensional (2D) gel electrophoresis.
CALLA molecules with molecular weights of
approximately 100,000 have been identified on cells from
lines of Burkitt's lymphoma, T-cell ALL and null cell ALL.
Five of six lines examined expressed a major protein
species with an average isoelectric point (pl) of 5.83.
Additional charged species, with pis ranging from 5.55 to
6.30, were observed in three of the six lines.
To study the contribution of carbohydrate side chains
to the observed charge heterogeneity, 2D gels are being
run on samples deglycosylated by treatment with tri
fiuoromethanesulfonic acid. Sequence relationships are
being probed by peptide mapping of partial enzymatic
digests using sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). In addition, preparative
quantities of CALLA, isolated by affinity chromatography
and SDS-PAGE, are being produced for analysis in the
gas-liquid solid phase microsequencer.
We hope to use the sequence data obtained to better
understand the structure and function of this common
leukemia antigen and its relationship to other antigens
expressed on normal cells and tumors. We expect these
data and the results of our comparative analyses to extend
our basic understanding of the role of such cell-surface
glycoproteins in normal cell function and to better enable
us to evaluate the efficacy of using such proteins as
targets in immunotherapeutic trials in vivo.
*Fred Hutchinson Cancer Research Center, Seattle, Washington.
37. MELANOMA SURFACE ANTIGEN p97 IS RELATED TO TRANSFERRlN
Investigators: Joseph P. Brown*, Rodney M. Bewick**, J:ngegerd Hellstrom•, Karl Erik Hellstrom*, Russell F. Doolittle•••, William J. Dreyer
This study, in which we have identified a human
melanoma cell-surfa~ protein as an iron receptor,
illustrates the power of the methodology and instru
mentation now available at Caltech (Brown et al., 1982).
A small amount of monoclonal antibody directed against
the cell-surface glycoprotein p97, which is found
predominantly on melanoma cells (Woodbury et al., 1980),
was added to a lysate of several grams of cultured cells.
The antigen-antibody complex, along with a slight excess
of the monoclonal antibody, was
lysate by passage through
Staphylococcus aureus protein A.
isolated from the crude
a column containing
After washing, the pH
was lowered to elute the monoclonal antibody and the
antigen. These proteins were separated on an SDS
acrylamide gel and stained with Coomassie blue. The
stained antigen was electrophoretically eluted and the
amino terminal sequence was determined in the new,
highly sensitive, gas-phase microsequenator (Hewick
et al., 1981). A computer search identified the amino acid
sequence as being closely related, but not identical, to
known members of the transferrin family of iron-binding
proteins. The cell-surface molecule was tested and found
to bind iron. Thus its function, previously unknown, was
determined by this approach. The amino terminal
sequence information has also been utilized to synthesize
DNA probes for use in isolation of the gene that codes for
this molecule and the control regions surrounding that
gene. The genes of normal cells will be compared with
those of melanomas to ascertain whether DNA sequences
provide any clues that might explain the high level of
expression of p97 on melanomas. At the same time, the
amino acid sequence information is being utilized to
synthesize a peptide that hopefully will be antigenic when
coupled to a suitable carrier molecule, a property that is
of potential clinical value. Monoclonal antibodies
directed against this molecule may also be useful in
diagnosis and therapy.
References: Brown, J. P., Hewick, R. M., Hellstrom, I., Hellstrom,
K. E., Doolittle, R. F. and Dreyer, W. J. (1982) Nature 296, 171-173.
Hewick, R. M., Hunkapiller, M. W., Hood, L. E. and Dreyer, W. J. (1981) J. Biol. Chem. 256, 7990-7997.
Woodbury, R. G., Brown, J. P., Yeh, M. Y., Hellstrom, I. and Hellstrom, K. E. (1980) Proc. Nat. Acad. Sci. USA 11, 2183-2187.
*Fred Hutchinson Cancer Center, Seattle, Washington. **Genetics Institute, Boston, Massachusetts. ***Department of Chemistry, University of California, San Diego.
38. PROTEIN CHEMICAL AND RECOMBINANT DNA FACILITY
Investigators: William J. Dreyer, Michael w. Htmkapiller, Rodney M. Hewick*, Charles E. Giffin**, Leroy E. Hood
The instruments and methods we have developed at
Caltech and at the Jet Propulsion Laboratory represent a
major advance in the speed and sensitivity with which
genes and their protein products can be isolated and
characterized.
We have built a new type of microsequenator for
peptides and proteins that uses (a) gas-phase reagents at
critical points in the Edman degradation, and (b) a
cartridge-style reaction cell (Hewick et al., 1981). This
new sequenator is particularly suitable for recombinant
DNA technology, where amino acid sequence information
on a very limited supply of protein (e.g., a few micro
grams eluted from a staiiled SDS gel) may be required.
Such information can be used to synthesize DNA probes
that are valuable for the isolation and cloning of rare
message genes. Also, an advanced type of automated
peptide synthesizer is under construction, and a very
effective automatic DNA synthesizer has been completed.
Both of these instruments are similar in design to the
protein sequencer. These will allow for the rapid
synthesis of substantial quantities of rare proteins and
peptides and for the synthesis of DNA probes and genes.
Both of these automated synthesizers make use of amino
acid sequence information.
A new instrument, which we expect to have in routine
operation within the next two years, is a new type of mass
spectrometer (Dreyer et al., 1974; Giffin et al., 1974;
Boettger et al., 1979). This mass spectrometer is small
35
and stable and is designed to be capable of being operated
by a relatively inexperienced operator. The key feature in
the development of this new mass spectrometer was the
development of an electro-optical ion detector array that
has the capacity to detect a single molecular ion. The
output of the electro-optical detector is ultimately fed to
a computer where the data are calibrated and analyzed.
This mass spectrometer can analyze PTH amino acid
derivatives in the femtogram range, thus making it
greater than a thousandfold more sensitive than present
HPLC instrumentation. Moreover, with the implemen
tation of an appropriate sample injection system and
computerized data processing, it should be possible to
have a sample turnaround time of less than 5 minutes on
the a mass spectrometer. Thus we envision in the future
multiplexing two or more gas-liquid solid phase micro
sequenators to a mass spectrometer and, accordingly,
being capable of carrying out a large number of analyses
each day using only minute amounts of proteins or
peptides.
References: Boettger, H. G., Giffin, C. E., Norris, D. D. and Dreyer,
W. J. (1979) In: Monograph on Image Detectors in Chemistry. Y. Talmi (Ed.), pp. 291-318. American Chemical Society, Washington, D.C.
Dreyer, W. J., Kupperman, A., Boettger, H. G., Giffin, C. E., Norris, D. D., Grotch, S. L. and Theard, L. P. (1974) Clin. Chem. 20, 998-1002.
Giffin, C. E., Norris, D. D. and Boettger, H. G. (1974) lnternat. J. Mass Spectrometry and Ion Physics 15, 437-449.
Hewick, R. M., Hunkapiller, M. W., Hood, L. E. and Dreyer, W. J. (1981) J. Biol. Chem. 256, 7990-7997.
Hood, L., Hunkapiller, M., Hewick, R., Giffin, C. E. and Dreyer, w. J. (1981) J. Supramol. Struct. & Cell. Biochem. 17, 27-36.
*Genetics Institute, Boston, Massachusetts. **Jet Propulsion Laboratory, Pasadena, California.
39. SPECULATIONS ON THE ROLE OF MOBILE GENES AND CELL-SURFACE MOLECULES IN EMBRYOGENESIS
Investigator: William J .. Dreyer
There is overwhelming evidence that antibodies must
have evolved from similar, more primitive protein
receptor systems. Antibody molecules are found in the
earliest vertebrates but homologous molecules have not
been detected in invertebrates. However, when one
compares the amino acid sequence of the heavy and light
chains of imrnunoglobulins from the earliest vertebrates
with antibodies of vertebrates that evolved much later, it
becomes evident that such a complex, polymorphic system
of molecules that display a high degree of homology
36
across all vertebrate species could not have arisen de nova
at the time of the emergence of vertebrates. During
differentiation, antibody molecules themselves first
appear as cell-surface receptors that serve as triggers for
further cellular division, differentiation and even further
gene cutting and splicing events (see Leder, 1982, for a
good review). The immediate evolutionary precursors of
antibody molecules are also likely to be found on cell
surfaces as part of an elaborate recognition system used
for sensing the cellular environment. Furthermore, the
very sophisticated gene cutting and splicing mechanisms
that program orderly genetic rearrangements during
embryogenesis of the immune system (see Huang and
Dreyer, 1978) are not likely to be unique to those tissues
or to have appeared in a creationary flash. Accordingly, I
feel quite confident that there must be other multigene
families that also use similar sophisticated genetic cutting
and splicing mechanisms to rearrange genes during normal
development. Many types of transposable elements and
similar genes are already well known in organisms such as
bacteria, yeast, corn, Drosophila, as well as in
vertebrates, including man (e.g., the retrovirus-like genes
of Abstract No. 35). It would be very surprising if similar
flip-flop and/or transposable elements do not function in
embryogenesis (see Patrusky, 1981, for a brief review).
More complex splicing events that use mechanisms
evolutionarily related to those that occur during the
embryogenesis of the immune system would be expected
to be useful for generating large families of cell-surface
receptors. Furthermore, descriptive biology dating back
to the previous century provides overwhelming evidence
that cell surfaces must display very sophisticated arrays
of receptors that we now refer to as area code molecules
(Hood et al., 1977). For example, the cells of early
embryos can be dissociated and upon reassociation,
specific cells move back into correct positions relative to
the rest of the cells. Also, cells marked with carbon
particles migrate through embryos to precise locations.
Such cellular migration occurs reproducibly under genetic
command. It seems correct to assume that cell-surface
receptors play a role in programmed cell migration and
placement as an embryo assembles itself. I have recently
summarized this view at a symposium in honor of C. B.
Anfinsen (Dreyer, 1982).
Taking the above into account, we have used three
basic approaches in our search for area code molecules:
(1) analysis of cell-surface molecules that are polymorphic
and/or may serve as receptors (see Abstract Nos. 33, 34,
36 and 37), (2) analysis of genes that are likely to be
involved in genetic rearrangements or gene splicing events
(see Abstract No. 35), and (3) the development of
instruments and methods that facilitate these studies (see
Abstract No. 38).
As may be seen in the abstracts that precede this, cell
surfaces of different tissues display complex assortments
of molecules whose functions are mostly unknown. At the
very least, our search for area code molecules will help
clear up some of these mysteries (as for example with the
characterization of melanoma antigen p97; Abstract
No. 37), and further our understanding of cell-cell
interactions.
References: Dreyer, w. J. (1982) Jn: Proceedings of the International
Symposium on the Contributions of Chemical Biology to the Biomedical Sciences. F. Goldberger and A. Schechter (Eds.), Academic Press, New York, in press.
Hood, L., Huang, H. V. and Dreyer, W. J. (1977) J. Supramolec. Struct. 7, 531-559.
Huang, H. V. and Dreyer, W. J. (1978) J. Immunol. 121, 1738-1747.
Leder, P. (1982) Scientific American 246, 102-115. Patrusky, B. (1981) Mosaic 12, 41-45.
PUBLICATIONS
Brown, J. P., Hewick, R. M., Hellstrom, I., Hellstrom, K. E., Doolittle, R. F. and Dreyer, W. J. (1982) Human melanoma-associated antigen p97 is structurally and functionally related to transferrin. Nature 296, 171-173.
Dreyer, w. J. (1982) Molecular evolution, antibody formation, and embryogenesis. In: Proceedings of the International Symposium on the Contributions of Chemical Biology to the Biomedical Sciences. F. Goldberger and A. Schechter (Eds.), Academic Press, New York, in press.
Hewick, R. M., Hunkapiller, M. W., Hood, L. E. and Dreyer, W. J. (1981) A gas-liquid solid-phase peptide and protein sequenator. J. Biol. Chem. 256, 7990-7997.
Hood, L., Hunkapiller, M., Hewick, R., Giffin, c. E. and Dreyer, W. J. (1981) Microchemical instrumentation. J. Supramolec. Struct. & Cell Biochem. 17, 27-36.
Hunkapiller, M. W., Hood, L. E., Hewick, R. M. and Dreyer, w. J. (1982) High sensitivity sequencing with a gas-phase sequenator. Jn: Methods in Enzymology. Vol. 91, Part I, Enzyme Structure, S. P. Colowick and N. 0. Kaplan (Eds.), in press.
Jennings, K. R., Kaczmarek, L. K., Hewick, R. M., Dreyer, W. J, and Strumwasser, F. (1981) Protein phosphorylation during afterdischarge in peptidergic neurons of Aplysia. J. Neurosci. 2, 158-168.
Roman, J. M., Hirsch, J., Readhead, C., Levy, D., DeOgny, L. and Dreyer, w. J. (1981) Heritable differences among gp70-like molecules on C3H ultraviolet light-induced fibrosarcomas. Transpl. Proc. 13, 1782-1786.
Professor: Leroy E. Hood Sherman Fairchild Distinguished Scholan James F. Crow Visiting Professor: Carol H. Sibley Visiting Associates: Martha W. Bond, Minnie McMillan,
Carlton H. Paul Ill, Marit P. Pecht, Irving L. Weissman Senior Research Fellows: Henry V. Huang, Michael W.
Hunkapiller, Roger M. Perlmutter, Carol Readhead Research Fellows: Richard H. Douglas, Robert S.
Goodenow, Johanna A. Griffin, Joan A. Kobori, Ellen B. Kraig, Anders Orn, Michael Steinmetz, Iwona T. Stroynowski, Martha C. Zuniga
Graduate Students: Stephen T. Crews, Kurt Eakle, Jay W. Ellison, Douglas A. Fisher, Tim Hunkapiller, Stuart K. Kim, Mitchell Kronenberg, Donna L. Livant, Arthur Roach, Beverly Taylor Sher, Gerald J.-M. Siu*, Yi Henry Sun, Astar Winoto
Member of the Professional Staff: Suzanna J. Horvath Research Staff: Bennett J. Berson, Paul K. Cartier m,
Michael Douglas, Vincent R. Farnsworth, Chin Sook Kim, Fred R. Larsen, Eva H. Lujan, Mary J. Macchi, Janet M. McNicholas, Karyl Minard, Stella Olive, Elizabeth A. Springer
Laboratory Staff: Maria A. De Bruyn, Bertha E. Jones, Jessie Walker
*Division of Chemistry and Chemical Engineering, California Institute of Technology.
SUpport: The work described in the following research reports has been supported by:
Ameriean Caneer Society The American-Scandinavian Foundation Earle C. Anthony Feliowship Applied Molecular Genetics, Inc. (AMGen) Arthritis Foundation Baylor College of Medicine Ethel Wilson Bowles and Robert Bowles
Professorship California Foundation for Biochemical Research Cancer Research Institute, Inc. Deutsche Forschungsgemeinschaft Fairchild Foundation Finkelstein Support for Interferon Research Fulbright Fellowship Hoag Foundation The Henry Kaiser Family Foundation MacArthur Foundation Louis B. Mayer Foundation Monsanto Co. Francis Mosley Fund for Cancer Research National Institutes of Health, USPHS National Research Council, Washington, D.C. National Science Foundation Prince Charitable Trusts Gordon Ross Medical Foundation Stanford University Sundry Donors for Cancer Research University of Southern California Weingart Foundation
Summary: Our laboratory is interested in multigene
families that encode and regulate various aspects of the
vertebrate immune response. These families include the
antibody gene families, the T-cell receptor gene families
and several gene families encoded by the major histo
compatibility complex. We use the mouse as a model
37
system and employ a variety of techniques including
protein chemistry, nucleic acid chemistry, and
immunology.
Antibodies are made up of two types of
polypeptides-light and heavy chains. These chains are
encoded by three multigene families-two encode light
chains (A and K) and the third encodes the heavy chain.
The light chains are encoded by three gene segments
termed variable (V), joining (J), and constant (C). The
heavy chains are encoded by three similar gene segments,
as well as a fourth-diversity (D). The antibody molecU]e
has two major categories of functions--pattern
recognition encoded by the variable domains and effector
functions encoded by the constant domains. Constant
domaihs are encoded by the constant genes, whereas the
remaining gene segments encode the variable regions.
Antibody genes employ two types of DNA rearrangement
during differentiation. First, in variable gene formation,
the V and J or V, D, and J gene segments are juxtaposed
to generate a contiguous coding region for the V L and V H
genes. Second, class switching leads to a DNA rearrange
ment whereby one heavy chain constant region is replaced
by a second heavy chain constant region during the
differentiation of the antibody-producing or B cell. In
addition, antibody heavy chain genes may undergo
alternative patterns of RNA splicing to generate
membrane and secreted for-ms of the corresponding
polypeptides. There are a variety of mechanisms for
generating diversity in antibody genes which include
(1) the existence of mU]tiple V, D, and J genes, (2) the
combinatorial joining together of any V with any J or D
gene segments, (3) two or three special types of somatic
mutation, and (4) the combinatorial association of light
and heavy chains.
We are currently studying several features of the
antibody gene system (Kim et al., 1981). First, we are
interested in the detailed organizational relationships of a
simple V H gene family-the Tl5 gene family with four
members in the inbred BALB/c mouse. Second, we are
interested in the evolution of the T15 gene family in
various inbred strains of mice, and substrains of mice,
rats, guinea pigs, and even humans. Third, we are
interested in the organization of the D gene segments and
their relationship to the J gene segments in the heavy
chain gene family. Fourth, we are interested in molecular
mechanisms which lead to class switching. Fifth, we are
interested in examining the organization and diversity
38
contained in the heavy chain gene family which includes
among its members the gene or genes responding to
immunization by the simple carbohydrate antigen dextran.
The dextran V H gene family is interesting and contrasts
sharply with the T15 gene family in that it is large (20-25
V H genes) and has a series of very closely related V H gene
members.
We also are attempting to clone the genes encoding
the T-cell receptor. We have employed a variety of
approaches assuming B- and T-cell receptors share
common VH genes. These approaches have failed. We are
now using T-cell hybridomas that synthesize T-cell
suppressor factors in an attempt to clone the corre
sponding genes.
The major histocompatibility complex of the mouse
encodes several families of cell-surface molecules that
are involved in self-nonself recognition. The class I gene
family includes the transplantation antigens and
hematopoietic differentiation antigens such as TL, Qa-2,3
and Qa-1. The class II gene family encodes the Ia
polypeptides or immune response genes. The class III gene
family encodes a variety of components that are involved
in the early activation sequence of the complement
pathway.
In the BALB/c mouse we have cloned most of the class
I genes (approximately 40) (Steinmetz et al., 1982),
determined their intron-exon organization, mapped many
of these genes and their corresponding gene clusters into
various regions of the major histocompatibility complex
by restriction enzyme site polymorphisms, and transferred
these class I genes into mouse L cells where they are
expressed as cell-surface molecules that can be examined
by immunochemical, serologic, and functional assays
(Goodenow et al., 1982). We also have nearly cloned the
complete class II gene region which appears to contain six
to seven genes. We are in the process of sequencing
several of these genes and using them for gene transfer
experiments to study their functions.
We are continuing to develop new types of micro
chemical instrumentation (Hood et al., 1981). We are now
in the process of refining an automated DNA synthesizer
and have just begun to build a peptide synthesizer. We are
continuing work that we hope will lead to automated
techniques for DNA sequencing. In collaboration with
Professor Dreyer and the Jet Propulsion Laboratory, we
are continuing to develop a highly sensitive mass
spectrometer for the analysis of amino acid derivatives.
These instruments in addition to the sensitive gas-phase
solid-state protein sequenator will constitute a micro
chemical facility with enormous potential for
manipulation of genes and proteins. Indeed, we are using
this facility to characterize proteins (and genes) that are
available in very small quantities such as the acetyl
choline receptor and various neurohormones.
References: Goodenow, R. S., McMillan, M., Orn, A., Nicolson, M.,
Davidson, N., Frelinger, J. A. and Hood, L. (1982) Science 215, 677-679.
Hood, L., Hunkapiller, M., Hewick, R., Giffin, C. E. and Dreyer, W. J. (1981) Microchemical instrumentation. ICN-UCLA Symposium. J. Supramol. Struct. & Cell Biochern. 17, 27-36.
Kim, S., Davis, M., Sinn, E., Patten, P. and Hood, L. (1981) Cell 27, 573-581.
Steinmetz, M., Winoto, A., Minard, K. and Hood, L. (1982) Cell 28, 489-498.
40. CHROMOSOMAL ARRANGEMENT OF ANTIBODY HEAVY CHAIN VARIABLE REGION GENES
Investigators: Stephen T. Crews, Elizabeth A. Springer
The heavy chain gene locus contains hundreds of heavy
chain variable region (V H) genes scattered over a large
region of chromosome 12 of the mouse. An understanding
of the function, evolution, and expression of the DNA
contained in this locus requires a knowledge of the
physical arrangement of these genes along the chromo
some. We have attempted to investigate the organization
of the VH locus by using recombinant DNA technology.
The system that we have chosen is the small family of
four V H genes (designated Vl, V3, Vll, and Vl3) that
comprise the T15 V H gene family. Utilizing both lambda
and cosmid recombinant DNA clones, we have isolated and
sequenced all four genes in this family (Crews et al.,
1981). These clones constitute a series of overlapping
DNA fragments containing the Tl5 genes. Restriction
mapping and hybridization studies have shown that these
overlapping clones fall into three clusters of DNA. The
first cluster contains the Vl and V3 genes, which are
separated by 15 kb of DNA. The Vll and Vl3 genes are
each found in a cluster of greater than 33 kb of DNA that
is unlinked to any other V H gene. Control experiments
have shown that our hybridization conditions can detect
all known V H genes, thus we can exclude the possibility of
distantly related V H genes also residing on these clones.
We have also isolated a large number of cosmid clones
bearing other, less related V H genes. Restriction mapping
has allowed us to place these clones into nine clusters of
nonoverlapping DN~. Overall, including the T15 VH gene
family, we have analyzed 21 different V H genes found on
12 gene clusters and encomJ;>assing 492 kb of DNA. We
have utilized this information to make several observa
tions about the organization of V H genes along the
chromosome. (1) Closely related V H genes can be
clustered (e.g., Vl and V3 are closely linked) although the
generality of this observation for the entire locus is still
unknown. (2) The spacing distance between V H genes is
variable; it can vary from 4 kb to greater than 30 kb. (3)
The average spacing distance is large, greater than 23 kb.
Assuming a minimum of 250 V H genes, the V H gene locus
may encompass over 5 million base pairs. We are
currently continuing molecular cloning experiments with
the goal of physically linking together all of the genes of
the T15 V H gene family.
Reference: Crews, S., Griffin, J., Huang, H., Calame, K. and Hood, L.
(1981) Cell 25, 59-66.
41. SEQUENCE ORGANIZATION OF THE T15 VH GENE FAMILY
Investigators: Gerald J. M. Siu•, Stephen T. Crews, Elimbeth A. Springer, Kathryn Calame**
We are interested in studying the structure, evoluti_on
and exJ;>ression of the heavy chain variable region (V H)
gene locus. Since the mouse genome contains hundreds of
V H genes, we have focused on a small family of four
closely related V H genes, called the T15 V H gene family
(Crews et al., 1981). Currently, large stretches of DNA
flanking these four genes are being sequenced utilizing
both the Ml 3-dideoxy sequencing method and the
chemical cleavage procedure of Maxam and Gilbert. This
information will allow a more complete understanding of
the structure of V H genes, identification of functionally
significant regions (as defined by conservation of
sequence), and the molecular events involved in the
evolution of these genes. Heteroduplex analysis of the
T15 family genes has revealed that there are extensive
stretcJ:tes of flanking sequence homology. Sequence
analysis of the regions where these flanking sequence
homologies end may allow a better understanding of the
mechanism of gene duplication. Finally, in the laboratory
of Dr. Kathryn Calame at UCLA, the caJ;> site (and
presumed site of initiation of transcription) has been
identified for one of the genes in this family. It lies
39
approximately 60 nucleotides upstream from the
methionine initiator codon of the signal peptide sequence.
There is a sequence, ATA, found 32 nucleotide pairs 51 to
the cap site that is characteristic of most eukaryotic
promoters. Examination of the sequences corresponding
to the promoters of each of the genes of this family as
well as other V H genes, in conjunction with functional
studies of their promoters, will allow a clearer picture of
the structure and function of variable region gene
promoters and the regulation and expression of antibody
genes.
Reference: Crews, s., Griffin, J., Huang, H., Calame, K. and Hood, L.
(1981) Cell 25, 59-66.
*Division of Chemistry and Chemical Engineering, California Institute of Technology. **Department of Biological Chemistry and the Molecular Biology Institute, University of California, Los Angeles.
42. EVOLUTIONARY ANALYSIS OF A SMALL IMMUNOGLOBULIN HEAVY CHAIN VARlABLE REGION (VH) MULTIGENE FAMILY
Investigators: Johanna A. Grifrm, Roger M. Perlmutter, Bennett J. Berson
T15 is a multigene family which in BALB/c mice has
four members that are from 86-96% homologous. The T15
gene segment after which the family was named encodes
the V H region of the antibody which binds J?hOSJ?hocholine
(PC). A gene segment, V3, located 15 kilobases 5' to Tl5
is a pseudogene. Two other gene segments, Vll and V13,
are probably functional but do not apr;>ear to be expressed
in a PC immune response and are not closely linked to the
Tl5-J;>seudogene comJ;>lex (Crews et al., 1981).
We have compared Southern blot hybridization
patterns of liver DNA from a number of inbred and wild
strains of mice and rats using a T15 cDNA probe. In a
majority of mouse strains, all four of the strongest
hybridizing bands are present, indicating that this gene
family arose before the strains diverged. There is a high
degree of conservation of the bands containing the T15
and V13 gene segments among mouse strains. Vll is
slightly more i;>olymorphic among different strains or
deleted in one strain, while the V3 pseudogene is deleted
in several strains. This variation probably reflects the
expansion and contraction of a family of closely related
genes over a period of probably no more than a million
years.
To analyze these findings in more molecular detail, we
selected the BlO.P mouse strain because it is thought to
40
be very distantly related to BALB/c, the strain from
which we originally sequenced the genes of the Tl5 family
(Crews et al., 1981). We prepared a sperm DNA genomic
library in the A bacteriophage L4 7 (Loenen and Bram mar,
1980). Using a T15 probe, we have isolated the T15 and
V3 gene segments. As in BALB/c mice, the T15 and V3
gene segments are linked in BlO.P mice. DNA sequence
analysis indicates that the T15 coding region in BlO.P
differs from T15 in BALB/c by 7 bases. Of these, four
would cause changes in the amino acid sequence. These
four phenotypic differences produce an amino acid
sequence in the BlO.P protein that is identical to the
protein sequence reported for the anti-PC antibody of a
closely related strain, C57/BL (Rudikoff and Potter,
1980). This similarity suggests that T15 is the V H gene
segment used in the immune response to PC in 810.P
mice.
The V3 pseudogene in BALB/c is nonfunctional for
several reasons. (1) There is a 4-base insertion in the
coding region. (2) It has a termination codon. (3) It has a
deletion in the spacer between the two conserved
rearrangement recognition sequences that would probably
preclude its rearrangement (Huang et al., 1981). (4) Part
of the leader sequence is deleted (G. Siu and S. Crews,
personal communication). The V3 gene segment of 810.P
does not have the 4-base insertion or the termination
codon within its coding region. However, it does have
deletions in the leader sequence and in the rearrangement
recognition region identical to those in BALB/c.
Therefore, the two deletion mutations must also have
occurred before strain divergence, while the insertion and
termination mutations in the BALB/c V3 must have
occurred more recently.
These analyses will allow us to gain some insight into
the evolution of the small Tl5 V H gene family, but more
importantly, they will expand our understanding of some
mechanisms involved in evolution in general.
References: Crews, S., Griffin, J., Huang, H., Calame, K. and Hood, L.
(1981) Cell 25, 59-66. Huang, H., Crews, S. and Hood, L. (1981) J. Mo!. Appl.
Genet. 1, 93-101. Loenen, W. and Brammar, W. (1980) Gene 10, 249-259. Rudikoff, S. and Potter, M. (1980) J. Immunol. 124, 2089-
2092.
43. SEQUENCE ORGANIZATION OF THE J558 GENE FAMILY JN BALB/c MICE
Investigators: Donna L. Livant, Garol Readhead, John B. Wall*
Dextran is the branched, storage polysaccharide found
in bacteria that contains a-1,3, a-1,4, and cx-1-6 linked
glucose in varying preparations (Sugii et al., 1981).
Protein sequence analysis of heavy chain variable regions
from 21 BALB/c hybridomas has shown that when a
BALB/c mouse is immunized with the cx-1,3 dextran,
B1355S, it expresses at least five different V H segments
in its immune response (Clevinger et al., 1981). One of
these V H segments corresponds to that of the myeloma
antibody J558, and was found in 16 of the 21 hybridoma
antibodies sequenced. We have, therefore, defined the
J558 V H gene as the prototype gene for the J558 gene
family. By studying the sequence organization of the J558
family, we hope to find out which of the five V H segment
variants found by Schilling et al. (1982) is encoded in the
germline, and which must have arisen by somatic
mutation. From our sequence and linkage studies, we also
hope to learn something about the structure of the V H
locus in general since several other V H families appear by
genome blotting experiments to be of similar size and
complexity (D. Livant, unpublished observations; R.
Riblet, unpublished observations).
In order to isolate the J558 gene family from the
BALB/c germline, we have screened approximately 1 x
106 clones from a BALB/c sperm DNA library (Davis
et al., 1980), and have selected the 20 most strongly
hybridizing clones for further study. Each of the 2 0
clones contains one, two, or three V H genes exhibiting
varying homology to J558. Jn all cases, at least one gene
per clone has significant 5'-flanking sequence homology to
the J558 gene. Also, these clones fall into five different
groups based on preliminary mapping data, suggesting that
the J558 VH gene family in BALB/c has 8-10 members.
Further evidence indicating that the J558 family has at
least eight members comes from the genome blotting of
Eco RI-digested BALB/c liver (germ!ine) DNA using the
J558 gene as a probe. About 25 Eco RI bands hybridize to
J558, 8-10 bands more strongly than all the rest.
We have sequenced 378, a member of the J558 gene
family having 5'-flanking sequence homology to J558, and
found it to be 93% homologous to the J558 gene. The
implied protein sequence of 37B has six amino acid
differences with respect to the J558 coding sequence.
Five of these involve single base changes. Four of the
amino acid changes are conservative with respect to
charge and size. The two changes leading to major
differences in charge and shape of Jhe amino acids
involved are located in the second hypervariable region.
The V H gene 37B resides on a clone having two other V H
genes, 37 A and 37C, neither of which has significant 5'
flanking sequence homology to the J558 gene. Also,
preliminary sequence data suggest that 37 A and 37C are
less homologous to J558 than 37B.
From these data, we conclude that the J558 family in
BALB/c mice probably consists of 8-10 members having
90% or greater homology as well as substantial flanking
sequence homology. Genes in this family are closely
linked to other V H genes, although in most instances the
neighboring V H gene does not appear to be a member of
the J558 family. How the observed clusters of two, three
and four V H genes are themselves linked awaits further
study.
References: Clevinger, B., Thomas, J., Davie, J., Schilling, J., Bond,
M., Hood, L. and Kearney, J. (1981) In: lmmunoglobulin Idiotypes. ICN-UCLA Symposium on Molecular and Cellular Biology. C. Janeway, E. E. Sercarz, H. Wigzell and C. Fred Fox (Eds.), Vol. XX, pp. 159-168. Academic Press, New York.
Davis, M., Calame, K., Early, P., Livant, D., Joho, R., Weissman, I. and Hood, L. (1980) Nature 283, 733-739.
Schilling, J., Clevinger, B., Bond, M., Davie, J. and Hood, L. (1982) Manuscript in preparation.
Sugii, s., Kabat, E., Shapiro, M. and Potter, M. (1981) J. Exp. Med. 153, 166-181.
*Undergraduate, California Institute of Technology.
44. MOLECULAR GENETICS OF ANTI-STREPTOCOCCAL ANTIBODIES
Investigators: Roger M. Perlmutter, Martha W. Bond, Moon Nahm*, Joseph M. Davie*
Group A streptococcal carbohydrate (GAC) is a
thymus-dependent antigen that elicits a highly restricted
antibody response in inbred mice entirely confined to IgM
and IgG3 classes (Perlmutter et al., 1977). Although
individual mice express IgG3 anti-GAC antibodies that
appear to be monoclonal by isoelectric focusing criteria,
it is unusual for two mice to produce identical antibodies.
Thus the murine anti-GAC antibody repertoire is quite
large. In addition, most of the electrophoretic diversity in
these antibodies resides in the heavy chains (Perlmutter
et al., 1978a).
As a first step in examining the manner in which a
single clone of anti-GAC antibody producing cells
41
becomes dominant during the course of the murine
immune response, we have initiated an analysis of V Hand
VL genes employed in the production of anti-GAC
antibodies. Fifty hybridomas with anti-GAC specificity
have been generated using hyperimmune A/J mice. N
terminal sequence analysis of four anti-GAC heavy chains
yielded a single sequence through residue 55, suggesting
that one V H gene predominates in generating these
antibodies. Four light chains that share a common
idiotype and isoelectric focusing pattern were found to
have almost identical primary structure with a single
variant residue at position 35. A fifth light chain that
lacks the common idiotype differs at seven positions in
the first 50 residues.
Recently, we have generated a genomic library from
A/J sperm DNA and have developed DNA probes that
should permit the enumeration and characterization of
anti-GAC antibody genes in the mouse. In this fashion we
hope to gain an appreciation of the extent to which
somatic mutation underlies the generation of this diverse
antibody response. Our research is also directed toward
an understanding of the rules that govern specific
expression of particular heavy and light chain genes, and
of the curious restriction of murine anti-carbohydrate
antibodies to the IgG3 subclass (Perlmutter et al., 1978b).
References: Perlmutter, R. M., Briles, D. E. and Davie, J. M. (1977) J.
Immunol. 118, 2161-2166. Perlmutter, R. M., Briles, D. E., Greve, J. M. and Davie,
J.M. (1978a) J. Immunol. 121, 149-158. Perlmutter, R. M., Hansburg, D., Briles, D. E., Nicolotti,
R. A. and Davie, J. M. (1978b) J. Immunol. 121, 566-572.
*Washington University School of Medicine, St. Louis, Missouri.
45. STRUCTURE AND EVOLUTION OF HUMAN IMMUNOGLOBULIN GAMMA CONSTANT REGION GENES
Investigators: Jay W. Ellison, Bennett J. Berson, Kathryn L. Galame*, Barbara Chapman**
Immunoglobulin G (IgG) molecules in humans are
divided into four subclasses based on the presence of
particular gamma heavy chain constant regions (CY
regions). These Cy regions (Cyl' Cy2' Cy3' and Cy4) are
encoded by distinct germline genes that are presumed to
be the products of gene duplication of an ancestral Cy
gene. Protein sequencing studies have shown that the
human gamma chain subclasses are about 95% homologous
42
to one another, implying that the corresponding genes
have diverged very recently in evolutionary history. We
are interested in studying structural features of human Cy
genes in order to gain insights into the evolution of the
human Cy gene family.
We have used a human cy3 cDNA probe to isolate
genomic C -containing fragments from a library of human y
DNA cloned in lambda Charon 4A bacteriophage. We have
completely sequenced three genes isolated from this
library, and have identified them as encoding cyl' cy2'
and cy4 regions (Ellison et al., 1981; Ellison and Hood,
1982; Ellison et al., 1982). A comparison of the nucleotide
sequences shows that these genes share about 95%
homology in the CH domain exons as well as noncoding
regions. The nucleotide sequence differences indicate
that these genes diverged from one another approximately
6-8 million years ago. An examination of hinge exons
shows that these coding regions have evolved more rapidly
than any other areas of the CY genes in terms of both
base substitution and deletion/insertion events. Coding
sequence diversity also is observed in areas of CH domains
that border the hinge. Our interpretation is that natural
selection favors the generation of diversity in the hinge
area of the proteins. Such diversity may be responsible
for the variations in effector functions observed for
different IgG subclasses.
Analysis of lambda clones has shown that the C Y2 and
cy4 genes are linked in human DNA, and are separated by
a distance of 17 kilobases. We hope to establish the
linkage relationships not only of the remaining CY genes,
but of all the genes in the human CH cluster. Toward that
end, we have constructed a cosmid library of human sperm
DNA, employing the strategy used in this lab to construct
a mouse cosmid library.
Several clone blot experiments, as well as restriction
maps of genomic clones, indicate that the homology
shared by the Cy genes extends over a large region of
DNA flanking the coding sequences. Electron microscopy
of heteroduplexes formed between different clones is
being done to examine the flanking sequence homology in
more detail.
In view of the recent divergence of the human Cy
genes, we thought it would be interesting to examine the
Cy gene families in a variety of primate species that
diverged from humans in the range of 5-25 million years
ago. Preliminary genomic blot experiments indicate that
multiple CY genes exist in all of these primates. We plan
to analyze these gene families in more detail.
References: Ellison, J., Berson, B. and Hood, L. (1982) Nucleic Acids
Res., in press. Ellison, J., Buxbaam, J. and Hood, L. (1981) DNA 1, 11-18. Ellison, J. and Hood, L. (1982) Proc. Nat. Acad. Sci. USA
79, 1984-1988.
*Department of Biological Chemistry, School of Medicine, University of California, Los Angeles. **Department of Biochemistry, University of California, Berkeley.
46. MOLECULAR ANALYSIS OF HUMAN B-CELL DIFFBRl!NTIATION USING B-CELL LEUKEMIAS AND IMMUNODEFICIENCil!S
lnvestiga tors: Martha C. Zuniga, Jay W. Ellison
We are studying human B-cell differentiation by
examining the programmed rearrangement of immuno
globulin genes in B-cell leukemias and B cells from
patients with well characterized immunodeficiencies. In
particular we are interested in examining the phenomenon
of heavy chain isotype switching with regard to the state
in B-cell development at which it occurs and the possible
role for abnormal switching in immunodeficiencies. These
studies are in collaboration with Dr. M. D. Cooper of the
Cellular Immunology Unit at the University of Alabama in
Birmingham, who is providing us with well characterized
human leukemia cells and lymphocytes from immuno
deficient patients. Using the Southern blotting technique
and purified, cloned human immunoglobulin gene probes
isolated in this laboratory, we are currently screening
DNAs from leukemic individuals for evidence of heavy
chain switching as detected by rearrangement of DNA at
heavy chain loci. The use of Southern blotting with
immunoglobulin gene probes in characterizing leukemias
and other immune disorders is also being pursued in
collaborative studies with Ors. Robert Cardiff and Fred
Meyers at the University of California at Davis. The goal
of these studies is to diagnose particular immune diseases
by the immunoglobulin gene organization of the affected
cells and to use this information in the treatment of
lymphoproliferative disorders.
47. REARRANGEMENT OF CLONED IMMUNOGLOBULIN GENES INTRODUCED INTO B CELL LINES
Investigators: Stuart K. Kim, Barbara J. Wold
Heavy chain switching, an antigen-driven differentia
tion event, is characterized by a DNA rearrangement in
which switch regions are recombined and the DNA
originating between them is deleted. Each CH gene has
unique repetitive switch (S) sequences to their upstream
sides that mediate class switching. We have used this
change in DNA topology to construct switching vectors
that, when introduced into B cells, can be used to detect
immunoglobulin CH gene rearrangement.
Specifically, a selectable marker, eco-gpt, has been
inserted between the S µ and S y 2b regions and cloned into
pBR322. The vector can be introduced into a B
lymphocyte, SP2, by DNA-mediated gene cotransfer using
G418 resistance conferred by a second selectable
marker-pNeo3. These cells will therefore be Nm+ eco-+ gpt • Hybridomas will then be made by fusing the SP2
parent to in vitro LPS-stimulated splenocytes that are
known to be undergoing CH switching. If CH switching
operates on the switching vector, the eco-gpt gene should + -be lost and should result in a Nm gpt phenotype. These
cells can then be selected and studied in three ways.
First, the mechanism of CH switching can be studied by
mapping· switch sites and by deletional analysis. Second,
factors which influence CH switching, such as antigen or
TH cells, can be identified. Third, the CH switching
mechanism may become amenable to somatic cell
genetics.
48. THE MOLECULAR NATURE OF THE GENE(S) ENCODING THE T-CELL RECEPTOR
Investigators: Mitchell Kronenberg, Ellen B. Kraig
Although T lymphocytes respond specifically to
antigenic stimulation, the nature of their recognition
structure has eluded characterization. The T-cell antigen
receptor clearly differs from the immunoglobulin
molecule that serves as the B-cell antigen receptor.
Cloned murine T-cell lines fail to rearrange or transcribe
a number of immunoglobulin gene segments (JL' CL' JH'
CH) in a productive manner. However, on the basis of
considerable evidence from serological studies, it has been
proposed that immunoglobulin heavy chain variable (V H)
gene segments are synthesized by T cells.
currently testing this hypothesis in two ways.
We are
First, it is possible that B and T cells responding to the
same antigen will employ homologous variable regions.
The immune response to the synthetic polypeptide OAT
(glutamic acid, alanine, tyrosine) is well characterized and
cloned B and T cells specific for GAT are available. We
have constructed a cDNA library from the poly(AJ' RNA
obtained from a OAT-specific B-cell hybrid. A clone
43
encoding part of the variable region of the OAT binding
protein has been isolated from this library and sequenced.
This clone will be used as a hybridization probe to analyze
RNA from the OAT-specific T cells.
Second, we have developE;!d methods for detecting a
wide spectrum of variable gene segments. We can
therefore test for synthesis of V H segments in T cells
without making any assumptions concerning the way B and
T cells responding to the same antigen employ these
segments. One method consists of low stringency
hybridization of cloned variable gene segments to colonies
containing cloned eukaryotic DNA. We have determined
that sequences only 60% homologous to our probe can be
detected. The second method (see Biology 1981, No. 69)
depends upon the use of a combination of two probes, a
synthetic oligonucleotide and a specifically primed cDNA.
Neither probe is completely specific for variable regions,
but a clone hybridizing to both should contain a variable
gene. Using a random primer, we have constructed
relatively large (>105 colonies) cDNA libraries from the
RNA of three different cloned T cells. These libraries are
currently being screened using the methods outlined
above.
49. MOLECULAR DISSECTION OF THE MOUSE MAJOR HISTOCOMPATIBILITY COMPLEX
Investigators: Michael Steinmetz, Astar Winoto, Janet M. McNicholas, Karyl Minard
The major histocompatibility complex (MHC) is a large
genetic region encoding various proteins involved in the
cellular interactions of the immune response. Class I
genes encode the classical transplantation antigens (K, D,
L, R) and two types of differentiation antigens (Qa, TL).
Transplantation antigens restrict the interactions between
cytotoxic T cells and virally infected target cells. Class II
genes code for the immune response associated antigens
(la) that regulate the interactions between helper T cells
and macrophages and B cells. A third class of proteins
also encoded by the MHC (class III genes) are certain
complement components that are not involved in cellular
interactions. The MHC of the mouse is located on
chromosome 17 and can be divided into the H-2 region
(about 0.3 cM , 600 kb) that encodes the transplantation
antigens, class II and class III molecules and into the Tla
region (about 1 cM , 2000 kb) that codes for the class I
differentiation antigens (Figure 1). Our aim is to clone
completely the H-2 region of the MHC in order to study
44
gene organization, evolution and function of this immuno
logically important gene complex. Since it is possible to
clone large eukaryotic DNA fragments (about 40 kb)
within cosmid vectors, we hope to achieve our goal in a
reasonable time.
Regions and ~
Subregions K A J E s ID, L, Rl (Qa-2,Qa-3) (Qo-1, Tlol
Genes
Class Il m I I I I I I I
Gene H-2 Complex
----~"----Tio---~
Distance 0.3cM ----'~----1.0cM --~
Figure 1. The linkage relationship of the H-2 and Tla loci on chromosome 17 of the mouse.
cDNA clones for class I genes (Steinmetz et al., 1981a)
were used for the isolation of class I genes from BALB/c
sperm DNA libraries constructed in phage A (Steinmetz
et al., 1981b) and in a cosmid cloning vector (Steinmetz
et al., 1982). From the cosmid library, 13 distinct clusters
ranging in size from 35 to 191 kb of DNA (a total of
837 kb of DNA) were isolated containing 36 class I genes.
To date, most of these class I gene clusters could be
assigned to genetically-defined regions of the MHC, using
either a transfection assay to screen for functional class I
genes that can be detected with defined monoclonal
antibodies (R. S. Goodenow et al., manuscript in
preparation) or Southern blot analysis of restriction
enzyme polymorphism in inbred and recombinant mouse
strains (Steinmetz et al., 1982; A. Winoto et al.,
unpublished). In this way, clusters for K, D, L, Qa and Tla
genes could be identified. We plan to complete our
mapping analysis for all 13 gene clusters and to use
chromosomal walking experiments in an attempt to link
some of the individual clusters.
In collaboration with C. Wake, E. Long and B. Mach
(University of Geneva), we have recently used a human
class II cDNA clone (encoding the DR ex chain) to isolate
the homologous mouse class II gene from our cosmid
library. Determination of the DNA sequence of this gene
has shown that it encodes the a chain of the 1-E molecule.
The four overlapping cosmid clones isolated define a
region of
middle.
about 60 kb of DNA with the E" gene in the
Southern blot analyses of recombinant mouse
strains with cloned restriction fragments confirm that the
gene maps into the E subregion of the I region whereas a
DNA segment 30 kb downstream of the E" gene maps into
the adjacent A subregion. For the first time, therefore,
two genetically defined subregions of the MHC have been
linked by molecular cloning (M. Steinmetz et al., manu
script in preparation).
We are presently doing chromosomal walking
experiments using the cloned region around the Ea. gene in
the middle of the H-2 region as a starting point. We have
so far walked about 30 kb of DNA both toward the K end
and the D end of the H-2 region. These clones will allow
us to study the J subregion that is believed to encode a
certain subunit of the elusive T-cell receptor.
References: Steinmetz, M., Frelinger, J. G., Fisher, D., Hunkapiller,
T., Pereira, D., Weissman, S. M., Uehara, H., Nathenson, S. and Hood, L. (1981a) Cell 24, 125-134.
Steinmetz, M., Moore, K. W., Frelinger, J. G., Sher, B. T., Shen, F. W., Boyse, E. A. and Hood, L. (198lb) Cell 25, 683-692.
Steinmetz, M., Winoto, A., Minard, K. and Hood, L. (1982) Cell 28, 489-498.
50. ASSIGNMENT OF COSMID CWNES ENCODING MOUSE TRANSPLANTATION ANTIGENS TO DEFINED REGIONS OF THE MOUSE MAJOR HISTOCOMPATIBILITY COMPLEX
Investigators: Astar Winoto, Michael Steinmetz
Sixty-four cosmid clones containing class I genes were
isolated from a BALB/c sperm DNA library with the
class I cDNA clones as probes (Steinmetz et al., 1982).
The cosmid clones were mapped with 10 different
restriction enzymes and, by restriction enzyme analyses,
were grouped into 13 clusters containing 36 class I genes
altogether. Cluster 1 could be assigned to the Qa2,3
region because it contained gene 27 .1 previously mapped
to this region (Steinmetz et al., 1981). This was
confirmed by Southern blot analysis with a low copy probe
isolated from a different part of the cluster. Cluster 2
contains the L gene, because it overlaps the insert in
lambda clone 27 .5 previously shown to encode the L d
antigen (Moore et al., 1982). To map the other clusters,
single or low copy fragments were isolated from the
cloned DNA. The fragments were ·identified by their lack
of hybridization to total mouse DNA used as a probe.
They were then subcloned into pBR322 or M13 mp8. When
no low copy fragment could be identified, a fragment
from the end of the cluster was isolated, cleaved into
smaller pieces with restriction enzymes and cloned into
Ml3. Clones containing low copy number DNA fragments
were then identified by positive hybridization to the end
fragment and negative hybridization to the total mouse
DNA.
With the above procedure, 14 low copy probes from
different clusters were obtained. These probes were used
to identify restriction enzyme site polymorphism between
inbred mice of differing haplotypes. These polymorphisms
were then mapped by Southern blot analyses of DNA from
congenic mouse strains exhibiting recombination in the
MHC. Clusters 3, 8 and 12 map to the Tia region and
cluster 6 maps to the Qa2,3 region. Cluster 9 maps to the
D, L, R or Qa2,3 region and cluster 7 has tentatively been
located to a region proximal to K. Cluster 10 has not yet
been mapped. Clusters 4 and 5 have been identified by
Doug Fisher to contain genes encoding TL antigens
because their restriction maps overlap with lambda clones
expressing these antigens. Transfection of cosmid clones
into mouse L cells has furthermore shown that the class I
genes in cluster 11 and 13 encode Kd and Dd transplan
tation antigens, respectively (R. Goodenow, personal
communication).
For clusters 2 and 13, hybridization probes were
isolated from the ends of the clusters and these probes
will be used for chromosomal walking experiments to link
the class I genes of the D end of the major histo
compatibility complex.
References: Moore, K. W., Sher, B. T., Sun, Y. H., Eakle, K. and Hood,
L. (1982) Science 215, 679-682. Steinmetz, M., Moore, K. w., Frelinger, J. A., Sher, B. T.,
Shen, F. W., Boyse, E. A. and Hood, L. (1981) Cell 25, 683-692.
Steinmetz, M., Winoto, A., Minard, K. and Hood, L. (1982) Cell 28, 489-498.
51. STUDIES OF GENES ENCODING H-2 MOLECULES OF THE np• HAPLOTYPE
Investigators: Mary J. Macchi, Johanna A. Griffin, Jeffrey A. Fre!inger*, Leroy E. Hood
The existence of inbred, congenic and recombinant
mouse strains has been an invaluable aid in the
examination of the proteins encoded by the major histo
compatibility complex. Thei;;e proteins are involved in
cell-cell recognition and interaction. Examination of the
transplantation antigens encoded within the complex has
revealed a wide array of polymorphisms, denoted by the
mouse haplotype. The H-2p haplotype appears to be
largely unrelated to the other inbred haplotypes, thus
maximizing polymorphic differences detected by allo
immunization. Hence, we are presently screening a
genomic DNA library made from the sperm of a BlO.P
mouse (H-2P) using a cloned cDNA sequence encoding a
45
mouse transplantation antigen (Steinmetz et al., 1981).
Appropriate clones will be characterized by restriction
enzyme mapping and DNA sequencing. DNA-mediated
gene transfer will be used to introduce the clones into
mouse fibroblast Ltk - cells (Goodenow et al., 1982).
We presently have a panel of monoclonal antibodies
and cytotoxic T-!ymphocyte clones (CTL) directed against
specificities of the H-2p haplotype. Using this battery of
test reagents, we can examine the expression of H-2
molecules on the surface of the transformed cells. We
will be able to determine with certainty the molecules
recognized by CTLs and monoclonal antibodies as well as
the number of antigenic specificities associated with each
molecule. By altering DNA in the recombinant clones
prior to gene transfer, we will be able to assign functions
to particular regions of the molecules.
References: Goodenow, R. s., McMillan, M., Orn, A., Nicolson, M.,
Davidson, N., Frelinger, J. A. and Hood, L. (1982) Science 215, 677-679.
Steinmetz, M., Frelinger, J. G., Fisher, D., Hunkapiller, T., Pereira, D., Weissman, S. M., Uehara, H., Nathenson, S. and Hood, L. (1981) Cell 24, 125-134.
*University of Southern California Medical Center.
52. STRUCTURE OF GENES ENCODING MOUSE TRANSPLANTATION ANTIGENS
Investigators: Kevin W. Moore*, Beverly Taylor Sher, Yi Henry Sun, Kurt A. Eakle, Robert S. Goodenow
The transplantation antigens, which are encoded by
genes in the major histocompatibility complex, play an
important role in the regulation of the immune response.
The inbred BALB/c mouse possesses at least four different
transplantation antigen molecules, Dd, L d, Rd, and Kd.
Dd and L d are closely linked and map in the D region of
the H-2 complex; Rd maps between Dd and the Tia region;
and Kd maps in the K region of the H-2 complex, about
0.3 cM from Dd.
Genomic clones encoding transplantation antigens and
other class I molecules have been isolated from an
amplified library of BALB/c sperm DNA cloned in the A
vector Charon 4A. The nucleotide sequence of the gene
found in A clone 27 .5 was determined by the dideoxy
sequencing technique (Moore et al., 1982). It corresponds
to the known partial amino acid sequence of the L d
molecule. The identification of gene 27 .5 as encoding L d
has been confirmed by gene transfer (Goodenow et al.,
1982).
46
Additional A genomic clones and clones isolated from a
cosmid library constructed from BALB/c sperm DNA
(Steinmetz et al., 1982) have been shown by gene transfer
to contain genes encoding the rest of the BALB/c
transplantation antigens {R. S. Goodenow et al., manu
script in preparation). In addition, genes encoding other
class I molecules that have not been previously defined
serologically have been identified (R. S. Goodenow et al.,
manuscript in preparation). We intend to determine the
nucleotide sequences of the genes on these clones. The
sequence data will help to define the different functional
domains and evolutionary history of the different class I
molecules. They will also facilitate the in vitro muta
genesis studies being carried out in our laboratory. In
addition, they will allow us to prepare synthetic poly
peptides corresponding to parts of each class I molecule's
sequence. Using these polypeptides, we can generate
specific antisera that can be used to study the expression
and functions of the different class I molecules.
References: Goodenow, R. s., McMillan, M., Orn, A., Nicolson, M.,
Davidson, N., Frelinger, J. A. and Hood, L. (1982) Science 215, 677-679.
Moore, K. W., Sher, B. T., Sun, Y. H., Eakle, K. A. and Hood, L. (1982) Science 215, 679-682.
Steinmetz, M., Winoto, A., Minard, K. and Hood, L. (1982) Cell 28, 489-498.
*DNAX, Palo Alto, California.
53. DNA SEQUENCE ANALYSIS OF THE Tia GENES OF THE BALB/c MOUSE
Investigators: Douglas A. Fisher, Marit P. Pecht
Thymus leukemia (TL) antigens are cell-surface glyco
proteins that are normally expressed only on thymocytes
of TL+ mice (Vitetta and Capra, 1978). Seven antigenic
specificities have been defined. Leukemias of TL+ mice
may express the thymocyte TL specificities, or may
express new TL antigens. TL antigens are also found on
leukemias of thymic origin in TL - mice.
Histocompatibility antigens (H-2), the T-cell
differentiation antigens Qal and Qa2, and TL antigens are
all structurally similar in that they consist of two 40,000
molecular weight polypeptide chains noncovalently
associated with two molecules of the same 12,000 MW
subunit, a2-microglobulin. The genes encoding these
antigens are also all closely linked on mouse chromosome
17.
Recently, two genomic clones isolated from a lambda
phage genomic library by hybridization with cloned H-2
cDNAs were shown to produce TL antigens when trans
formed into mouse L cells (R. S. Goodenow, unpublished
results). We have begun DNA sequence analyses of these
clones with the aim of defining the gene structure of TL
antigens and clarifying the relationship between H-2 and
Tla genes. The Tla system may prove to be an excellent
system in which to study gene expression, since the genes
are expressed normally in limited cell types as well as
aberrantly in leukemia cells. In addition, TL expression
may be regulated in cell culture in response to thymic
hormones (Goldstein et al., 1980; Umiel et al., 1982).
With TL-specific probes isolated from Tla genes, unlike
the crossreacting H-2 probes, it should be possible to
analyze TL genes and their RNAs in normal and leukemic
cells, as well as in different haplotypes of mice.
References: Goldstein, G., Kung, P. C., Post, P. W. and Lau, c. Y.
(1980) Thymus, Thymic Hormones, and T-Lymphocytes. Proc. Serono Syrnp., F. Aiuti and H. Wigzell (Eds.), Vol. 38, pp. 195-199.
Umiel, T., Shilder, D., Pecht, M. and Trainin, N. (1982) Submitted for publication.
Vitetta, E. S. and Capra, J. D. (1978) Adv. in Immunol. 26, 147-173.
54. IDENTIFICATION OF THE CODING FUNCTIONS OF THE CLONED GENES OF THE H-2 AND Tia REGIONS OF THE MOUSE BY DNA-MEDIATED GENE TRANSFER
Investigators: Robert S. Goodenow, Minnie McMillan, Margery A. Nicolson*, Iwona Stroynowski, Norman Davidson*
DNA-mediated gene transfer has been used to
determine the coding functions of the cloned BALB/c
class I genes encoding the major histocornpatibility or H-2
antigens and the related products of the Tla complex.
Coding assignments for the cloned class I genes, obtained
from a lambda (Steinmetz et al., 1981) and cosmid
(Steinmetz et al., 1982) library, were made on the basis of
the identification of the products of the transferred genes
in mouse L cells. For example, the H-2 L d gene was
identified by demonstrating that thymidine kinase
negative (tk-) L cells transformed with A27.5 DNA and the
Herpes viral tk gene reacted with monoclonal antibodies
specific for the L d transplantation antigen (Goodenow
et al., 1982a). The immunoprecipitated products were
shown to be virtually indistinguishable from the L d
molecules on BALB/c spleen cells by two-dimensional gel
electrophoresis.
The cloned genes encoding other serologically defined
class I antigens such as Kd and Dd have also been
identified (Goodenow et al., 1982b). In addition, a number
of novel genes have apparently been detected based on the
association of their products with the 12,000 dalton
subunit, s2-microglobulin, which is noncovalently
associated with class I molecules.
The cloned genes of the Tla complex have also been
identified by gene transfer. The products of these genes
are related to the histocompatibility antigens although
these genes are only expressed on certain subpopulations
of lymphoid cells. The Qa-2,3 and several genes encoding
the thymus-leukemia or TL antigen found on thymocytes
have been identified. Moreover, serological analysis of
the products of the transferred TL genes suggests that the
expression of one of these genes may account for the
additional TL molecules found on certain leukemic cells.
Therefore, gene transfer abrogates or circumvents the
regulation of gene expression that occurs in vivo.
Finally, a process resembling homologous
recombination may occur during the transformation of
mouse L cells with subcloned fragments of the class I
genes to yield complete products. This process is
currently being analyzed in order to reveal the molecular
basis for this phenomenon.
References: Goodenow, R. S., McMillan, M., Orn, A., Nicolson, M.,
Davidson, N., Frelinger, J. A. and Hood, L. (1982a) Science 215, 677-679.
Goodenow, R. s., McMillan, M., Nicolson, M., Davidson, N. and Hood, L. (1982b) Nature, submitted for publication.
Steinmetz, M., Moore, K. W., Frelinger, J. G., Sher, B. T., Shen, F .-W ., Boyse, E. A. and Hood, L. (1981) Cell 25, 683-692.
Steinmetz, M., Winoto, A., Minard, K. and Hood, L. (1982) Cell 28, 489-498.
*Division of Chemistry and Chemical Engineering, California Institute of Technology.
55. STUDIES ON THE GENE STRUCTURE, EXPRESSION AND FUNCTION OF MOUSE CLASS I TRANSPLANTATION ANTIGENS
Investigator.: Iwona Stroynowski
Two cloned genes encoding products of the BALB/c
murine major histocompatibility or H-2 complex were
recently sequenced. Clone 27 .1 is believed to be a
pseudogene that maps to Qa-2,3 region (Steinmetz et al.,
1981). Clone 27.5 contains a functional H-2 L d gene,
which is expressed on the cell surface after DNA transfer
47
into mouse fibroblast L cells (Goodenow et al., 1982a). Its
product can be recognized as a restriction element by
cytotoxic T cells in lymphocytic choriomeningitis virus
(LCMV) (Orn et al., 1982).
Hybrid genes have been constructed that contain
sequences from 27.1 and 27.5 DNA. Currently, these 11shuffled11 genes are being introduced into c 3H L tk - cells
by cotransformation with the herpes viral thymidine
kinase ( tk) gene as described previously (Goodenow et al.,
1982a) in order to examine the expression of these hybrid
genes. The transformation products will be studied using
radioimmunoassays, two-dimensional gel protein analysis
and cytotoxic T cell reactions. The objective of this
approach is to determine which L d domains are recognized
by virus-restricted and allogeneic cytotoxic T cells.
Using the same approach for shuffling other class I
genes, e.g., Kd and Dd (Goodenow et al., 1982b) with L d,
we hope to establish whether cytoplasmic and trans
membrane domains of transplantation antigens are
essential in T-cell killing.
In addition, in vitro mutagenesis will be used to modify
5' and 3' gene sequences as well as intron sequences of
27.5 DNA to localize the transcriptional regulatory
elements essential for the characteristic constitutive
expression of this gene in almost all tissues of the mouse.
References: Goodenow, R. S., McMillan, M., Orn, A., Nicolson, M.,
Davidson, N., Frelinger, J. A. and Hood, L. (1982a) Science 215, 677-679.
Goodenow, R. S., McMillan, M., Nicolson, M., Davidson, N. and Hood, L. (1982b) Nature, submitted for publication.
Orn, A., Brayton, P. R., Woodward, J. G., Goodenow, R. S., Harmon, R. C., Frelinger, J. A. and Hood, L. (1982) Nature 297, 415-417.
Steinmetz, M., Moore, K. W., Frelinger, J. G., Sher, B. T., Shen, F.-W., Boyse, E. A. and Hood, L. (1981) Cell 25, 683-692.
56. GENERATION OF RECOMBINANT HISTOCOMPATIBILlTY ANTIGEN GENES AND ANALYSIS OF THEIR EXPRESSION IN MOUSE LCELLS
Investigators: Iworut Stroynowski, Martha C. Zuniga, Anders Orn
A major goal of the studies on H-2 genes in this
laboratory is to elucidate the relationship between the
structures of molecules encoded in the murine major
histocompatibility complex and their functional inter
actions with other molecules. In particular it is important
to identify the domains on H-2 antigens that interact with
48
the T-cell receptor for antigen in allogeneic T cell killing
and in H-2 restricted killing of virally infected cells by
cytotoxic T cells. In an effort to answer these questions
we are generating recombinant genes in vitro using
standard molecular techniques (Shortle et al., 1981) on
genes cloned and sequenced in this laboratory. By
deleting exons and by shuffling exons of different class I
genes (e.g., second exon of H-2Kd with second exon of
H-2Dd), we are constructing recombinant genes of defined
structure. These genes are being cotransformed with the
Herpes simplex virus thymidine kinase gene into tk -
mouse L cells (H-2k). After selection in HAT medium,
transformants are assayed for presence of H-2d gene
products by radioimmunoassay (Goodenow et al., 1982).
Cloned transformants are tested as targets for allo
reactive cytotoxic T lymphocytes (Woodward et al., 1982),
and after viral infection, as targets for virus-specific T
killer cells (Orn et al., 1982). These experiments should
enable us to identify the class I molecule domains
recognized by T cells. Using these methods we are also
trying to determine the role of the transmembrane and
cytoplasmic portions of the class molecule in the
function of the antigen, in particular to determine the
role of the cytoplasmic domains in the normal display of
H-2 antigens on the cell surface. Finally this approach is
being applied to the a2-microglobulin gene in an effort to
determine its sites of interaction with the class I H-2
antigen and its role in H-2 restriction.
References: Goodenow, R. S., McMillan, M., Orn, A., Nicolson, M.,
Davidson, N., Frelinger, J. A. and Hood, L. (1982) Science 215, 677-679.
Orn, A., Brayton, P. R., Woodward, J. G., Goodenow, R. S., Harmon, R. C., Frelinger, J. A. and Hood, L. (1982) Nature 297, 415-417.
Shortle, D., Di Maio, D., Nathans, D. (1981) Ann. Rev. Genet. 15, 265-294.
Woodward, J. G., Orn, A., Harmon, R. C., Goodenow, R. s., Hood, L. and Frelinger, J. A. (1982) Proc. Nat. Acad. Sci. USA, in press.
57. EXPRESSION OP HISTOCOMPATIBILITY GENES IN MOUSE TERATOCARCINOMA CELLS
Investigators: Martha C. Zuniga, Anders Orn
A teratocarcinoma is a tumor which occurs as the
result of the transplantation of a normal mouse embryo
(one- to seven-day-old) to an extrauterine site in a histo
compatible host of either sex (Stevens, 1970). Stem cell
lines have been derived from such tumors that share
biochemical and morphological properties with pluripotent
embryonic cells, and that, in some cases, are able to
differentiate to various cell types in vitro. Moreover,
embryonal carcinoma cells (ECC) derived from
teratocarcinomas, when injected into normal mouse
blastocysts, can contribute to the resulting tissues of the
fully developed normal mouse derived from such a mosaic
embryo (Mintz and Illmensee, 1975). Hence the
teratocarcinoma is believed to be a good in vitro model
system for studying normal mammalian embryonic
development.
Teratocarcinomas and ECCs, like their normal
embryonic counterparts in cleavage stage embryos, do not
express histocompatibility antigens (Solter and Knowles,
1979). We are currently studying the expression of
histocompatibility genes in teratocarcinomas using cloned
genes isolated in this laboratory from BALB/c (H-2d)
libraries. The approach we are using is to cotransform tk -
teratocarcinoma F9 (H-2b) cells (Gmilr et al., 1980) with
the Herpes simplex virus thymidine kinase gene and the
H-2d gene of interest, to select transformants in HAT
medium, and to detect expression with anti-H-2 antigen
antibodies by radioimmunoassay. Using this approach we
hope to answer several questions. (1) Will the
introduction of foreign DNA overcome normal cellular
controls and permit the constitutive expression of H-2
genes in undifferentiated teratocarcinoma cells?
(2) Does expression of a H-2 class I gene require the
concomitant introduction of a cloned a 2-microglobulin
gene or is the endogenous 13 2-microglobulin gene
expressed under these conditions? (3) If induction of
differentiation is required for expression of these genes,
then are H-2 genes of the b and d haplotypes coordinately
expressed, or are they independently regulated?
Ultimately, we also want to study H-2 gene expression in
chimeric mice derived from normal mouse blastocysts
injected with pluripotent ECCs (Martin, 1981) or terato
carcinoma cells transformed with defined H-2 genes.
References: Gmiir, R., Solter, D. and Knowles, B. B. (1980) J. Exp.
Med. 151, 1349-1359. Martin, G. R. (1981) Proc. Nat. Acad. Sci. USA 78, 7634-
7638. Mintz, B. and Illmensee, K. (1975) Proc. Nat. Acad. Sci.
USA 72, 3585-3589. Solter, D. and Knowles, B. B. (1979) Curr. Top. Dev. Biol.
13, 1395. Stevens, L. c. (1970) Develop. Biol. 21, 364-382.
58. ffiOCHEMICAL ANALYSES OF MOUSE L CELLS TRANSFORMED WITH CLASS I GENES OF THE MOUSE MAJOR HISTOCOMPATIBILITY COMPLEX
Investigator: Minnie McMillan
Genes encoding the H-2L d and H-2Kd class I transplan
tation antigens have been identified using indirect
immunoprecipitation and two-dimensional gel electro
phoresis.
The lysates from biosynthetic radiolabeled
transformed mouse L cells were incubated with mono
clonal antibodies specific for L d (or Kd) molecules. The
antibody-antigen complexes were first isolated using
Staphylococcus aureus and then analyzed on two
dimensional gels. The resulting electrophoretic patterns
were shown to be virtually identical to those obtained for
L d (or Kd) molecules isolated from normal splenic
lymphocytes.
In order to characterize new class I gene products for
which specific monoclonal antibody reagents do not as yet
exist, antisera directed against either a2-microglobulin or
against the transformed cells themselves were used for
the immunoprecipitations. Anti-S2-microglobulin serum
proved to be an inefficient reagent for precipitating most
class I gene products, presumably because the noncovalent
association of a2-microglobulin, the invariant chain of all
known class I gene products, with the polymorphic heavy
chain of the class I molecule, is relatively weak.
Preliminary two-dimensional gel analysis of the
immunoprecipitate from the transformant 8-1 using an
alloantiserum raised against 8-1 showed a pattern of
closely-related spots characteristic of a transplantation
antigen, as well as several other unrelated spots not
present in "control" immunoprecipitates.
59. CHEMICAL CHARACTERIZATION OF la ANTIGENS OF THE MOUSE MAJOR HISTOCOMPATIBILITY COMPLEX
Investigator: Minnie McMillan
The Ia antigens are encoded in the I region of the
major histocompatibility complex {H-2) of the mouse.
The I region controls a series of phenotypic traits which
are intimately involved in the immun~ response. By
recombinational analysis the I region has been divided into
five subregions I-A, I-B, I-J, 1-E and I-C.
The only gene products from the I region that have
been directly identified are the Ia antigens. These
molecules are expressed predominantly on B .lymphocytes
49
and are integral membrane proteins that appear to be
highly polymorphic by serological analysis. They are
composed of at least two subunits of approximate
molecular weights 35,000 (<:<) and 28,000 <el, which
coimmunoprecipitate with a third invariant polypeptide,
>!, of approximate molecular weight 30,000. Distinct la
molecules are encoded by the I-A and 1-E subregions.
I have demonstrated that this invariant polypeptide is
not a precursor of the Aa' Aa, Ea or Ea polypeptides and
that its interaction with Ia polypeptides varies with
haplotype. I have also shown that the polypeptides we
have previously characterized are contaminated with very
little, if any, invariant protein. Further, I have used a
high-pressure liquid chromatography tryptic peptide map
technique to formally map the genes encoding Aa.' Aa and
Ee to the I-A subregion using recombinant and F 1 hybrid
mice (McMillan et al., 1981).
These experiments are carried out in collaboration
with Dr. H. O. McDevitt (Stanford Medical School,
Stanford, California), Dr. D. B. Murphy (Yale Medical
School, New Haven, Connecticut) and Dr. J. A. Frelinger
(USC Medical School, Los Angeles, California).
Reference: McMillan, M., Frelinger, J. A., Jones, P. P., Murphy, D.
B., McDevitt, H. 0. and Hood, L. (1981) J. Exp. Med. 153, 936-950.
60. DEVELOPMENT OF PROTEIN MICROSEQUENCING METHODOLOGY
Investigators: Michael W. Hunkapiller, Paul K. cartier m We have continued the development of protein and
peptide microsequencing techniques (see Biology 1981,
No. 79). This work has included refinement of the gas
phase Edman sequenator developed in collaboration with
Professor William Dreyer. Improvements in reagent
methods and changes in both reagents and operating
programs have improved the yields of several amino acid
derivatives that are produced by the Edman degradation
and thereby increased the sensitivity of the sequencing
experiments. This has provided greater reliability in the
assignment of several amino acids in the low picomole
range.
We have also begun development of an automated C
terminal protein sequencing method to complement the
Edman N-terminal method. No generally useful C
terminal sequencing method is currently available
although a variety of chemical approaches has been tried.
50
They have been limited by an inability to selectively
remove excess reagents and cleaved amino acids from the
protein while keeping the remainder of the protein in the
reaction vessel. We hope to overcome this problem by
adapting the gas-phase Edman sequenator to allow for
delivery of gas-phase reagents to effect the thiocyanate
c-terminal cleavage that results in removal of the C
terminal amino acid as the thiohydantoin.
61. STRUCTURAL ANALYSIS OF THE ACETYLCHOLINE RECEPTOR
Investigator: Miehe.el W. Hunkapiller
The acetylcholine receptor (AcChR) is a prototype for
many membrane-bound proteins that function in the
nervous system by transiently altering the electrical
properties of the cell membrane in response to chemical
transmitters such as acetylcholine or to drugs. Molecular
characterization of AcChR can serve as a model for
interactions of small molecules with membrane-bound
proteins and also for transport of ions across a biological
membrane in response to these interactions.
By employing microsequencing techniques developed in
our laboratory, we have determined the amino acid
sequences for extended stretches from the amino termini
of the subunits of AcChR from the electric ray Torpedo
californica, the electric eel Electrophorus electricus, and
fetal calf. These studies reveal that AcChR from these
diverse species share a common subunit structure
(consisting of two identical and three structurally related
polypeptides) and that all of the AcChR subunits have
arisen from a common ancestral gene early in the
development of neuromuscular systems.
The shared ancestry of the different subunits, revealed
by the amino acid sequence homology, suggests that each
of the four subunits plays a functional role in the
receptor's physiological action. We are using quantitative
amino acid sequence analysis to examine the
stoichiometry of binding of several snake venom toxins to
Torpedo AcChR. This can reveal whether binding sites of
these antagonists exist only on the smaller two subunits or
whether, at least for some toxins, there are functional
binding sites on some or all of the others.
These studies are being carried out in collaboration
with Professor Michael Raftery and Dr. Bianca Conti
Tronconi of the Division of Chemistry and Chemical
Engineering at the California Institute of Technology.
62. GENERALIZED RECOMBINATION IN B. COLI
Investigators: Henry V. Huang, Lisa L. Flitz*
We measured the dependence of the efficiency of
generalized recombination on the homology of parental
sequences in E. coli. The parental sequences used are a
series of BALB/c mouse V H gene segments that are
67-100% homologous to each other. One v 8 gene
segment (405 bp) is cloned in a plasmid containing a supF
gene, and is allowed to recombine in vivo with the
identical V H gene segment, or any of a number of other
V H gene segments, residing in amber-marked phages.
We find a dramatic decrease in the frequency of
recombinants when the parental sequences are non
identical. The frequency of recombinants is 3 x 10-4
when parental sequences are 9096 homologous, compared
to 1.5 x 10-2 for 100% homology. At 78% homology, the -6 frequency of recombination drops to 7 x 10 . We
interpret the strong effects of parental homology on
recombination as arising from the requirement of the
recombination enzymatic machinery for short sequence
identities or minimal efficient processing segments
(MEPS). The size of the MEPS is estimated from these
results, using the known sequences of the V H gene
segments, to be 40-60 bp.
A more direct measurement of the MEPS is to use a
series of 10096 homologous sequences of various lengths.
We find that a 405 bp long sequence gives 10-3 frequency
of recombinants, while a 214 bp long sequence gives 10-5
-9 and a 21 bp long sequence gives 10 frequency of
recombinants. The 44 bp frequency is consistent with the
405 bp frequency if we assume that the MEPS is 42 bp
long: the 405 bp sequence contains 354 MEPS, while the
44 bp long sequence contains only two MEPS, thus giving
about 102 fold difference in frequency of reoombinants.
It is not possible to rationalize the 21 bp frequency in a
similar manner, and we interpret the low frequency as
being due to the decreased efficiency of the enzymatic
machinery when forced to process sequences that are
shorter than optimal.
Our approach also allows us to recover and
characterize two of the four recombinant DNA strands in
the recombination intermediate. The sequence difference
between the parental sequences serves as a fine-scale
genetic marker in this approach. The extent of the
heteroduplex region in the intermediates can be estimated
from sequence analyses of two recovered strands from
recombination between non-identical parental sequences.
The results show that heteroduplex regions can be as short
as 28 bp, and maximally 110 bp.
In summary, generalized recombination seems to
operate efficiently on short, perhaps 20-60 bp, sequence
identities, and recombination between parental sequences
that are 88% homologous result in heteroduplex regions
only 28-110 bp long.
We are now testing which E. coli recombination
pathway is responsible for the observed results, and if
different recombination pathways have different require
ments for parental sequence length and homology.
*Undergraduate, California Institute of Technology.
63. MINIPLASMIDS, MICROPLASMIDS, REPLICON MODULES
Investigator: Henry V. Huang
have constructed a 1895 bp plasmid, pMT21
(Morpheus Thanatos) that consists of a Col El replicon, an
ampicillin-resistance gene, and a 47 bp polylinker that
contains the following unique restriction sites: Sac II,
Eco RI, Sma I, Barn HI, Sal I, Pst I, Bgl II, Xba I and
Hind III. The many tandem restriction sites make this
plasmid convenient for directional cloning. Its small size
results in a minimal number of vehicle fragments that
might interfere with analyses of cloned sequences.
I have also used the same polylinker, minus the Sac II
site, in a microplasmid. The microplasmid, n AN7
(Arcadian Nomius) is 885 bp in size, has a Col El replicon
with high copy number, and is amplifiable. The selective
marker is the supF gene. This plasmid is useful for the
supF-amber phage recombination method for library
screening devised by Brian Seed (in press).
During the construction of nAN7, I have isolated a 649
fragment containing the entire Col El replicon flanked by
Hind ill and Cla I restriction sites. This small replicating
module should be useful in a number of applications.
64. GENES FOR REPRODUCTIVE HORMONES OP APLYSIA
Investigators: Arthur Roach, Felix Strumwasser, Leroy E. Hood
Most animals employ peptide hormones to regulate
some aspects of their reproduction. One can isolate
several neuroactive peptides from tissues of the sea hare,
Aplysia, which in the pure form can induce or alter the
animal's reproductive behavior. Several of these peptides
have been sequenced in our and other laboratories (Chiu
51
et al., 1979; Heller et al., 1980; Schlesinger et al., 1981).
A family of genes coding for some of these peptides has
recently been cloned from a genomic Aplysia library
(Scheller et al., 1982). Initial results suggest that several
active peptides may be processed from a single large
precursor protein.
We have succeeded in constructing a cDNA library
from messenger RNA molecules isolated from the atrial
gland of Aplysia, the site of abundant production of
several of these peptides. Initial screening has allowed us
to select several clones for further study--primarily,
sequence determination. Through a combination of
sequence data and processing studies, we hope to under
stand the pathways of synthesis of some of these peptides,
as well as learn about other possible members of this
peptide family.
References: Chiu, A. Y., Hunkapiller, M. W., Heller, E., Stuart, D. K.,
Hood, L. E. and Strumwasser, F. (1979) Proc. Nat. Acad. Sci. USA 76, 6656-6660.
Heller, E., Kaczmarek, L. K., Hunkapiller, M. w., Hood, L. E. and Strumwasser, F. (1980) Proc. Nat. Acad. Sci. USA 77, 2328-2332.
Scheller, R. H., Jackson, J. F., McAllister, L. B., Schwartz, J. H., Kandel, E. R. and Axel, R. (1982) Cell, in press.
Schlesinger, D. H., Babirak, S. P. and Blankenship, J. E. (1981) In: Symposium on Neurohypophyseal Peptide Hormones and Other Biologically Active Peptides. D. H. Schlesinger (Ed.), Elsevier/North Holland Publishing Co.
PUBLICATIONS
Alexander, A., Steinmetz, M., Barritault, D., Frangione, B., Franklin, E. c., Hood, L. and Buxbaum, J. (1982) Gamma heavy chain disease in man: cDNA sequence supports partial gene deletion model. Proc. Nat. Acad. Sci. USA, in press.
Chapman, B. s., Hood, L. E. and Tobin, A. J. (1982) Amino acid sequences of the e: and cxE globins of Hbe, a minor early embryonic hemoglobin of the chicken. J. Biol. Chem. 257, 643-650.
Chapman, B. S., Hood, L. E. and Tobin, A. J. (1982) Minor early embryonic chick hemoglobin M amino acid sequences of the e: and cxD chains. J. Biol. Chem. 25'1, 651-658.
Clevinger, B., Thomas, J., Davie, J., Schilling, J., Bond, M., Hood, L. and Kearney, J. (1981) Anti-dextran antibodies: Sequences and idiotypes. In: Immunoglobulin Idiotypes. C. Janeway, E. E. Sercarz, H. Wigzell and C. Fred Fox (Eds.). ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. XX, pp. 159-168. Academic Press, New York.
Ditto, M. D., Chou, J., Hunkapiller, M. W., Fennewald, M. A., Gerrard, S. P ., Cozzarelli, N. R., Hood, L. E., Cohen, S. N. and Casadaban, M. J. (1982) The amino terminal sequence of the Tn3 transposase protein. J. Bacterial., submitted for publication.
52
Early, P. and Hood, L. (1981) Allelic exclusion and nonproductive immunoglobulin gene rearrangements. Cell (Minireview) 24, 1-3.
Early, P., Nottenburg, c., Weissman, I. and Hood, L. (1982) Immunoglobulin gene rearrangements in normal mouse B cells. Mol. Cell. Biol., in press.
Ellison, J. w., Berson, B. J. and Hood, L. E. (1982) The nucleotide sequence of a human immunoglobulin Cyl gene. Nucleic Acids Res., in press.
Ellison, J., Buxbaum, J. and Hood, L. (1981) Nucleotide sequence of a human immunoglobulin Cy4 gene. DNA 1, 11-18.
Ellison, J. and Hood, L. (1982) Linkage and sequence homology of two human immunoglobulin y heavy chain constant region genes. Proc. Nat. Acad. Sci. USA 79, 1984-1988.
Frelinger, J. G., Hood, L. E. and Wettstein, P. J. (1981) Analyses of RT! products using two-dimensional polyacrylamide gels. Transplant. Proc. 13, 1360-1363.
Goldstein, A., Fischli, w., Lowney, L. I., Hunkapiller, M. and Hood, L. (1981) Porcine pituitary dynorphin: Complete amino acid sequence of the biologically active heptadecapeptide. Proc. Nat. Acad. Sci. USA 78, 7219-7223.
Goodenow~ R. S., McMillan, M., Orn, A., Nicolson, M., Davidson, N., Frelinger, J. A. ang Hood, L. (1982) Identification of a BALB/c H-2L gene by DNAmediated gene transfer. Science 215, 677-679.
Hewick, R. M., Hunkapiller, M. W., Hood, L. E. and Dreyer, W. J. (1981) A gas-liquid solid phase peptide and protein sequenator. J. Biol. Chem. 256, 7990-7997.
Hood, L. (1982) Antibody genes. Arrangements and rearrangements. In: Molecular Genetic Neuroscience. F. 0. Schmitt, S. J. Bird and F. E. Bloom (Eds.), Chapt. 7, pp. 75-82. Raven Press, New York.
Hood, L. E. (1982) Antibody diversity. In: Science Year. B. Merz (Ed.), World Book-Childcraft International, Inc., Chicago, in press.
Hood, L., Griffin, J., Crews, S., Huang, H., Kronenberg, M. and Kim, S. (1981) Antibody and MHC genes. In: Immunoglobulin Idiotypes. C. Janeway, E. E. Sercarz, H. Wigzell and C. Fred Fox (Eds.), ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. XX, pp. 805-824. Academic Press, New York.
Hood, L., Hunkapiller, M., Hewick, R., Giffin, C. E. and Dreyer, W. J. (1981) Microchemical instrumentation. ICN-UCLA Symposium. J. Supramol. Struct. & Cell. Biochem. 17, 27-36.
Hood, L., Steinmetz, M. and Goodenow, R. (1982) Genes of the major histocompatibility complex. Cell (Minireview) 28, 685-687.
Huang, H., Bond, M. w., Hunkapiller, M. W. and Hood, L. E. (1982) Cleavage at tryptophanyl residues with dimethylsulfoxide/hydrochloric acid and cyanogen bromide. Meth. Enzymol., submitted for publication.
Huang, H., Crews, S. and Hood, L. (1981) An immunoglobulin Va pseudogene. J. Mo!. Appl. Genet. 1, 93-101.
Huang, H., Crews, S. and Hood, L. (1981) Diversification of antibody genes through DNA rearrangements. In: Advances in Experimental Medicine and Biology, Vol. 137, The Reminant Immune System. J.E. Butler (Ed.), pp. 475-488. Plenum Press, New York and London.
Huang, H., Crews, S. and Hood, L. (1981) The arrangement, rearrangement, and diversification of antibody genes. In: Frontiers in Immunogenetics. w. H. Hildeman (Ed.), pp. 63-74. Elsevier North Holland, Inc., New York, New York.
Huang, H. and Hood, L. (1982) The expression of antibody genes. In: Advances in Comparative Leukemia Research 1981. D. S. Yohn and J. R. Blakeslee (Eds.), pp. 169-172. Elsevier Biomedical Press, New York.
Hunkapiller, M., Hewick, R. M., Dreyer, W. J. and Hood, L. E. (1982) A new protein microsequenator using gas phase Edman reagents. In: IVth International Conference on Methods in Protein Sequence Analysis. The Humana Press Inc., Clifton, New Jersey, in press.
Hunkapiller, M. W. and Hood. L. E. (1981) Microsequence analysis of polypeptides using automated Edman degradation. In: Chemical Synthesis and Sequencing of Peptides and Proteins, Vol. 17, Developments in Biochemistry. T.-Y. Liu, A. Schechter, R. Hendrikson and P. Condliffe (Eds.), pp. 111-118. Elsevier North Holland, Inc., New York.
Hunkapiller, M. W. and Hood, L. E. (1982) Analysis of phenylthiohydantoin amino acids by ultrasensitive gradient HPLC. Meth. Enzymol., in press.
Hunkapiller, M. W., Hood, L. E., Hewick, R. M. and Dreyer. W. J. (1982) High sensitivity sequencing with a gas phase sequenator. Meth. Enzymol., in press.
Hunkapiller, T., Huang, H., Hood, L. and Campbell, J. (1982) The impact of modern genetics on evolutionary theory. In: Perspectives on Evolution. R. Milkman (Ed.), pp. 164-189. Sinauer Associates, Inc., Sunderland, Massachusetts.
Hunkapiller, M. W., Lujan, E., Ostrander, F. and Hood, L. E. (1982) Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Meth. Enzymol., in press.
Hunkapiller, M. W., Strader, C. D., Hood, L. E. and Raftery, M. A. (1982) Subunit stoichiometry of Torpedo californica acetylcholine receptor. Proceedings Conference on Taste and Olfaction (March 1980). R. K. Cagin (Ed.), in press.
Johnson, N., Slankard, J., Paul, L. and Hood, L. (1982) The complete V domain amino acid sequences of two myeloma inulin-binding proteins. J. Immunol. 128, 302-307.
Kehry, M. R., Fuhrman, J. S., Schilling, J. W., Rogers, J., Sibley, C. H. and Hood, L. E. (1982) Complete amino acid sequence of a mouse mu chain: Homology among heavy chain constant region domains. Biochemistry, in press.
Kenner, G. W., Moore, s., Ramachandran, K. L., Ramage, R., Dockray, R. A. G., Hood, L. and Hunkapiller, M. (1981) Porcine big gastrin: Sequence, synthesis, and immunochemical studies. Bioorg. Chem. 10, 152-160.
Kim, S., Davis, M., Sinn, E., Patten, P. and Hood, L. (1981) Antibody diversity: Somatic hypermutation of rearranged Ya genes. Cell 27, 573-581.
Kronenberg, M., Kraig, E., Horvath, S. J. and Hood, L~ E. (1982) Cloned T cells as a tool for molecular geneticists: Approaches to cloning genes which encode T-cell antigen receptors. In: Isolation, Characterization and Utilization of T Lymphocyte Clones. C. G. Fathman and F. Fitch (Eds.), Academic Press, New York, in press.
Martens, C. L., Moore, K. W ., Steinmetz, M., Hood, L. and Knight, K. L. (1982) Heavy chain genes of rabbit IgG: Isolation of a cDNA encoding y-heavy chain and identification of two genomic Cy genes. Proc. Nat. Acad. Sci. USA, in press.
Moore, K. w., Sher, B. T., Sun, Y. H., Eakle, K. and Hood, L. (1982) DNA sequence of a gene encoding a BALB/c mouse Ld transplantation antigen. Science 215, 679-682.
Nicholson, B. J., Hunkapiller, M. w., Grim, L. B., Hood, L. E. and Revel, J.-P. (1981) Rat liver gap junction proteins: Properties and partial sequence. Proc. Nat. Acad. Sci. USA 78, 7594-7598.
Orn, A., Brayton, P. R., Woodward, J. G., Goodenow, R. S., Harmon, R. C., Frelinger, J. A. and Hood, L. (1982) Product of a transferred H-2Ld gene acts as a restriction element for LCMV-specific killer T cells. Nature 297, 415-417.
Steinmetz, M., Frelinger, J. G., Fisher, D. A., Moore, K. W., Sher, B. T. and Hood, L. (1981) Isolation and characterization of cDNA clones encoding mouse transplantation antigens. In: Developmental Biology Using Purified Genes. D. Brown and C. Fred Fox (Eds.), ICN-UCLA Symposium on Molecular and Cellular Biology, pp. 173-188. Academic Press, New York.
Steinmetz, M., Moore, K. W., Frelinger, J. G., Sher, B. T., Shen, F.-w., Boyse, E. A. and Hood, L. (1981) A pseudogene homologous to mouse transplantation antigens: Transplantation antigens are encoded by eight exons which correlate with protein domains. Cell 25, 683-692.
Profes;or: Norman H. Horowitz Sherman Fairchild Distinguished Scholar: David R. Stadler Visiting Associate: L. Elizabeth Bertani Research Fellow: Hans Lennart Adler Member of the Professional Staff: Gisela W. Charlang Research Staff: Bradford Ng
Support: The work described in the following research reports has been supported by:
Fairchild Foundation National lnstitutes of Health, USP HS University of GOteborg, Sweden
Summary: Last year we described the identification of
some highly active siderophores produced by Aspergillus
nidulans and Penicillium chrysogenum. At the time of
writing, identification of two of the compounds was not
finished. The work has since been completed, and both
A-13 and P-13 are identified as fusigen B, or fusarinine B,
a linear trimer of the ornithine derivative, fusarinine.
These results are published in the paper listed at the end
of this report.
This report will be the last from the Horowitz group,
since Horowitz is retiring at the end of this academic
year. The subject of our research in recent years-the
relation between water and iron .requirements in
fungi-arose out of the Mars explorations in which some
of us were involved in the '60s and '70s. Before that, we
worked on the genetic determination of enzyme synthesis
in Neurospora, a subject that is now of historical interest
only.
53
Steinmetz, M., Winoto, A., Minard, K. and Hood, L. (1982) Clusters of genes encoding mouse transplantation antigens. Cell 28, 489-498.
Wettstein, P. J., Frelinger, J. G. and Hood, L. (1981) Serological and biochemical characterization of rat (RTl) class II molecules with restricted mouse anti-Ia sera. Immunogenetics 13, 93-107.
Wettstein, P. J., Frelinger, J. G. and Hood, L. (1981) Serological and biochemical analyses of rat class II molecules with anti-la sera. Transplant. Proc. 13, 1364-1366.
Woodward, J. G., Harmon, R. C., Orn, A., Brayton, P. R., McLaughlin-Taylor, E., Goodenow, R. S., Hood, L. and Frelinger, J. A. (1982) Biological properties of class I MHC molecules expressed after DNA-mediated gene transfer. In: B and T Cell Tumors: Biological and Clinical Aspects. E. Vitetta and C. Fred Fox (Eds.), ICN-UCLA Symposia on Molecular and Cellular Biology. Academic Press, New York, in press.
Woodward, J. G., Orn, A., Harmon, R. C., Goodenow, R. S., Hood, L. and Frelinger, J. A. (1982) Specific recognition of the product of a transferred major histocompatibility complex gene product by cytotoxic T lymphocytes. Proc. Nat. Acad. Sci. USA, in press.
We are the last group in the Biology Division still using
Neurospora crassa as its principal research organism. It is
interesting to recall that the first cultures of Neurospora
at Caltech were brought here in 1928 by T. H. Morgan,
who received them from B. o. Dodge before Morgan left
Columbia University. Dodge told Morgan that Neurospora
would be important for genetics some day. This prophecy
was fulfilled when Beadle and Tatum used Neurospora to
produce the first "biochemical mutants"-auxotrophs in
modern terminology. Neurospora was uniquely suited for
this pioneering investigation, since at the time it was the
only microorganism whose genetics and nutritional
requirements were understood. Investigation of the many
auxotrophic mutants that were eventually obtained in
Neurospora led to the realization that a specific relation
ship exists between genes and enzymes, a relationship
summarized in the phrase "one gene, one ·enzyme." This
discovery became one of the foundation stones of
molecular biology. The illustrations on the back cover of
this Annual Report show the procedure used by Beadle and
Tatum to isolate the first mutants.
65. ORNITIHNE SYNTHESIS BY AN ORNITIHNEDEFICIENT TRIPLE MUTANT OF NEUROSPORA
lnvestigators: Gisela CharlBng, Bradford Ng
A mutant blocked in all pathways of ornithine
synthesis should be unable to produce siderophores, since
54
the latter contain three molecules of ornithine. Such a
mutant has been constructed by Davis (1970). The triple
mutant arg-5, ota, aga lacks enzymes in all known
pathways leading to ornithine. It has been reported that
this mutant is unable to make siderophores (Winkelmann,
1973).
A different picture has errierged from our studies,
however. Using the very sensitive bioassay for
siderophores developed in our laboratory, we find that
after four transfers on agar medium under stringent
ornithine-free conditions, the conidia of the triple mutant
still contain ferricrocin at approximately 5% of the wild
type level.
Under conditions of iron deficiency, siderophore
production is strongly derepressed in N eurospora, as in
other fungi. Under these conditions, siderophore--and
therefore ornithine-production is also increased in the
triple mutant. We find that the triple mutant produces
nearly 6 micromoles of ornithine, as siderophores, per
g dry weight of mycelium on an Fe-deficient medium.
This is about 296 as much siderophore ornithine as wild
type produces under the same conditions.
These results suggest either that an alternative
pathway to ornithine synthesis exists which is derepressed
under Fe-starvation conditions, or that the mutant is
slightly leaky-not enough to be detected under ordinary
conditions, but enough to show a response under the strong
derepression effected by Fe deficiency.
References: Davis, R. (1970) J. Bacterial. 102, 299-305. Winkelmann, G. (1973) Arch. Mikrobiol. 88, 49-60.
66. THE FUNCTION OF CELLULAR SIDEROPHORES
Investigators: N. H. Horowitz, Gisela Charlang, Bradford Ng
Fungi make two classes of siderophores, cellular and
extracellular. In a given species, the two are chemically
different substances, although structurally related;
however, the cellular siderophore of one species can be
the extracellular siderophore of another. Extracellular
siderophores are secreted into the environment during
growth, where they are known to solubilize and transport
iron. Cellular siderophores remain in (or on) the cell
through the life of the cell. We have identified a small
fraction of the cellular siderophores of conidia as
essential for conidial germination. This fraction appears
to be bound to the cell surface, where it functions in the
transport of iron at a critical point in the life cycle.
Most cellular siderophores are not involved in conidial
germination, however, and one presumes that they have
some other cellular function. We have tested the
hypothesis that they have an essential role in the synthesis
of heme; for example, they might transport iron into the
mitochondria, where heme synthesis occurs, and release it
at a critical point. Experiments showed that 3H
ferricrocin is taken up by mitochondria isolated from
exponentially growing cultures of wild type and of the
triple ornithine-deficient mutant described in the previous
abstract. Active transport is not involved, since uptake is
not inhibited by azide.
Further tests were done with the triple mutant.
Although the mutant produces up to 5% of the wild-type
level of siderophores, it might be expected to show a
deficiency of cytochromes if cellular siderophores have
the role suggested. Because of its block in ornithine
synthesis, the mutant requires exogenous arginine and
putrescine for growth. If heme synthesis does not occur,
respiration would be expected to proceed through the CN
insensitive flavoprotein pathway, and cytochromes should
be absent. The mutant was grown in several different
media, and the mycelium was examined with a hand
spectroscope following treatment with ascorbic acid to
reduce cytochromes. In all cases, the characteristic
absorption bands of cytochromes a, b, and c were visible
in the mutant. The bands were not noticeably fainter than
those of wild-type controls.
These results suggest that the cellular siderophores do
not have a specific function in heme synthesis. Quite
possibly, their function is that of iron storage in the cell.
The results also show that, except for ungerminated
conidia, siderophores are not required for iron uptake by
Neurospora. The latter has been known for some time.
Citrate, among other substances, can transtmrt iron for
growing mycelia; and ferrous iron is taken up by a
separate, and unknown, mechanism.
67. SEARCH POR SIDEROPHORE RECEPTORS IN NEUROSPORA
Investigators: Hans Lennart Adler, Gisela Charlang
The uptake of siderophores by Neurospora crassa shows
saturation kinetics and is inhibited by metabolic poisons
and SH-reacting agents. It is also competitively inhibited
by structurally similar siderophores indicating a receptor
mechanism on the cell membrane. Previous results from
this laboratory indicate that conidia release surface
(membrane?)-bound siderophores at low water activity,
presumably a response to prevent the spores from
germinating under conditions of low water availability. In
an effort to identify the siderophore receptor(s) of the
plasma membrane, we have taken advantage of our
recently isolated uptake-deficient mutants. The electro
phoretic pattern of membrane proteins from wild type and
mutants has been compared on SDS gels and we are
proceeding to use a radioactive crosslinking reagent
(Schwartz et al., 1982) for affinity labeling of the
receptor(s). One of the siderophores, Coprogen B, has
been coupled to an activated agarose gel and we are
presently trying to find suitable conditions for affinity
chromatography of solubilized rece1.>tor(s).
Reference: Schwartz, M. A., Das, o. P. and Hynes, R. 0. (1982) J.
Biol. Chem. 257, 2343-2349.
68. UTILIZATION OF TRIACETYLFUSIGEN FOR moN TRANSPORT
Investigators: Gisela Charlang, Bradford Ng
Aspergillus nidulans produces several extracellular
siderophores. In young cultures, fusarinines A and B
predominate; in older cultures (6 days or more)
triacetylfusigen is the major siderophore. While the
fusarinines are very active in our bioassay, triacetyl
fusigen shows no activity at all. A. nidulans also makes
two intracellular siderophores, triacetylfusigen and
ferricrocin.
It has puzzled us for some time why an organism would
produce great quantities of a siderophore both inside the
mycelium and into the medium and yet be unable to use it.
We have evidence now that suggests that triacetylfusigen
can, indeed, be used as a siderophore by A. nidulans, even
though it is inactive under the conditions of our bioassay.
The evidence comes from two kinds of experiments.
(1) We have done U[>take studies of tritiated triacetyl
fusigen (TAF) and find that it is trans[>Orted into the cells
by an azide-inhibitable process. Amounts of 3H-TAF
transported are much less than amounts of 3H-ferricrocin
(FC), which is active in the bioassay, when measured
under identical conditions. The highest values recorded
for 3H-TAF were about 0.89 nmol/mg dry weight of 16-hr
mycelium in 5 min of incubation, compared to 2.6 nmol of 3H-FC. We found no competition between TAF and FC
U[>take. (2) We have found that a 1.>artially [>Urified
55
extract of 4-day-old mycelium will hydrolyze the ester
bonds of TAF to release the monomers, acetylfusarinine,
liberating the iron in the process.
A third &[>[>roach tested the idea that TAF might
function in iron transfer to ferricrocin, which then would
deliver iron to wherever it was needed. 55Fe-TAF was
incubated with an equimolar amount of desferri FC and
samples were analyzed on silica gel TLC at hourly
intervals. After an early (1 hr) transfer of 25% of the 55Fe to FC, the final equilibrium favored TAF with 90%
of the isotope remaining with (or returning to) TAF. The
reverse experiment showed an initial 50:50 distribution
and a final 45 (FC):55 (TAF) equilibrium. Evidently iron
entering the cell with TAF will not readily transfer to FC.
69. ISOLATION OF AN AUTONOMOUSLY REPLICATING PLASMID IN NEUROSPORA CRASSA
Investigators: L. Elizabeth Bertani, David R. Stadler
For many years Neurospora crassa has been an
organism of choice for the study of basic genetic and
biochemical phenomena in eukaryotes. There have been
surprisingly few published attempts, however, to investi
gate the organism at the molecular level, using
conventional recombinant DNA techniques. Such an
investigation seems warranted, as Neurospora diverged
from other fungi at a relatively early time and thus might
be expected to have developed original features in the
organization and regulation of its genetic material.
To date, there are no known plasmids-either naturally
occurring or laboratory synthesized-that can multiply
autQnomously in Neurospora. Therefore, as a first step,
we are trying to isolate or construct such a plasmid
vector. On the one hand, we are screening a series of
wild-type strains-some of N. crassa and some of N.
intermedia-that have been isolated from different areas
of the world, for the presence of covalently-closed,
circular structures by gel electrophoresis and-with the
hell.> of Sue Celniker of the Chemistry Division-by
electron microscopy.
At the same time, we are preparing a library of
5-20 kb fragments of Neurospora DNA in a hybrid
bacterial vector (generously provided by Mary Case,
University of Georgia) that already contains a cloned
Neurospora gene that can be selected either in E. coli
(Alton et al., 1978) or in the fungus itself (Case et al.,
1979). This DNA will be used to transform a suitable
56
strain of Neurospora. Potential independently-replicating
structures will be selected by pooling the DNA from the
transformants and using it to transform again the same
strain, under the assumption that only replicating
structures will be able to transform in the second round.
In addition, we will test for the presence of a cytoplasmic
factor by forcing heterokaryon formation between the
initial transformants en masse and a tester strain that can
be examined later to see if it has acquired the selected
character.
Assistant Professor: Elliot M. Meyerowitz Visiting Associate: Elliott S. Goldstein Research Fellow: Leslie S. Leutwiler Graduate Students: Madeline A. Crosby, Thomas E.
Crowley, Mark D. Garfinkel, Robert E. Pruitt Research Staff: Anne M. Villeneuve Laboratory Staff: Phillip A. Patten
Support: The work described in the following research reports has been supported by:
Arizona State University National Institutes of Health, USPHS National Science Foundation Alfred P. Sloan Foundation
Summary: Our research is directed toward understanding
the molecular mechanisms of organismal development.
Most of our experiments use the laboratory fly, Drosophila
melanogaster, but this year we have also initiated several
projects with Arabidopsis thaliana, a green plant.
Two different systems are being studied in the fly
work. Most of our effort is devoted to the 68C polytene
chromosome glue puff. This is a chromosomal locus that
contains the coding sequences for three RNAs that are
coordinately transcribed in third instar larval salivary
glands. We have shown that two of these RNAs are
messenger RNAs, coding for two of the polypeptides that
comprise the complex glue that fly larvae secrete from
their salivary glands just before puparium formation.
Preliminary evidence indicates that the third RNA codes
for yet another glue polypeptide. This glue serves to
attach the pupa to its substrate during metamorphosis.
Our interest in the locus derives from the fact that its
expression is directly regulated by the steroid hormone
ecdysone; we wish to determine the mechanism by which
Refer'"1ces: Alton, N. K., Hautala, J. A., Giles, N. H., Kushner, S. R.
and Vapnek, D. (1978) Gene 4, 241-259. Case, M. E., Schweizer, M., Kushner, S. R. and Giles, N.
H. (1979) Proc. Nat. Acad. Sci. USA 76, 5259-5263.
PUBLICATIONS
Charlang, G., Horowitz, R. M., Lowy, P. H., Ng, B., Poling, S. M. and Horowitz, N. H. (1982) The extracellular siderophores of rapidly growing Aspergillus nidulans and Penicillium chrysogenum. J. Bacteriol. 150, 785-787.
Charlang, C. and Ng, B. (1982) Ornithine synthesis by an ornithine-deficient triple mutant. Neurospora Newsl., in press.
the steroid coordinately affects the levels of the three
RN As. In the past year our goals fell into two classes:
determination of the level at which ecdysone affects RN A
quantity, and identification of the sequences necessary for
steroid action at the 68C locus. Pulse-labeling experi
ments told us that the steroid acts at or very soon after
the transcriptional level; genetic experiments showed that
a maximum of 20,000 base pairs of contiguous DNA is
required for normal tissue-specific, steroid-dependent
expression of all three RNAs. This 20 kb (see Figure 1)
divides into two regions: the 5 kb that includes the three
RNA coding regions, and the region to the left of this.
Studies of the DNA sequence of the 5 kb cluster region
showed some sequences shared between the three
transcribed regions that may be involved in control of
transcript level. Examination of the 20 kb locus in several
different species of Drosophila demonstrated that the
region to the left of the cluster is remarkably conserved
in nucleotide sequence, and thus is at least a candidate for
a region that is important for control of the 68C gene
cluster.
The second Drosophila system being studied is the eye.
Development of the eye from an undifferentiated
epithelium to a highly ordered and regular structure
results from the processes of pattern formation and
differentiation, neither of which are understood at the
molecular level. A number of known mutations prevent
normal eye development in Drosophila; we have started to
analyze these mutations to determine the precise develop
mental processes that they affect.
The final set of projects currently under way uses a
green plant, Arabidopsis thaliana. This tiny member of
the mustard family has been selected for stuc;lies of
genetic control of gene expression because it possesses a
number of useful properties for such studies, including a
very small genome size for a plant, ease of culture, ability
to grow in great numbers in a small space, ability to grow
as tissue culture cells, short generation time, and ease of
classical genetic manipulation. This year we initiated
culture of the plant, began the molecular characterization
of the genome, and started the construction of a number
of mutant strains that will be necessary for our further
studies.
rs
57
70. EFFECT OF f!-ECDYSONE ON RNA METABOLISM IN DROSOPIDLA SALIVARY GLANDS
Investigator: Thomas E. Crowley
Chromosomal puffs on Drosophila salivary gland
polytene chromosomes are sites of RNA synthesis (Bonner
et al., 1977). The steroid hormone B-ecdysone causes
regression of the 68C puff in cultured salivary glands
(Ashburner, 1973). To determine if the effect of the
steroid is on RNA transcription, and to find the levels of
ecdysone that produce effects on puff expression, the
amount of newly synthesized RNA in salivary glands was
analyzed, using hybridization of newly-made RNA to
rt vin Lsp-2
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s s I
'---' I kb
Figure 1. The 68C glue puff. At the top is a depiction of the segment of the salivary gland polytene chromosome set that contains the 68C puff, with the vertical arr3w indicating the position of the puff. Below this are bars indicating the extent of two chromosomal deficiencies. Df(3L}vin defines the maximal rightward extent of the DNA sequence required for 68C puff function; Df(3L}vin4 entirely removes the puff. The hatched bar shows the right breakpoint of the chromosomal inversion In(3LJHR15, which defines the maximal leftward extent of the puff. The bottom of the figure shows a restriction map of the 20,000 base pair region between the inversion and deficiency breakpoints. Different letters indicate cleavage sites of different restriction endonucleases (X; Xho I, R ; Eco RI, B; Barn HI, H; Hind III, S; Sal[). The horizontal arrows indicate the extent and direction of transcription of the three steroid-controlled RN As coded at the puff locus. The group II RNA is 360 nucleotides in length, group III is 320 nucleotides and the group N RNA extends for 1100 nucleotides. The three RN As are expressed coordinately in the salivary glands of third instar Drosophila larvae.
58
filter-bound cloned DNA encoding the 1100 nucleotide (nt)
group IV RNA from 68C as an assay for the rate of
appearance of new 68C RNA, and an RNA not from 68C
as a control. Salivary glands were cultured in the
presence of various concentrations of S-ecdysone and
RNA was pulse-labeled with 3H-adenosine. RNA was then
extracted and hybridized to each of the cloned, filter
bound DNAs. The hybridized RNA was then eluted from
the DNA filters and the radioactivity of the RNA
quantitated. Maximal incorporation of label into the 68C
RN A occurred in salivary glands which had been cultured
in 10-9 M S-ecdysone. The relative amount of label in
this RN A was decreased threefold by increasing the
concentration of B-ecdysone in the culture medium to
10-5 M. The amount of label incorporated into the
non-68C RNA was not affected by the steroid. The
specific activity of total salivary gland RNA did not
decrease with increasing B-ecdysone concentrations, but -5 actually increased. Therefore, B-ecdysone at 10 M
causes a decrease in the rate at which the 68C RNA
enters the cytoplasm, or causes an increase in the
degradation rate of the RNA in the cytoplasm.
The concentration of S-ecdysone in Drosophila
salivary glands increases near the end of the third larval
instar (Maroy et al., 1980). The 68C puff begins to regress
at this stage. The 1100 nt 68C RNA (group IV RNA) is
thought to encode Sgs 3, a glue protein that is secreted
from the salivary gland at the end of the third larval
instar (see Abstract No. 72). It is likely that the synthesis
of this RN A decreases near the end of the third larval
instar as a consequence of increased ecdysone titer.
References: Ashburner, M. (1973) Devel. Biol. 35, 47-61. Bonner, J. J., Berninger, M. and Pardue, M. L. (1977) Cold
Spring Harbor Symp. Quant. Biol. 42, 803-814. Maroy, P., Koczka, K., Fekete, E. and Vargha, J. (1980)
Dros. Inf. Serv. 55, 98-99.
71. TRANSCRIPT MAPPING OF THE 68C RNAs
Investigator: Mark D. Garf'mkel
The three transcripts of the 68C glue gene cluster are
each interrupted by a single intervening sequence. The
evidence that leads to this conclusion derives from the use
of three methods: nucleotide sequencing of cloned cDNA
inserts, nuclease digestion of end-labeled genomic
restriction fragments hybridized to salivary gland poly(A) +
RNA, and sequence determination of cDNA synthesized
with reverse transcriptase, using salivary gland RNA as a
template and cloned restriction fragments as gene
specific primers.
The 5' exon of each mRNA is approximately 55
nucleotides long. Forty-five contiguous nucleotides (nt)
are about 80% homologous among the three mRNAs. The
homologous sequences encode the first 10 amino acids of
each polypeptide and consensus splicing donor sites. The
intervening sequences are each about 70 nt long, conform
to the GT-AG rule, and are not homologous to each other.
In mRNAs II and III, the 3' exons begin with 38-nt-long
conserved regions; in mRNA IV this conserved region is
only 20 nt long. The two conserved sequences that are
joined together in each mature mRNA encode the amino
terminal hydrophobic region of each polypeptide. We have
not yet determined if the nucleic acid sequences are
conserved solely to preserve the amino-terminal protein
sequence, or if they have functional significance at the
RN A or DNA levels.
72. COMPARATIVE SEQUENCE ANALYSIS OF DROSOPHILA GLUE PROTEINS
Investigators: TI!omas E. Crowley, Martha W. Bond
A 5400 dalton polypeptide has been identified as the
product of the 320 nucleotide (nt) group III RNA from the
68C locus. The protein was labeled in cultured salivary
glands with 35s-Cys and 3H-Pro or 3H-Lys, and purified
by preparative two-dimensional electrophoresis. A partial
amino acid sequence was determined using the spinning
cup Edman rnicrosequenator designed and built at Caltech
(Hunkapiller and Hood, 1980). This partial amino acid
sequence matches that predicted by the group III 68C
RNA. This protein has an isoelectric point between pH 7
and pH 9 and is part of the glue secreted from the larval
salivary glands at the time of pupariation.
Sgs-3, a large, heavily glycosylated glue protein, has
been mapped to the 68C locus by genetic means (Akam 35 et al., 1978). This protein was labeled with S-Cys,
purified and sequenced as described above. The position
of four Cys residues was determined; they can be aligned
with the amino acid sequence predicted by the 1100 nt
group IV 68C RNA. The sequence of this RNA also
predicts that its protein product should be approximately
40% threonine. The Formosa strain of Drosophila
melanogaster produces an Sgs-3 that migrates differently
on acid-urea gels than the Sgs-3 of the Oregon R wild
type strain (Korge, 1977). Oregon R and Formosa salivary
gland proteins were labeled in vivo with 3H-Thr and
analyzed on acid-urea gels. For each strain, only one 3 major band was detected by fiuorography. The H-Thr
bands coincided with the gel position of the Sgs-3 protein
from each strain. Thus, Sgs-3 is probably encoded by the
68C group IV RN A.
To identify the protein product of the group II RNA, a
5000 dalton salivary gland protein was labeled in vivo with 35s-Cys and 3H-Lys, purified and sequenced as described
above. Preliminary results indicate that this protein is
probably encoded by the group II 68C RN A. This protein
has an isoelectric point between pH 5 and pH 6 and is part
of the salivary gland glue.
References: Akam, M. E., Roberts, D. 8., Richards, G. P. and
Ashburner, M. (1978) Cel! 13, 215-225. Hunkapil!er, M. W. and Hood, L. E. (1980) Science 207,
523-525. Korge, G. (1977) Devel. Biol. 58, 339-355.
73. SEQUENCE ANALYSIS OF 68C PUFF DNA
Investigator: Mark D. Garfinkel
A detailed analysis of 6746 bp of DNA sequence from
the 68C3-5 glue gene cluster has been completed. The
analysis has been aided by fine-structure transcript
mapping (see Abstract No. 71), by protein sequencing (see
Abstract No. 72), and by the DNAMST data base system
(see Biology 1981, No. 81).
In the course of this work, I determined exactly the
point of insertion of the 9.2 kb roo transposable element
found at 68C in some Oregon R stocks (Meyerowitz and
Hogness, 1982). Clones from a roo-free and from a roo
containing chromosome were sequenced. Comparison of
the two sequences placed the roo insertion at 763 bp to
the left of the 3' terminus of mRNA II (the arrowhead on
the restriction map in Figure 1). Since 68C puff
expression is not affected by the insertion, we may
conclude that if sequences to the genetic left of the
cluster region are relevant to cluster expression, then
they may be displaced 9.2 kb further to the left without
consequence.
The limits of each transcript have been mapped to
single-nucleotide precision (see Abstract No. 71). The
Goldberg-Hogness box (TATAAA box) is the only sequence
element found adjacent to all three mRNA 5' terminal
sites. The inverted gene pair-mRNAs II and III-have 5'
termini separated by only 550 base pairs. Flanking these
mRNAs, the DNA sequences are highly homologous for
nearly 100 bp upstream of each mRNA. These similarities
59
suggest that genes II and III may be coordinately regulated
in a manner independent of gene IV.
Conceptual translation of the sequences of each
mature mRNA species revealed a modular design for the
probable protein products. All three mRNAs encode
amino-terminal hydrophobic regions of approximately 25
amino acids and cysteine-rich carboxy-terminal segments
of 50 amino acids. Gene IV differs from the others by the
insertion of a third segment that, roughly speaking,
separates gene IV's copies of the other two elements. At
the DNA level, the third element is a tandem repetition of
a 15 base pair unit and simple variants of the unit.
Different D. melanogaster strains diffe~n the length of
the gene IV mRNA. This difference may be due to
variation in the number of repeated units. The origin of
this tandem repetition is unknown; it has some sequence
similarity to the inverted repeat element that flanks the
gene II and gene ill mRNAs (Biology 1981, No. 85).
The 68C3-5 cluster region thus contains three genes
that appear to be related to each other by gene
duplication and divergence. The divergence may include
both single base substitutions and more involved inversions
and insertion-deletion events.
Referenee: Meyerowitz, E. M. and Hogness, D. S. (1982) Cell 28,
165-176.
74. TWO WAYS IN WIDCH THE 68C PUFF IS NOT CONTROLLED
Investigator: Elliot M. Meyerowitz
Expression of several RN As in eukaryotic tissues has
been shown to be regulated by DNA rearrangements;
levels of other RN As have been shown to be modulated by
DNA amplification. DNA extracted from whole adult
flies, embryos, and isolated salivary glands of one
Drosophila melanogaster strain was digested with
restriction endonucleases and the resulting fragments
separated by electrophoresis in an agarose gel. The DNA
in the gel was denatured, and the gel pattern blotted to a
nitrocellulose filter. Hybridization of various 32 P-labeled
cloned chromosomal fragments of the 68C puff to the
filter-bound genomic DNA was followed by auto
radiography. The resulting autoradiograms revealed no
differences in size or relative intensity of comparable 68C
puff DNA segments from any of the three sources. Thus,
no DNA amplification or DNA rearrangement in the 68C
puff was detected in salivary gland, the expressing tissue.
60
75. MOLECULAR LIMITS OF THE 68C GLUE PUFF
Investigators: Elliot M. Meyerowitz, Madeline A. era.by
The length of chromosomal DNA required for normal
function of the 68C glue puff can be determined by
finding how close to the cluster chromosomal rearrange
ment breakpoints can occur without affecting puff
function. Last year (Biology 1981, No. 88) we reported
that Df(3L)vin3, a deficiency that removes DNA from the
right of the gene cluster, starting less than 2000 base
pairs from it, has no effect on puff activity. Thus DNA
2 kb and more to the right of the cluster is not required
for normal puff function. In the past year a leftward , molecular limit for the cluster has been set by
ln(3L)HR15, which takes sequences starting between 10
and 14 kb to the left of the cluster and moves them to a
location thousands of kb away. The molecular breakpoint
of In(3L)HR15 was indicated by anomalies in genome blot
analyses of the inverted strain when probed with labeled
genomic clones representing the DNA 10 to 14 kb to the
left of the gene cluster; proof that the breakpoint is in
this region came from hybridization of cloned DNA
including this region, labeled with tritium, to the polytene
chromosomes of an In(3L)HR15 homozygous strain. Auto
radiography revealed that the signal resulting from this
hybridization was present at both ends of the inversion,
and therefore that the labeled probe included the 68C
breakpoint of In(3L)HR15. In(3L)HR15 homozygous fly
stocks apparently have normal 68C puff activity, thus
DNA more than 14 kb to the left of the gene cluster need
not be in the vicinity of the cluster for normal action.
The extent of DNA required at 68C for function of this
multi-transcript polytene chromosome puff is therefore
less than or equal to 20,000 base pairs.
76. ANALYSIS OF THE 68C CLUSTER IN DROSOPHILA SPECIES OTHER THAN D. MELANOGASTER
Investigator: Elliot M. Meyerowitz
The melanogaster species subgroup of the genus
Drosophila includes Drosophila melanogaster, D. simulans,
D. mauritiana, D. erecta, D. arena, D. yakuba and
D. teissieri. Approximately 30 kb of contiguous
chromosomal DNA, homologous to the D. melanogaster
68C glue puff, has been cloned from D. simulans, D.
erecta and D. yakuba, and the restriction map of these
three sets of clones has been compared to that of the
homologous D. melanogaster DNA. All four species show
little if any restriction site conservation in the 5 kb glue
gene cluster region of the cloned DNA; they also show
considerable divergence to the right of the cluster (toward
the centromere). The 10 kb immediately to the left of the
cluster is remarkably conserved, however, showing no
difference between D. melanogaster and D. simulans, and
restriction site differences indicative of only 2-4% DNA
sequence divergence between D. melanogaster and the
other species. The reason for the high conservation of
this region is unknown; one possibility (among many) is
that it serves an important function in regulation of the
68C glue gene cluster.
11. MUTAGENESIS OF THE 68C REGION
Investigators: Madeline A. Crosby, Anne M. Villeneuve
To facilitate genetic analysis of the 68C region, we
have been carrying out a series of mutagenesis experi
ments designed to detect lethals and deficiencies in a
large region flanking 68C.
In the region from 68A3 to 69Al-2, we have induced
approximately 100 lethals. These have been roughly
mapped by complementation tests with overlapping
deficiencies which extend into the 68 region. The
distribution of these lethals is very unusual. In the regions
from 68A3 to 68Bl-2 (6-7 bands) and from 68C9-11 to
69Al-2 (approximately 25 bands), many lethals have been
found, distributed over both regions. However, in the
region from 68Bl-2 to 68C9-11, an area of 12-14 bands,
no lethals have been found. This is the region in which the
68C glue gene cluster is located.
We have started complementation analysis of the
newly-induced lethals; several complementation groups
are represented two or more times, but we have not yet
saturated the area. This is verified by the fact that we
have not found a lethal which is allelic to a known lethal
gene at 68C9-10, 1(3)v4.2.
We have been using two different mutagens in this
screen, the alkylating agents EMS (ethylmethane
sulfonate) and ENU (ethylnitroso urea). Thus far, the
pattern of lethal distribution is very similar for both
mutagens. Both of these mutagens are believed to create
predominantly point mutations. We are presently testing
DEB (diepoxybutane) which is believed to induce small
deletions.
We were recently given a deficiency that extends into
the 68C region from the left, Df(3L)lxd6, with the right
breakpoint at 68Cl-4. We have, in addition, several
deficiencies which extend into 68BC from the right. One
in particular, Df(3L)vin 4, extends through 68C and breaks
at 68Bl-2 (Figure !). Since our lethal mutagenesis results
indicate that there may be no lethals in the region of
overlap between these two deficiencies, we decided to
test if animals that are Df(3L)Jxd6 /Df(3L)vin 4 survive.
Such animals do survive, despite the fact that they are
totally deficient for approximately 5 bands. They look
normal, but are semi-sterile. It appears that this huge
chromosomal region has no essential function in the
development of the fly.
78. CHARACTERIZATION OF MUTATIONS AFFECTING DEVELOPMENT OF THE DROSOPIDLA EYE
Investigators: Elliot M. Meyerowitz, Anne M. Villeneuve
The adult Drosophila eye is composed of a regular
hexagonal array of 700 to 800 ommatidia. Each
ommatidium is itself a regular array of several different
specialized cell types. This highly structured tissue
develops from an undifferentiated epithelium called the
eye-antenna! imaginal disc, which is present in the larval
stages of development. Various observations and experi
ments (see Ready et al., 1976) make it clear that two
processes are involved in eye development. First, cells
learn what sort of cell to differentiate into, presumably
by receiving messages from neighboring eye cells
(certainly not from inheriting this information from their
ancestors); and then the individual cells differentiate to
the appropriate cell type. A number of mutations are
known to cause abnormal eye development. As a first
step in comprehending which of the two processes, pattern
formation or differentiation, is affected by each
mutation, somatic recombination spots containing tissues
mutant for each of these loci are being induced in
otherwise wild-type eyes, and the phenotype of the spot
and its surrounding tissue studied. (For dominant
mutations, the somatic recombination spot is wild type, in
an otherwise mutant eye.) Each spot is simultaneously
marked with an autonomous eye color mutation and an eye
development mutation. If the mutation affects
differentiation and not pattern formation, the eye
development mutant phenotype on the spot should
coincide with the autonomous eye color;: marker. If
communication between neighboring cells is the process
blocked by the eye development mutation, differentiation
of genotypically wild-type cells adjacent to the
genotypically mutant patch should be abnormal, due to the
61
mutant neighbors of these genetically wild-type cells
being unable to send signals of positional information to
them.
Fifteen different eye development mutations have
been studied in this way, fourteen are autonomous in their
effect and thus affect differentiation of individual cells.
One mutation is nonautonomous. Further study will show
whether or not it affects communication between cells in
the developing eye or a diffusible factor required for
differentiation of individual eye cells.
Reference: Ready, D. F., Hanson, T. E. and Benzer, S. (1976) Devel.
Biol. 53, 217-240.
79. PRELIMINARY CHARACTERIZATION OF THE GENOME OF ARABIDOPSlS THALlANA
Investigators: Robert E. Pruitt, Leslie s. Leutwiler
The small crucifer Arabidopsis thaliana has many
advantages as an experimental organism with which to
study plant molecular biology. It has a short life cycle
which can be completed in four weeks, only five
chromosomes, and a genome that is approximately the
same size as Drosophila (approximately 2 x 108 nucleotide
pairs). In addition, large populations can easily be grown
in a small controlled environment. In order to learn more
about the Arabidopsis genome a recombinant A library was
constructed from Arabidopsis genomic DNA, partially
digested with Eco RI and cloned in the 1- vector Sep6.
Random clones were picked from this library and the
insert DNAs analyzed in the following manner: the clones
were digested with Eco RI, subjected to agarose gel
electrophoresis, and blotted to nitrocellulose filters.
These filters were probed with 32P-labeled Arabidopsis
DNA. The intensity of the resulting signal was used to
determine in a crude manner which clones clearly
contained repetitive DNA and which might be unique.
Those clones which appeared to be unique were then used
as probes of genome blots to help confirm their unique
ness. Of ten clones examined, eight appear to be unique,
one appears to contain some moderately repetitive DNA,
and one appears to be composed of highly repetitive DNA.
Neither of the repetitive clones represents chloroplast
DNA sequences, because when either clone is used to
probe a genome blot, many bands not corresponding in size
to those present in the clone are seen. A chloroplast DNA
clone would be expected to hybridize only to a small
number of bands corresponding to the chloroplast
62
sequences cloned. It is interesting that eight out of ten
clones appear to contain only unique DNA and one of the
remaining two appears to contain only highly repetitive
DNA. Since the average length of these clones is 13 kb,
this would indicate that the Arabidopsis genome may have
a long period sequence interspersion pattern. We are
currently extending this analysis to more clones and also
Professor: Herschel K. Mitchell Senior Research Associate: Peter H. Lowy Senior Research Fellow: Nancy S. Petersen Research Fellow: Donald J. Silveri Graduate Students: Antonio A. Reyes, Loveriza A.
Sarmiento Research Staff: Joan Roach
Support: The work described in the following research reports has been supported by:
Biomedical Research Support Grant (NIH) California Foundation for Biochemical Research Josephine v. Dumke Fund National Institutes of Health, USPHS The Rockefeller Foundation
Summary: We are interested primarily in mechanisms of
regulation of gene expression, particularly in relation to
the programs of differentiation and development. We use
Drosophila for the most part as biological material and
have made increasing use of wing development in pupal
stages for a variety of purposes. The reason for this
choice is that wing cells differentiate in remarkable
synchrony in the absence of cell division. This permits
studies that directly relate molecular biology and
morphogenesis. We have been able to correlate expression
of actin genes with particular cell activities related to
differentiation of cell hairs. In addition we have defined a
number of steps in hair morphogenesis that occur at the
same time that particular sets of proteins are synthesized.
We have continued studies on mechanisms of action of
heat shock proteins and their relation to phenocopy
production and teratogenesis. We have extended work on
the multihair phenocopy to two bithorax mutants with the
conclusion that individual cells or groups in synchrony
react to heat shock on a time scale based on a
predetermined program.
Investigations on the pupation process in Drosophila
have now shown clearly that protein components synthe
sized in the prepupal salivary glands are transported into
will be carrying out other studies using Arabidopsis in the
future.
PUBLICATION
Meyerowitz, E. M. and Rogness, D. s. (1982) Molecular organization of a Drosophila puff site that responds to ecdysone. Cell 28, 165-176.
the intercuticle space where they evidently function in
separation of the prepupal cuticle from the pupal case.
80. DEVELOPMENTAL ABNORMALITIES IN DROSOPHILA INDUCED BY HEAT SHOCK
Investigators: Herschel K. Mitche!J., Nancy S. Petersen
Phenocopies in Drosophila, as induced by heat shock,
have proven to be valuable as tools for studies of the
molecular events in morphogenesis (Mitchell and Lipps,
1978; Mitchell and Petersen, 1981; Petersen and Mitchell,
1981). We have recently summarized information on
conditions for induction of phenocopies by heat shock of
Drosophila pupae, and have constructed a temporal map of
sensitive periods for abnormalities that can be produced in
more than 90% of the treated animals. Most of the 34
abnormal phenotypes considered are concerned with
alterations in differentiation of epithelial cells and most
of them resemble known mutants.
References: Mitchell, H. K. and Lipps, L. S. (1978) Cell 15, 905-918. Mitchell, H. K. and Petersen, N. S. (1981) Devel. Biol. 85,
233-242. Petersen, N. S. and Mitchell, H. K. (1981) Proc. Nat.
Acad. Sci. USA 78, 1708-1711.
81. THE MORPHOGENESIS OF CELL HAIRS ON DROSOPIHLA WINGS
Investigators: Herschel K. Mitchell, Joan Roach, Nancy S. Petersen
We have shown previously that patterns of mRNA and
protein synthesis change rapidly during differentiation of
wings in pupal stages of Drosophila (Mitchell and
Petersen, 1981). During this time cell hairs are produced,
drastic changes in cell shape occur and cuticulin and
cuticle are deposited, all in the absence of further cell
division.
We have now completed experiments that yielded a
detailed timetable for the morphogenetic events that
occur in sequence as each wing cell differentiates to
produce a cell hair. Since this is done in synchrony among
about 30,000 cells, it is feasible to relate gene expression
directly to hair construction. We have observed that five
abundant proteins synthesized in the time interval of hair
extrusion and cuticulin deposition (33-39 hr). There is a
maximum in the rate of actin synthesis at the time of
greatest cell movement and there are several candidates
for proteins that are deposited as fibers within the
structure of the hairs. Components that may become part
of the structure of adult cuticle have also been observed.
Of the various proteins whose synthesis is turned on and
off during hair differentiation, only actin has been
identified. We expect that some of the others are
structural materials and this is under investigation.
Reference: Mitchell, H. K. and Petersen, N. S. (1981) Devel. Biol. 85,
233-242.
82. GRADIENTS OF DIFFERENTIATION IN WILD-TYPE AND BITHORAX MUTANTS OF DROSOPHILA
Investigators: Herschel K. Mitchell, Nancy S. Petersen
Cell hairs are normally produced on many areas of
Drosophila during metamorphosis. We have shown that
the cells in each area respond on their own predetermined
time scale to a heat shock that induces the differentiation
of abnormal hairs (the multihair phenotopy). In general a
gradient of sensitive periods goes from anterior to
posterior on dorsal structures. Hair differentiation occurs
after cell division has ceased and it occurs synchronously
within an area. For example, hairs are extruded within a
two-hour period or less on about 28,000 of the 30,000 cells
of the developing wing. Thus the wing provides sufficient
material in synchrony for studies of RN A and protein
synthesis.
In order to ascertain whether the heat shock sensitive
periods in the gradient are due to position of the cells in
the animal or a predetermined program in the cells, we
have looked at heat shock sensitivity in two mutants that
carry different combinations of genes in the bithorax
complex. In one of these, halteres are replaced by
complete wings, and in the second the halteres are
replaced by very small wings. Anterior normal wings and
haltere replacements were compared with respect to
periods sensitive to heat shock and changing patterns of
63
protein synthesis. In normal animals, haltere cells respond
four hours later than wing cells. In the mutants, large
posterior wings responded on wing time and small
posterior wings on haltere time except for small cell
patches which responded to heat on wing time. Quite
clearly the gradient observed in hair differentiation by
epithelial cells is due to predetermination of a program of
gene expression rather than position in the animal.
83. EFFECTS OF HEAT SHOCK ON MESSENGER RNA SYNTHESIS, STABILITY, AND TRANSLATION IN DIFFERENTIATING DROSOPHILA WINGS
Investigators: Nancy S. Petersen, Herschel K. Mitchell
Heating at 40-41° induces stage-specific develop
mental defects in Drosophila pupae. These defects can be
prevented by a lower temperature (35°} pretreatment that
induces heat shock gene expression but does not shut off
normal protein synthesis. In order to investigate the
molecular basis for this protection phenomenon, we have
chosen to look at the multihair phenocopy on wings.
Biochemical analysis is simplified by the facts that wing
tissue is almost entirely one cell type and the cells
differentiate synchronously. Cell division already has
been completed at the time each wing cell differentiates
to produce one hair. However, either mutation or heat
shock can alter the normal course of hair formation to
produce multiple hairs or branched hairs. The multihair
phenocopy is induced by heating during the process of hair
formation at 38 hr after puparium formation. During this
time there is a rapidly changing program of protein
synthesis. The changes in protein synthesis reflect in
changes in the concentrations of major mRNAs. A heat
shock sufficient to induce the multihair defect shuts off
both RNA and protein synthesis.
We have looked at protein synthesis and mRNA
content of normally developing wings and of wings that
were heat shocked at 38 hr either by a single 40.8"
treatment or by heating first at 35° and then at 40.8°. We
have fotmd that heat shock interrupts the developmental
program of protein synthesis and that the delay in
recovery of the developmental program is much less when
the 40.8° heat shock is preceded by a 35° treatment. The
effect on recovery of new RNA synthesis is less
pronounced. The remarkable effect of heat shock on
mRNA is to stabilize messages that would normally decay
as part of the developmental program. The resumption of
mRNA decay is coincident with the synthesis of new
64
mRNAs in the developmental program. The decay of
specific messages and the appearance of new messages
occur sooner in wings that received at 35° pretreatment.
This indicates that there are very specific controls for the
synthesis and decay of messages during development and
that the decay of normal messenger RNA is probably not
the cause of the phenocopy defect. The multihair
phenocopy is probably caused by the noncoordinate
resumption of two morphogenetic processes involving
protein synthesis. The prevention of phenocopies by the
35° pretreatment may be due to an effect of one or more
of the heat shock proteins on the ability of cells to
recover normal protein synthesis.
84. CHANGl!S IN ACTIN GENE EXPRESSION DURING WING DEVELOPMENT
Investigators: Nancy S. Petersen, Beverley J. Bond, Herschel K. Mitchell, Norman Davidson*
During pupal development, there are rapid changes in
the proteins that are being synthesized in wings. One of
the proteins whose synthesis changes is actin. We have
looked at actin messenger RNA at three different times
during wing development: 44-48 hours, 52-60 hours and
78-84 hours after puparium formation. The first and last
periods correspond to periods of peak actin synthesis while
there is at least tenfold less actin synthesis in the 52-60
hour period. Wing tissue is ideal for studying temporal
regulation of gene expression during development since it
consists mainly of one cell type that differentiates quite
synchronously.
The peak of actin synthesis between 44 and 48 hours
after puparium formation is associated with striking
changes in cell shape and with the movement of cell hairs
from lying flat at the edges of cells to an upright position
in the center of cells. We would like eventually to be able
to show how the expression of the actin genes is related to
these changes in cell shape.
There are six actin genes in Drosophila. The coding
sequences for the different actins are almost identical;
however, the intervening sequences and the 51 and 3'
noncoding regions of the mRNAs are quite different
(Fyrberg et al., 1981). We have used cloned sequences
specific for the 3' noncoding portion of each message to
detect expression of actin genes during the 44-48 hour
peak, the 52-60 hour minimum and the 78-84 hour period.
At 44-48 hours, three of the six actin genes are expressed,
Al, A3 and A5. There is very little actin message in RNA
made from 52-60 hour pupae. Actin A2 and A6 genes are
expressed in the later time period. This suggests that the
different actin genes may have specific functions in cells
and that the regulation of their levels of synthesis may be
important during development. We are planning to
investigate the role of actin gene expression in wing
development further by looking at in situ hybridization of
specific actin probes to developing wing tissue in order to
confirm the expression of more than one gene in the same
cell and in order to correlate the expression of specific
genes with specific changes in cell shape.
Reference: Fyrberg, E. A., Bond, B. J., Hershey, N. P., Mixter, K. s.
and Davidson, N. (1981) Cell 24, 107-116.
*Division of Chemistry and Chemical Engineering, California Institute of Technology.
PUBLICATIONS
Bond, B., Petersen, N. S., Mitchell, H. K. and Davidson, N. P. (1982) Expression of actin genes during wing development in Drosophila. Manuscript in preparation.
Buzin, C. and Petersen, N. S. (1981) Major Drosophila heat shock proteins resolve into multiple components on 2-D gels. J. Mol. Biol., in press.
Chomyn, A. and Mitchell, H. K. (1982) Synthesis of the 84,000 dalton protein in normal and heat shocked Drosophila cells as detected by specific antibody. Insect Biochem. 12, 105-114.
Li, G., Petersen, N. S. and Mitchell, H. K. (1982) Induced thermotolerance and heat shock prot~in synthesis in CHO cells. Int. J. Radiation, Oncology and Biol. Phys. 8, 63-67.
Mitchell, H. K. and Petersen, N. S. (1981) Rapid changes in gene expression in differentiating tissues of Drosophila. Devel. Biol. 85, 233-242.
Mitchell, H. K. and Petersen, N. S. (1982) Developmental abnormalities in Drosophila induced by heat shock. Devel. Genetics, in press.
Mitchell, H. K. and Petersen, N. S. (1982) Gradients of differentiation in wild-type and bithorax mutants of Drosophila. Devel. Biol., submitted for publication.
Mitchell, H. K. and Petersen, N. S. (1982) Heat shock induction of abnormal morphogenesis. Cold Spring Harbor Publication, in press.
Mitchell, H. K., Roach, J. and Petersen, N. S. (1982) The morphogenesis of cell hairs in Drosophila wings. J. Cell Biol., submitted for publication.
Petersen, N. S. and Mitchell, H. K. (1982) Heat shock proteins. In: Biochemistry of Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. X. G. A. Kerkut and L. I. Gilbert (Eds.), Pergamon Press, Oxford, in press.
Petersen, N. S. and Mitchell, H. K. (1982) Effects of heat shock on gene expression during development: Induction and prevention of the multihair phenocopy in Drosophila. Cold Spring Harbor PUblica tion, in press.
Associate Professor: James H. Strauss Jr. Visiting Associate: Lynn Dalgarno Senior Research Fellow: Ellen G. Strauss Gosney Researeh Fellow: Charles M. Rice III Researeh Fellow: John R. Bell Graduate Students: Jeffrey T. Mayne, Jing-hsiung
James Ou Special Graduate Students: Carlos F. Arias-Ortiz, Susana
Lopez-Charreton Research Staff: Peggy Feyen, Edith M. Lenches Laboratory Staff: Jeannette Johnstone
SUpport: The work described in the following research reports has been supported by:
Australian National University Biomedical Research Support Grant (NIH) Charles B. Corser Fund for Biological Research E. S. Gosney Fund National Institutes of Health, USPHS National Science Foundation National University of Mexico
Summary: We wish to understand the molecular biology
of replication of Sindbis virus. Although not pathogenic to
man, this alphavirus is a close relative of many other
alphaviruses that are important human or veterinary
pathogens. In addition, the virus provides a useful model
membrane system. Two virus-encoded glycoproteins are
made after infection that migrate from the site of
synthesis in the endoplasmic reticulum through the Golgi
to the cell surface. The virus matures when the
nucleocapsid, formed in the cytoplasm, buds through the
modified cell plasma membrane and acquires an outer
membrane layer.
The following reports describe various approaches we
are using to study Sindbis virus in particular and alpha
viruses in general. Our recent projects have stressed
comparative approaches, such as looking for conserved
nucleotide sequences in the alphaviruses that may serve as
control regions in replication, comparing the amino acid
sequences of the structural proteins of different alpha
viruses to deduce evolutionary relationships between
members of this virus group, and determining the amino
acid substitution responsible for the temperature-sensitive
phenotype of a number of mutants. Further progress has
been made in studying the nonstructural proteins of
Sindbis, both by determining the RNA sequence of regions
of the genomic RNA encoding these polypeptides and in
producing antibodies from synthetic peptides whose amino
acid sequence is deduced from the nucleotide sequence.
85. THE 3'-NONCODING REGIONS OF ALPHAVIRUS RNAs CONTAIN REPEATING SEQUENCES
Investigators: Jing-llsiung James Ou, Dennis W. Trent•, James H. Strauss
65
We ha Ve compared the 3' terminal noncoding sequences
of the RNAs from 10 alphaviruses and found this region to
be composed of distinct domains in terms of base
composition, degree of sequence conservation, and
sequence organization. The first 50-60 nucleotides
adjacent to the 3'-terminal poly(A) tract are extremely
AU rich (up to 90% A+U). Of these, the first 19
nucleotides are highly conserved, and we postulate that
this conserved sequence serves as a replicase recognition
signal. For strains of Venezuelan, Western, and Eastern
equine encephalitis viruses, Highlands J virus and Sindbis
virus, only the sixth nucleotide of this sequence shows any
variation. This conserved region is slightly more variable
for Semliki Forest virus and Middelburg virus. The
remainder of the AU-rich region shows only limited
homology among viruses and may contain signals for
polyadenylation. Upstream from the AU-rich domain,
between 60 and 300 nucleotides from the poly(A) tract,
there are repeated sequences in each viral RNA. These
repeats are up to 60 nucleotides in length and can be
either tandemly or nontandemly arranged. The repeated
sequences show considerable conservation among closely
related viruses, in contrast to the nonrepeated sequences
i_n this region which contain little homology.
*Centers for Disease Control, Center for Infectious Diseases, Vector-Borne Diseases Division, Fort Collins, Colorado.
86. SEQUENCE STUDIES OF SEVERAL ALPHAVIRUS GENOMIC RNAs IN THE REGION CONTAINING THE START OF THE SUBGENOMIC RNA
Investigators: Jing-llsiung James Ou, Charles M. Rice, Lynn Dalgarno, Ellen G. Strauss, James H. Stra...,
The sequence of the region of the 49S genomic RNA
which contains the 5'-end of the subgenomic 268 RNA and
the 5'-flanking sequences in 498 RNA were determined for
several alphaviruses. A highly conserved sequence of 21
nucleotides was found that includes the first two
nucleotides of 26S RNA and the 19 nucleotides preceding
it. We propose that the complement of this sequence in
the minus strand is the recognition site used by the viral
transcriptase for initiation of transcription of 26$ RNA.
The COOH-terminal sequence of the nonstructural poly
protein precursor, which is translated from 498 RNA, has
66
been deduced for each virus. These protein sequences are
highly homologous, but the particular triplets used for a
given amino acid have diverged markedly between viruses,
indicating that virus evolution is quite rapid at the
nucleotide level. Clusters of in-phase stop codons for the
nonstructural polyprotein were found in the nontranslated
region of 268 RNA in each case. The length of
untranslated sequence at the 5'-end of 268 RNA is
between 48 and 51 nucleotides, depending on the virus.
ST. COMPARATIVE STUDil!S OF THE 5'-TERMINAL SEQUENCES OF SEVERAL ALPHAVIRUS GENOMIC RNAs AND A FAMILY OF THEIR DEFECTIVE INTERFERING RNAs
Investigators: Jing-hsiung James OU, Ellen G. Strauss, James H. Strauss
By developing sequencing strategies we have
determined the 5'-terminal sequences of several alpha
virus genomic RNAs and a family of their defective
interfering (DI) RNAs. A highly conserved sequence and
several secondary structures formed by the 5'-terminal
sequences which might be important in alpha.virus
replication were discovered. Comparative studies also
enabled us to deduce the NH2-terminal sequences of their
nonstructural polyproteins.
88. CONSTRUCTION OF SINDB!S VIRUS DEFECTIVE INTERFERING RNAs IN VITRO
Investigators: Charles M. Rice, Henry V. Humig
As mentioned in Abstract Nos. 85, 86 and 87, it
appears that at least three regions of the alphavirus 498
genomic RNA are important in the replication of the
genome and transcription of the 3'-terminal subgenomic
268 RNA. Two of these regions, the 5' and 3' ends of the
498 RNA, are found in defective interfering (DI) RNAs
(truncated and rearranged RNAs of viral origin that
replicate only in the presence of a helper virus and thus
compete with the helper genome for the replicational
machinery) and are thought necessary for production of
full-length(-) and(+) 498 RNA. A third region (called the
junction region), adjacent to the start of 268 RNA, may
encode the recognition site for initiation of 268 tran
scription from the 498 (-) strand template. To study the
replication and transcription of these RNAs, we are
presently constructing cDN A clones of these three regions
as well as clones containing full-length DNA copies of
Sindbis virus 498 RNA, and several DI RNAs. Once
constructed, we will attempt to reintroduce these viral
sequences into tissue culture cells either as DNA or RNA
and study their ability to replicate (or transcribe sub
genomic RNA) in the presence or absence of an
appropriate helper virus. Along with current methods for
in vitro mutagenesis, such a system will enable us to
correlate structural features of the genome with their
function in RN A replication and transcription.
89. EVOLUTION OF ALPHAVIRUSES
Investigator: John R. Bell
Historically, morphological characteristics and
serological specificity have been the most important
criteria for the taxonomic classification of viruses, both
to assign viruses to families and to assess the relationships
between members of a given family. Recently, additional
measures such as the mode of replication and both protein
and nucleic acid homologies have been used for classifica
tion. These molecular criteria have been most useful in
redefining the relationships between members of a given
family or genera. In particular, two viruses of a given
group that show no serological cross-reaction may have
extensive homology at the level of amino acid sequence.
Furthermore, by studying amino acid replacements in the
structural proteins of a group of related viruses it is
possible to trace their evolution, in the same way that
comparative sequence data of cytochromes, for example,
have been used to trace evolution of higher organisms.
With this in mind, we have determined, in collabora
tion with Dr. Dennis Trent and Mr. Richard Kinney of the
Centers for Disease Control, Fort Collins, Colorado, the
terminal amino acid sequences of the two glycoproteins of
six related alphaviruses and analyzed the results in
conjunction with the published data for Sindbis and
Semliki Forest virus proteins. This comparison shows that
the alphavirus proteins share regions and features of
homology. Particularly noteworthy is the conservation of
cysteine residues. Since cysteine residues are involved in
disulfide bridges linking distal regions of polypeptides,
their conservation implies that the overall three
dimensional conformation of the proteins is also
conserved. The two glycoproteins show differing degrees
of diversity, with E2 being the more variable. Within the
eight E2 sequences examined there are small deletions as
well as examples of amino acid replacements at almost
every Joeation with the exception of the cysteines. This
would imply that considerable variation in the primary
sequence of the polypeptide can be accommodated
without destroying protein function. The overall
homology among the proteins is readily apparent and
supports the currently accepted hypothesis that all alpha
viruses have evolved from a common ancestor.
The protein sequence data are currently being further
analyzed by computer programs that are designed to
elucidate evolutionary relationships. Although the
analysis is not yet complete, it is clear that this analysis
will greatly add to our understanding of the evolution of
alpha viruses.
90. SEQUENCE ANALYSIS OF ROSS RIVER VIRUS 265 RNA
Investigators: Lynn Dalgarno, Charles M. Rice
Ross River virus (RRV), an alphavirus first isolated in
Australia, is capable of causing periodic outbreaks of
human disease, characterized by polyarthritic symptoms.
Strains of RRV that differ in pathogenicity have been
isolated in nature, and wild strains can be altered in
pathogenicity by passage in tissue culture. The purpose of
this study was to examine at the molecular level the
evolutionary relationships between RRV and other alpha
viruses with different geographical distributions, such as
Sindbis virus and Semliki Forest virus (SFV), and also to
begin collecting sequence data that should eventually
allow the identification of changes in the RRV genome
that cause the observed differences in pathogenicity
between strains. Thus far we have determined the
nucleotide sequence of the subgenomic 268 RNA of RRV
that encodes the virus structural proteins. The RNA
sequence was determined without the use of molecular
cloning by chemical sequence analysis of end-labeled
Hae m digested cDN A fragments. These sequences were
translated by computer into the three possible reading
frames. The correct coding phase was identified by (1) a
lack of termination codons, and (2) homology with the
corresponding Sindbis virus and SFV structural protein
sequences. By comparison with the known sequences of
the 268 RNA, we could unambiguously order the RRV
Hae III cDNA fragments. The overlapping regions were
then determined by sequencing the appropriate double
stranded cDNA restriction fragments. The complete
sequence reveals that the genomic organization of the
RRV structural protein genes is identical to that of
Sindbis virus and SFV. However, the 3'-untranslated
region of RRV, 524 nucleotides, is more than 200
nucleotides longer than the corresponding region in Sindbis
67
or SFV. Examination of structural protein sequences of
these viruses revealed that RRV is much more closely
related to SFV (75% homology between the structural
proteins) than to Sindbis virus (47% homology). In
addition, during the sequence analysis of RRV, a variant
RNA population was detected that contained a 21-
nucleotide deletion that encoded seven amino acids near
the NH2 terminus of the virion glycoprotein E2. This
variant could be plaque-purified from the original RRV
stock, and differed phenotypically from the parental
strain by producing smaller plaques.
91. INTRACELLULAR TRANSPORT OF SINDBJS VIRUS GLYCOPROTEINS
Investigators: Carlos F. Arias-Ortiz, Edith M. Lettches
We have sequenced the cDNA corresponding to the
genes encoding the viral glycoproteins of two
temperature-sensitive mutants of Sindbis virus, tslO and
ts23, which are defective in the transport of their
glycoproteins from the rough endoplasmic reticulum to
the plasma membrane at the restrictive temperature.
This defect is reversible and the proteins are transported
after shiftdown to the permissive temperature, implying
that the signal for the transport is specified by the protein
itself. We also determined the sequence of revertants of
these mutants, and by comparing the deduced amino acid
sequence of the mutants with that of the parental strain,
Sindbis HR, and with the sequence of the revertants, we
determined that ts23 contained two mutations in glyco
protein El, while tslO had a single mutation in the same
glycoprotein, as expected since they belong to the same
complementation group.
El is a glycoprotein of 439 amino acids. In tslO, the
lysine at position 176 is changed to glycine. In ts23,
alanine106 is replaced by threonine and arginine267 by
glutamine. Thus, two of the changes involved the loss of a
positive charge. In the tslO revertant, glycine172 goes to
arginine, restoring the charge but not the exact amino
acid. In the ts23 revertant, the original amino acids are
restored. The results of this study suggest that the idea
that a linear domain in the protein is responsible for its
correct intracellular transport may be too simplistic, and
that the three-dimensional conformation of the protein
(perhaps involving interactions between widely separated
regions of the amino acid sequence) may be very
important for transport. We cannot rule out a primary
sequence being responsible for transport, whose
68
accessibility or conformation can be altered by mutation
in other regions of the molecule.
92. ANALYSIS OP THE GLYCOSYLATION OP SEVERAL ALPHAVIRUSl!S
Investigators' Jeffrey T. Mayne, John R. Bell
Elucidation of the complete protein sequences (from
cDNA) of the structural proteins of several alphaviruses
(Sindbis virus, Semliki Forest virus, and Ross River virus)
provides detailed knowledge of their potential asparagine
linked carbohydrate attachment sites. This enables us to
investigate the specificity and extent of glycosylation of
these proteins.
We have purified the structural proteins from Sindbis
virus grown in chick embryo fibroblasts and analyzed the
glycopeptides produced from tryptic digests by gel
filtration, high pressure liquid chromatography, and
protein sequenation. We found that in both El and E2,
both of which contain two potential glycosylation sites,
the first glycosylation site contained complex carbo
hydrates and the second one contained simple
carbohydrates. Preliminary data indicate that these sites
are essentially completely glycosylated. In addition, E3
has only one potential carbohydrate attachment site,
which contains a complex carbohydrate.
It is known that Sindbis virus grown in BHK cells has a
different glycosylation pattern, in that E2 contains almost
no simple carbohydrate. Using the above method, we
should be able to determine whether the second
glycosylation site is unglycosylated or contains a complex
carbohydrate.
We want to investigate further the conservation and
variation of the glycosylation of the potential sites in
different alphavirus glycoproteins. Such comparisons will
help to define the necessary common characteristics of
glycosylation sites in membrane proteins and may
elucidate those features of a protein that determine
whether it is glycosylated with simple or complex
carbohydrate chains.
93. STUDIBS ON A SMALL GLYCOPROTEIN PRODUCED BY SINDBIS VIRUS
Investigators: Jeffrey T. Mayne, Charles M. Rice, Ellen G. Strauss
The two best studied alphaviruses are Sindbis virus and
Semliki Forest virus (SFV). One of the major differences
between the two viruses is the presence of E3 (a s·mall,
heavily glycosylated protein) in the membrane of the SFV
virion and its absence in the Sindbis virion. In SFV, E3 is
produced at a late stage of virus maturation when PE2 is
cleaved to produce E2 and E3. But in the Sindbis
infection, PE2 is cleaved to produce E2 only, and the
fragment equivalent to E3 is released into the culture
fluid. We have previously purified and characterized this
protein extensively (see Biology 1981, No. 113).
Careful analyses of the carboxy terminus of E3 have
shown that at least the carboxy terminal -Lys-Arg is
absent from greater than 98% of the population of E3.
Thus E3 is analogous to some other pro-proteins that seem
to be cleaved by a trypsin-like activity followed by a
carboxypeptidase B-like activity. Further labeling studies
confirm that this cleavage is not the rate limiting step in
virus maturation and that E3 is not even transiently
associated with the virion.
94. SEQUENCING OP THE REGION OP THE SINDBIS GENOME ENCODING THE NONSTRUCTURAL PROTEINS
Investigator: Ellen G. Strauss
The genome of Sindbis virus is a single stranded RNA
molecule of approximately 12,000 nucleotides that is
capped at the 5' end and polyadenylated at the 3' end.
Early in the infection cycle, the infecting genome RNA
serves as messenger RNA for the virus-encoded non
structural proteins. The structural proteins are translated
from a subgenomic message that contains roughly one
third of the genome and is coterminal with the 3'
terminus. This subgenomic RN A has been completely
sequenced (Rice and Strauss, 1981, 1982). We are
currently extending these results to determine the
sequence of the remainder of the genome, primarily by
direct chemical sequencing of restriction fragments of
single stranded cDNA synthesized using virion RNA as
template. We have now determined more than 90% of the
sequence of Hae III fragments and linked many of them
together by means of partial digests and sequencing of
Taq I fragments. Experiments to complete the ordering of
these fragments and to deduce the protein sequence of the
nonstructural proteins are in progress.
References: Rice, C. M. and Strauss, J. H. (1981) Proc. Nat. Acad. Sci.
USA 78, 2062-2066. Rice, C. M. and Strauss, J. H. (1982) J. Mo!. Biol. 150,
315-340.
95. STUDY OF THE NONSTRUCTURAL PROTEINS OF SINDBIS VIRUS
Investigators: SUsana Lopez-Charreton, Jolm R. Bell
The study of the nonstructural polypeptides of Sindbis
virus, i.e., the proteins that are involved in the replication
and transcription of the genomic RNA, has been hampered
by the fact that these proteins are produced in only
catalytic amounts during infection; the nonstructural
proteins are translated as a single polyprotein which is
then cleaved to yield three or four polypeptides.
Recently, the cDNA sequence corresponding to the
region that encodes the nonstructural proteins has been
partially determined by E. G. Strauss, and with these
nucleotide sequences the amino acid sequences of some
regions of the polyprotein have been predicted.
As an approach to study the nonstructural poly
peptides, we used these predicted amino acid sequences to
chemically synthesize (by solid state methods) polypeptide
subsets corresponding to the 11 carboxy terminal amino
acids, and a 12 amino acid long region situated 250 amino
acids away from the NH2-terminus of the polyprotein.
These peptides were coupled to BSA as a protein carrier
and injected into rabbits to raise antibodies.
We hope that the resulting antibodies will recognize
some of the nonstructural viral proteins and their
precursors in infected cells or in a cell-free translation
system directed by the viral mRNA, allowing us a clear
identification of these proteins.
These antibodies will also offer a new approach in the
study and isolation of the polypeptides involved in the
viral replicase functions, in which we are particularly
interested.
96. FLA VIVIRUS PROTEINS
Investigators: John R. Bell, James H. Strauss
The members of the flavivirus genus of the Togavirus
family include many important human pathogens,
including yellow fever virus and dengue virus. Originally,
the fl.aviviruses were thought to be related to the
alphaviruses on the basis of similar morphology and mode
of transmission. Members of both genera are small
enveloped viruses with an icosahedral nucleocapsid and
one molecule of single stranded RNA of plus polarity as
their genome. Both alphaviruses and flaviviruses replicate
in their vertebrate hosts and their invertebrate vectors.
However, at the molecular level these two groups are
quite dissimilar in the polypeptide composition of the
69
virions and in their translation and replication strategies.
Flaviviruses have one species of nucleocapsid protein 13.5
kilodaltons, a glycosylated envelope protein of roughly
60 K and a small envelope polypeptide of approximately
8 K.
We have begun to characterize the structural proteins
of St. Louis encephalitis virus, in collaboration with Dr.
Dennis Trent and Richard Kinney from the Centers for
Disease Control in Ft. Collins, Colorado. We have
determined the N-terminal amino acid sequence of the
three polypeptides for 16 to 55 amino acids, using an
automatic protein sequenator developed by Professor
Leroy Hood. None of the sequences obtained show any
detectable homology with any of the alphavirus proteins.
The genomic RNA appears to be the only viral message
in flavivirus infected cells, but whether the viral genes
are translated as a single polyprotein precursor that is
processed by proteolytic cleavages or whether the
individual proteins are the result of independent initiation
and termination events is unclear. Our sequence studies
show that methionine is not found at the N-terminus of
any of the St. Louis encephalitis proteins, nor are the
proteins blocked by N-terminal acetylation. These results
favor the model with a polyprotein and posttranslational
cleavage. However, the nucleotide sequence of the
genome will be required to unambiguously locate initiation
codon(s) in the RN A.
We plan to extend this project with a similar study of
the N-termini of the structural proteins of two additional
flaviviruses, dengue virus and yellow fever virus.
PUBLICATIONS
Bell, J. R. and Strauss, J. H. (1981) In vivo NHrterminal acetylation of Sindbis virus proteins. J. Biol. Chem. 256, 8006-8011.
Bell, J. R., Rice, C. M., Hunkapiller, M. W. and Strauss, J. H. (1982) The NH2-terminus of PE2 in Sindbis virus infected cells. Virology, in press.
Monroe, S. S., Ou, J.-H., Rice, c. M., Schlesinger, S., Strauss, E. G. and Strauss, J. H. (1982) Sequence analysis of cDNAs derived from the RNA of Sindbis virions and of defective interfering particles. J. Virol. 41, 153-162.
Ou, J.-H., Rice, c. M., Dalgarno, L., Strauss, E. G. and Strauss, J. H. (1982) Sequence studies of several alphavirus genomic RNAs in the region containing the start of the subgenomic RNA. Proc. Nat. Acad. Sci. USA, in press.
Ou, J.-H., Strauss, E. G. and Strauss, J. H. (1982) Comparative studies of the 5'-terminal sequences of several alphavirus RNAs. J. Mol. Biol., submitted for publication.
70
Ou, J.-H., Trent, D. w. and Strauss, J. H. (1982) The 3'noncoding regions of alphavirus RNAs contain repeating sequences. J. Mo!. Biol. 156, 719-730.
Rice, c. M. and Strauss, J. H. (1982) Synthesis, cleavage, and sequence analysis of DNA complementary to the 26S messenger RNA of Sindbis virus. J. Mo!. Biol. 150, 315-340.
Rice, c. M. and Strauss, J. H. (1982) Association of Sindbis virion glycoproteins and their precursors. J. Mo!. Biol. 154, 325-348.
Rice, C. M., Bell, J. R., Hunkapiller, M. w., Strauss, E.G. and Strauss, J. H. (1982) Isolation and characterization of the hydrophobic COOH-terminal domains of the Sindbis virion glycoproteins. J. Mo!. Biol. 154, 355-378.
Assistant Professor: Barbara J. Wold Research Staff: Amanda E. Milgram, Charles Reel
Support: The work described in the following research reports has been supported by:
Charles B. Corser Fund for Biological Research National Institutes of Health, USPHS The Alfred P. Sloan Fund for Basic Research
Summary: We are interested in the biology of
mammalian cell-surface receptors. Most of our studies
employ the low density lipoprotein (LDL) receptor as a
mqdel system. Our interests include the mechanism(s) of
receptor regulation, the cell biology of receptor-mediated
endocytosis and the structure of the receptor itself. The
experimental approach employs various molecular
techniques in oonjunction with somatic cell genetics.
Our efforts this year were mainly directed toward
developing techniques and obtaining reagents necessary in
order to study the LDL receptor and its cell biology at the
molecular level. These include: (1) development of a
selection system that renders cells in culture dependent
upon a functional LDL receptor; (2) development of
permanent cell lines bearing mutations in LDL receptor;
and (3) model studies demonstrating that a significant
amount of homologous recombination occurs during DNA
transformation in animal cells and that this can be
utilized for rapid fine-structure mapping of cloned
selectable genes. We are also working to isolate the LDL
receptor gene.
In a related project we are attempting to isolate the
hydroxymethy!glutaryl CoA reductase (HMG CoA reduc
tase) gene. This enzyme catalyzes the rate-limiting step
in de novo cholesterol biosynthesis, while the LDL
receptor governs acquisition of cholesterol from the
Strauss, E. G. and Strauss, J. H. (1982) Replication strategies of the single stranded RNA viruses of eukaryotes. In: Current Topics in Microbiology and Immunology. Springer-Verlag, Berlin, submitted for publication.
Strauss, E. G., Tsukeda, H. and Simizu, B. (1982) Mutants of Sindbis virus. IV. Interspecific complementation and phenotypic mixing between temperature-sensitive mutants and wild-type Sindbis and Western equine encephalitis viruses. J. Gen Viral., submitted for publication.
extracellular environment. We are interested in under
standing the integration of reductase and LDL receptor
regulation that ultimately results in homeostasis with
respect to cellular cholesterol content.
97. A SELECTION SYSTEM FOR LDL RECEPTOR FUNCTION
Investigator: -... J. Wold
In any system where a genetic approach is to be
employed, a selection technique that permits identifica
tion and subsequent isolation of the rare variant from a
large population of individuals is a powerful experimental
tool. Therefore we have developed a biochemical
selection system that permits isolation of the rare LDL
receptor-plus (LDL R+) cell from a population of
receptor-negative cells. This system is to be used in
several studies. The first is to identify LDL R + revertants
of various receptor-negative and receptor-defective cell
lines. Our objective is to locate both true revertants and,
of special interest, second-site mutants. These should
help us to gain insight into receptor structure-function
relationships and identify additional gene products
involved in receptor function. The second is to perform
complementation analyses by either cell fusion between
different receptor mutants or complementation via DNA
mediated gene transfer. In each case the complemen
tation event can be identified by survival under LDL R
selection.
The selection strategy depends on the fact that
cholesterol is required by all animal cells as a component
of the cell membrane. This requirement can be satisfied
by either of two independent pathways. One is entirely
intracellular and results in the de novo synthesis of
cholesterol from acetate. The rate-limiting step in this
J?athway is the reduction of hydroxymethylglutaryl CoA to
mevalonic acid, a reaction catalyzed by HMG CoA
reductase. The second pathway provides for the
acquisition of cholesterol from the extracellular environ
ment via the receptor-dependent endocytosis of low
density liJ?OJ?rotein (LDL), the J?rinciJ?al cholesterol
transport moiety in serum.
The key element in the biochemical selection for LDL
receptor function is the drug mevinolin, which is a potent
competitive inhibitor of HMG CoA reductase. For some
time it has been known that mevinolin effectively blocks
reductase activity in vivo, thereby blocking the de novo
biosynthesis of cholesterol (Goldstein et al., 1979), and
leaving the LDL receJ?tor J?athway as the only J?OSSible
cholesterol source. Under such circumstances, it is
expected that cells possessing functional LDL receptors
could survive via the catabolism of LDL while receptor
negative cells should die due to cholesterol starvation.
However, it proved necessary to consider one additional
feature of the HMG CoA reductase selection. If reduc
tase is 100% blocked by mevinolin, there will be no
mevalonic acid to supply quantitatively minor
noncholesterol anabolic pathways (reviewed in Brown and
Goldstein, 1980), and at least one of these mevalonate
end-products is essential for cell growth (James and
Kandutsch, 1979; Queeney-Haneeus et al., 1979). There
fore, at high mevinolin dosages, cells will die regardless of
how much cholesterol they have, because they are starved
for a noncholesterol mevalonate end-product. This minor
requirement can be satisfied by adding back a small
amount of mevalonate to the cells, such that the minor
pathways are served, but inconsequential quantities of
cholesterol are synthesized.
The final LDL receptor selection conditions were
found to be DMEM (Dulbecco's modified Eagle's medium)
J?lUs 10% liJ?id-deJ?leted fetal calf serum J?lus 40 µM
mevinolin J?lUS 165 µM mevalonate J?lus 2 µg/ml J?Urified
LDL. Receptor-plus cells grow in this medium while
receptor-negative cells die, and it has been shown that the
selection is dependent on LDL, as expected.
References: Brown, M. S. and Goldstein, J. L. (1980) J. LiJ?id Res. 21,
505-517. Goldstein, J., Helseson, J. A. S. and Brown, M. S. (1979) J.
Biol. Chem. 254, 5403-5409. James, M. J. and Kandutsch, A. A. (1979) J. Biol. Chem.
254, 8442-8446. Queeney-Haneeus, V., Wiley, M. and Siperstein, M. (1979)
Proc. Nat. Acad. Sci. USA 76, 5056-5060.
98. HOMOLOGOUS RECOMBINATION IN ANIMAL CELLS
Investigators: Barbera J. Wold, Charles Reel, Steven McKnight*
71
We are utilizing the cloned Herpes virus thymidine
kinase (tk) gene as a model system to study homologous
recombination in animal cells. The objectives of this
study are: (1) to develoJ? a technique for raJ?id maJ?J?ing of
mutations of functional significance in cloned selectable
genes; (2) to develOJ? a model system for studying the
substrate specificities and mechanisms of recombination
in animal cells; and (3) to attempt to develop conditions
under which homologous recombination between donor
sequences and their chromosomal homologues is
sufficiently frequent that it can be used for site-directed
introduction of cloned or synthetic sequences into their
proper chromosomal locations.
The experimental approach is to transform cells with a
combination of two nonfunctional mutant genes that share
a small defined stretch of homologous sequence. These
mutant genes have been chosen so that a single
recombination event in the region of homology should
regenerate a wild-type gene as one of its products. Cells
in which such a recombinant gene has been produced and
expressed can be identified by their ability to survive
selective growth conditions that require the wild-type
gene product. In these experiments, we used two mutant
tk genes constructed by Steven McKnight (Figure 1). Each
clone contains a portion of the tk gene including 217 bases
of honi.ology in the center of the protein coding region.
When cells are grown in media containing HAT (hypo
xanthine, aminopterin and thymidine), they will survive if,
and only if, they have a functional thymidine kinase
activity. Tk-minus mutants such as the mouse Ltk - cell
line will survive HAT selection if a wild-type tk gene is
introduced via DNA-mediated gene transfer. Initial
control experiments in this study showed that neither of
the cloned fragments of the HerJ?es tk gene could by itself
transform tk - cells, but a mixture containing both
plasmids generates tk + transformants. Southern blotting
analysis of the tk + transformants confirmed the presence
of an intact tk gene, which must be the result of a
homologous recombination event in the recipient cell
during DNA transfer. Further experiments investigated
the effect of donor J?lasmid toJ?ology on transfer
frequency and showed that linear molecules transform
three to fivefold better than supercoils. Linearization at
72
the region of homology (in this case the Hind III sites)
enhanced the frequency approximately tenfold over
linearization at the other sites, suggesting that initial
homologous strand invasion may be facilitated by a cut in
the region of shared sequence. We are presently exploring
the substrate specificity of the recombination in greater
detail. Under these conditions we have observed >2 x
102 recombinants/5 x 105 recipient cells/217 base; of
homology. These frequencies can be compared with
internal controls for transfer of a second unrelated, intact
marker to measure the fraction of all DNA transformants
that have undergone a recombination event between the
tk markers. Preliminary data for Hind III linearized
molecules show that one of every 10 cells transformed has
included a functional tk recombinant. For the purposes of
mapping mutations in a selectable function, the technique
at its present stage of development will be useful. Thus a
library of sequential 3' and 5' deletions in the gene of
interest will be constructed and transformations
conducted with pairs consisting of the complete mutant
gene to be analyzed plus an in vitro deletion fragment
from the wild-type gene. By noting which wild-type
deletion fragments are capable of complementing the
unknown mutant, the lesion of interest can be localized to
within 200 bases. Additional experiments are directed
toward quantitating and enhancing the frequency of
recombination between donor DNA and sequences residing
in the animal cell chromosome. Various copy numbers of
the M'l.24 plasmid have been introduced into cells, and
these are to be transformed with the l!.3'1.94 clone.
Recombinants, if present, will be detected by their
survival in HAT and analyzed by subsequent Southern
blotting of DNA from the transformants.
*Assistant Professor, Fred Hutchinson Cancer Research Center, Seattle, Washington.
~§~1~~~------- F Figure 1. Two cloned deletion mutants of the Herpes virus thymidine kinase gene are shown. Herpes-derived DNA sequence is indicated by the solid line and pBR322 sequence is indicated by the dotted line. The boxed region corresponds to homologous Herpes tk sequence present in both clones. H = Hind III restriction sites; B = Bam HI restriction sites.
99. ISOLATION AND CHARACTERIZATION OF CELL LINES RESISTANT TO IIlGH LEVELS OF MEVINOLIN
Investigators: Amanda E. Milgram, Barbara J. Wold
Mevinolin is a competitive inhibitor of the enzyme 3-
hydroxy-3-methylglutaryl CoA reductase (HMG CoA
reductase). We have selected a number of independent
human and rodent cell lines resistant to progressively
higher doses of rnevinolin in the presence of standard
media (Dulbecco's modified Eagle's medium) supplemented
with lipid-depleted fetal calf serum. The parental cell
lines are sensitive to 0.5-1 µM mevinolin under these
Culture conditions, and the majority of cells are killed
within 2 weeks. With variable frequency (10-3-10-5),
resistant cells appeared. These were then subjected to a
three to fivefold higher drug dose, survivors selected, and
the cycle repeated. At present, we have two cell lines
resistant to 100 times the normal killing dosage. This
resistant character reflects a correspondingly higher level
of HMG CoA reductase. Our working hypothesis is that
these cells have undergone gene amplification at the HMG
CoA reductase locus. Our purpose in generating these cell
lines is twofold: (1) it is likely that the exceedingly high
levels of enzyme activity derive from a correspondingly
high level of reductase mRNA, and we are attempting to
capitalize on this in our efforts to isolate cDNA and
chromosomal clones of the reductase gene(s); and
(2) HMG-CoA reductase activity is, under normal
circumstances, regulated over 300-fold in response to
different growth conditions. We are interested in studying
the regulation of reductase in cells with different doses of
the gene. Additional studies will focus on the conse
quences of altered reductase gene doses to the regulation
of the LDL-receptor pathway with the hope of shedding
light on the nature of the coordination of the two
cholesterol pathways.
CELLULAR BIOLOGY AND BIOPHYSICS
Howard C. Berg
Charles J. Brokaw
John J. Hopfield
Elias Lazarides
Jean-Paul Revel
Professor: Howard c. Berg Visiting Associate: I. Richard Lapidus Research Fellows: Shahid M. M. Khan, Akira Ishihara Graduate Students: Markus Meister*, Paul W. Meyer,
Jeffrey E. Segall Special Graduate Student: Steven M. Block Research Staff: M. Patricia Conley, Robert D. Smyth
*Division of Physics, Mathematics and Astronomy, California Institute of Technology.
Support: The work described in the following research reports has been supported by:
Biomedical Research Support Grant (NIH) National Institutes of Health, USPHS National Science Foundation Gustaws and Louise Pfeiffer Research Foundation
Summary: We are interested in the behavioral biology of
bacteria and other unicellular microorganisms.
Flagellated bacteria possess a remarkable motile
system based on a reversible rotary motor linked by a
flexible coupling to a thin helical propeller. The motor
derives its energy from protons driven into the cell by
chemical gradients or electrical fields. The direction of
rotation of the motor depends, in part, on signals
generated by sensory systems, the best studied of which
analyzes chemical stimuli. The nature of these signals is
not known. We would like to understand how the motor
works, what the signals are that control its direction of
rotation, and how these signals are processed by the
chemical sensory system.
The rotation of the motor can be observed directly by
fixing the helical propeller to a glass slide: the cell body
spins alternately clockwise (CW) and counterclockwise
(CCW). These cells are called tethered cells. If chemical
attractants or repellents are added to the medium
surrounding tethered cells, chemotactic responses can be
elicited.
Some bacteria have no recognizable organelles of
locomotion, yet move steadily when in contact with solid
surfaces. We would like to understand the mechanism for
this gliding motility.
The unicellular green alga Chlamydomonas, a eukary
otic cell with two anterior flagella, is able to track a
source of light. We are studying the coupling between the
antenna, a quarter-wave optical stack,. and the flagella.
Another project deals with the genetics of this organism.
The spore-bearing stalk of the fungus Phycomyces
grows away from solid objects. It is thought to have some
kind of chemical radar. We are studying the physics of
this avoidance response.
100. ADAPTATION IN E.COLI CHEMOTAXIS
Investigator: Steven M. Block
75
Bacteria, in their sensory behavior, exhibit a feature
that is common to most living organisms-they adapt.
Cells are able to ignore the ambient level of sensory input
and respond to changes about that level. This sensitivity
to the time-derivative of the input may be termed "range
adjustment." There exists a region where the organism is
most sensitive, with a saturated output occurring for
stimuli that are too large and a threshold for stimuli that
are too small. This may be considered as "range
compression.'' Range adjustment and compression occur
for a wide array of sensory modalities across all
phylogeny. I am studying the details of the adaptation
process using E. coli as a model system.
In my experiments, tethered cells are subjected to
programmed changes in the concentration of the non
metabolizable attractant e>-methylaspartate. The cells
are maintained in a flow cell that is part of a miniaturized
constant-flow perfusion apparatus. The attractant con
centration in the flow can be changed in a continuous and
controlled manner by an electronic pump programmer that
is set up to produce exponential, stepwise, linear, and
sinusoidal variations. The behavior of the tethered cells is
recorded on videotape through phase-contrast optics.
Later, the pattern of reversals of the spinning cells is
transferred to a strip-chart record that is digitized and
analyzed by computer.
In a chemically isotropic environment, tethered E. coli
spin in either direction for roughly equal periods of time,
the probability of a reversal at any given instant being
roughly constant. Accordingly the distributions of clock
wise and counterclockwise rotation intervals are
approximately exponential (obey Poisson statistics). In
response to exponential ramps up, the cells bias their
reversals in such a way as to lengthen the time spent in
the counterclockwise (run) mode and shorten the time
spent in the clockwise (tumble) mode. The amount of bias
is directly related to the steepness of the exponential
ramp. In response to ramps down, cells show the reversed
bias; however, the st6epness of the down-ramp must be
much greater in order to give the same degree of bias.
Up-adaptation and down-adaptation, therefore, exhibit a
fundamental asymmetry. In response to sinusoidal
changes in concentration, cells are able to "adapt out"
variations with periods on the order of 1000 sec. For
faster periods, the CCW /CW bias of the cell varies in an
76
oscillatory fashion, with frequency-dependent phase and
amplitude variations. For frequencies about 5 x 10-3 Hz,
the response of the cell begins to saturate (clip). If the
system is treated as approximately linear, a Bode plot is
obtained that is typical of a first-order high-pass filter
with a 3 dB point at 3 x 10-3 Hz. This corresponds to a
temporal change of the same magnitude as that which
half-saturates the response during exponential ramps.
Cells continue to show Poisson-type behavior even under
stimulation. They do not appear to respond to either the
concentration per se or to time-derivatives of the
concentration higher than first-order.
A closer examination of adapted cells, as well as cells
exposed to ramps, shows that the CW-CCW distributions
are not strictly exponential: there is a characteristic
absence of short events (corresponding to times less than
about 0.4 sec) which cannot be accounted for by
systematic errors in the analysis. The adapted distribu
tions can be fit by a convolution of two exponential
processes, with one time constant of 0.8 sec that accounts
for the exponential "tail" of the distribution, and another
of 0.16 sec that yields the paucity of short events. It is
intriguing that the shorter time is very close to the
latency observed by Segall and Manson (see their report,
"Signal Processing Times ... 11). Since a convolution of two
exponential processes is exactly what one expects for a
cascade of two first-order reactions, we are modeling the
system in terms of a sequence of steps that lead to motor
reversal, using components known from genetics and
biochemistry to be a part of the chemosensory pathway.
101. SIGNAL PROCl!SSING TIMl!S IN E.COLI CHEMOTAXIS
Investigators: Jeffrey E. Segall, Michael D. Manson*
We are using iontophoretic pipettes to deliver pulses-of
attractants and repellents to tethered cells in order to
learn more about how cells process chemotactic stimuli
(Segall et al., 1982). With wild-type cells the mean
latencies are 0.2 to 0.3 sec following stimulation with cx
methylaspartate (a-MeAsp, an attractant) or benzoate (a
repellent). With Cl-MeAsp, this latency changes by less
than a factor of 2 over a hundredfold range of stimulus
strengths. The .latencies are much longer than the
stimulus delivery times measured with the negatively-
charged dye fluorescein. Control experiments with
mutants that fail to respond to specific chemoattractants
indicate that we are dealing with bona fide chemotactic
responses.
We have analyzed several generally nonchemotactic
mutants. Cells defective in adaptation via methylation
that contain deletions of the che R and che B genes have
normal latencies. However, the latencies of strains
containing che Z mutations are very long, ca. 2 sec.
Future work will involve a study of the temperature
dependence of the latency and of pulses of attractant
followed by pulses of repellent, designed to determine if
the latency is due to a series of steps or a single
reversible step.
Reference: Segall, J. E., Manson, M. D. and Berg, H. C. (1982) Nature,
in press.
*FakultB.t ftir Biologie, UniversitB.t Konstanz, West Germany.
102. THE CHEMOTACTIC IMPULSE RESPONSE
Investigators: Jeffrey E. Segall, Steven M. Block
One useful method of characterizing a sensory system
is a measurement of its response to a very brief
stimulus-the impulse response. For a sufficiently short
impulse, the duration of the response ceases to correspond
to that of the stimulus and instead reflects the intrinsic
kinetics of the system under study. We are using short
( <0.1 sec) pulses of an attractant or repellent generated
iontophoretically to stimulate tethered E. coli. The cells
are monitored with a microscope equipped with an
electro-optical device that extracts information about
their si;>eed and direction of rotation. Data records are
collected and digitized off-line as a two-valued function
of time (denoting CW or CCW rotation). Many such
records are put into register, and a graph is generated of
the probability that a cell will rotate in the CCW (run)
direction as a function of time following the stimulus.
The change in the rotational bias of the cell is the impulse
response.
When wild-type cells are pulsed with an attractant, the
response is biphasic: within about 0.2 sec of stimulation,
the cells increase their CCW bias for about a second and
then decrease this bias for a period that lasts somewhat
longer (Figure 1). For pulses of repellent, the shape of the
impulse response is inverted (Figure 1). For both kinds of
pulses, the response is independent of the direction of
rotation of the cell when the pulse is given. Moreover,
the kinetics of the impulse response does not seem to be
strongly correlated with the initial CCW /CW bias of the
cell, the nature of the attractant, or the temperature.
The biphasic nature of the response is characteristic of
systems that show adaptive behavior, i.e., that respond
only to changes in the intensity of a stimulus. In the
terminology of the systems analyst, the bacterium
behaves as a differentiator or high-pass filter. Its
"memory time" is comparable with the time during which
the impulse response has an appreciable value, a time of
order 1 or 2 sec (Figure 1). It should be noted that
previous work on E. coli has demonstrated adaptation
times of order hundreds of seconds. This long-term
adaptation is mediated, at least in part, by a protein
methylation-demethylation system. It constitutes a form
of "long-term" memory. The "short-term" memory
described here has not been seen before. Bacteria
swimming up a gradient must be able to estimate changes
in concentration occurring over periods of time of order
100
~ u u
0 - 1 sec c: <ll 100 u
... <ll Q.
0
pulse
77
1 sec if they are to bias their random walk successfully, so
it is not surprising that processes with time constants of
this magnitude are observed.
We also are studying mutants, in order to identify the
gene products responsible for the various portions of the
impulse response. We find that che Z mutants have an
increased latency, as well as a much longer initial lobe to
the impulse response. However, cells that are missing the
methylation-demethylation enzymes (cells with che R and
che B deletions) have normal impulse responses, although
their response thresholds are higher. These results
suggest that the che Z gene product is involved in the
excitation phase of chemotaxis, while , the che R-che B
gene products are not. The availability of a number of
other chemotaxis-deficient mutants will allow us to
investigate, by physiological means, how the various
components of the chemosensory pathway interact to give
rise to excitation, adaptation, and motor response.
time
Figure 1. Impulse responses of tethered bacteria. Top: to a pulse of attractant. Bottom: to a pulse of repellent. Cells were followed over 20-sec periods. with the stimulus applied at t = 5 sec. This process was repeated many times, and records were accumulated of the direction of rotation (CW or CCW) as a function of time. These records (over 150 in number) were placed in register and averaged to yield the probability of CCW rotation (percent cells rotating CCW). The attractant used was aspartate or its non-metabolizable analog o.-methylaspartate; the repellent used was benzoate. The pulses were of duration 0.1 sec or less.
78
103. CHEMIOSMOTIC COUPLING TO THE FLAGELLAR MOTOR AND MEMBRANE ATPase OF STREPTOCOCCUS
Investigators: Shahid M. M. Khan, Howard C. Berg
We have found that the torque generated by the
flagellar rotary motor and the rate at which ATP is
synthesized by the membrane ATPase do not change when
cells of Streptococcus strain V4051 are powered by a
deuteronmotive force rather than a protonmotive force.
Therefore, although the protonmotive force determines
rates, protonation-deprotonation reactions are not rate
limiting. The torque is constant over a wide temperature
range. Its generation does not involve the formation and
breakage of chemical bonds. ATP is synthesized at a rate
that varies exponentially with inverse temperature, but
the activation enthalpy does not change with proton
motive force. The motor and the ATPase appear to be
reversible engines driven by simple acid-base dissociation.
These considerations have led us to a model for the
flagellar rotary motor (Figure 2) in which gated channels
(channel complexes), elastically connected to the S-ring,
move from site to site along the periphery of the M-ring.
The probability that a channel complex advances or
retreats depends on whether sites in contact with the
external medium or the cytoplasm are protonated or
deprotonated.
The relative motion of sites and channel complexes is
subject to the following two constraints: 1) A site cannot
move past the center of a channel complex, which we
assume to be hydrophilic, unless the site is protonated.
2) A site cannot move away from a channel complex into
the surrounding hydrophobic region of the membrane
unless the site is unprotonated. Thus, when the motor is
driven by an inward-direction proton flux, the complexes
advance only when the sites, o, in contact with the outer
channels are protonated and the sites, i, in contact with
the inner channels are unprotonated. When these
conditions are met, protons move from the external
medium through the outer channels, onto the M-ring,
around to the adjacent inner channels, and through these
channels into the cytoplasm. The motor reverses when
the gating particles flip to the other stable position, a
transition that requires coordinate control. This
transition interchanges the labels o and i.
(a)
Binding site~
A-
• I I I I I
' ( b) ' I
~"' --- ',, , ,,. .... ' I -' ' \
I ,' \ \ t 1 I J I \ I I \ \. I I ' ' ,, ,, ', .... __ .... ,,."
.... ___ ....
Rod
i~ o B I
bl
' I
I B
( c)
' I 1 Channel I
S-ring I
-!c_________,--~c__,--_---.~IE'" M-ring tcytoplasmic Gating
membrane particle 10 nm
Figure 2. A model for the flagellar rotary motor of a gram-positive bacterium, drawn to scale. (a) The M-ring sectioned through its center in a plane parallel to that of the cytoplasmic membrane. (b) The motor viewed from the side, section AA. (c) A third view of a channel complex, section BB. The M-ring is embedded in the cytoplasmic membrane .and attached to the rod. which leads to the proximal hook and the flagellar filament (not shown). The S-ring is attached to the cell wall. Channel complexes are distributed around the periphery of the Mring, as shown in (a) and attached to the periphery of the S-ring, as shown in (b). The number of complexes is not critical; two are shown in (a). Each complex has two channels that penetrate the cytoplasmic membrane. The outer or inner part of each channel is blocked by a gating particle, shown in (b) and (c). The gating particle toggles between two stable positions, blocking the outer part of one channel and the inner part of the other channel, or vice versa. Each channel leads to a proton binding site on the periphery of the M-ring, as shown in (a) and (b). The number of sites is not critical; 32 are shown in (a). The distance between adjacent sites is d. For a description of the operation of the motor, see the text.
104.. DYNAMICS OF THE FLAGELLAR MOTOR OF STREPTOCOCCUS
Investigator: Akira lshihara
An energized tethered cell rotates at a speed that is
inversely proportional to the viscosity of the external
medium: its flagellar motor generates a constant torque.
What happens when an external torque is applied and the
motor is forced to stop? What is the stall torque?
If a tethered cell is starved, it stops rotating: the
motor is fixed in one orientation. How large an external
torque is required to force the motor to rotate? Will a
motor driven in this way pump protons?·
In order to answer these questions, we are attempting
experiments in which external torques are applied
magnetically. We have found that magnetic particles can
be attached to the cell bodies with poly-D-lysine. The
stall torque can be measured by the angular deviation of
these magnets in a fixed magnetic field. Rotation can be
driven by rotating magnetic fields. These experiments
should further our understanding of flagellar motor
function.
105. EFFECT OF EOSIN ON MOTILITY OF STREPTOCOCCUS
Investigator: M. Patricia Conley
Photodynamic dyes are known to affect motility in a
variety of bacteria. In our attempts to identify chemical
groups involved in flagellar motor function (reported on
these pages last year), we found that motility is inhibited
when tethered cells of Streptococcus strain V 4051 are
exposed to eosin plus light. The speed of rotation
becomes progressively slower, until ce~ stop completely.
A similar time course is observed for energized cells and
cells driven by an artificially induced protonmotive force,
i.e., by a potassium diffusion potential. Eosin not
activated by light has no effect. Cells that have stopped
spinning cannot be induced to start again, after the
reagent has been removed, by supplying an energy source
or imposing a diffusion potential. However, if cells are
exposed to eosin plus light until their speed is partially
reduced, further inhibition of motility can be blocked by
placing a filter in the light path. Such cells continue to
rotate at the reduced rate. These results suggest that
photooxidation changes the structure of the flagellar
motor and that it reduces the number of independent
force generators. Experiments in progress are designed to
rule out the alternative possibility that photooxidation
reduces the protonmotive force. If this _alternative can be
ruled out, then it will be of interest to compare the
physiological properties of motors photooxidized to
varying extents. Can a flagellar motor run on a single 11piston11?
106. MECHANISM OF GLIDING MOTILITY
Investigators: L Richard Lapidus, Howard C. Berg
79
Cytophaga strain 067 is a gram-negative, rod-shaped
organism about 0.5 µm in diameter and 4 µm long. The
cells glide singly on glass at speeds in excess of 1 µm/sec.
They move in the direction of their long axes, stop,
hesitate or back up, and occasionally pivot rapidly about
one pole (sometimes completing more than one
revolution). They actively propel polystyrene latex
spheres along their surfaces. We have used video
techniques to analyze these maneuvers quantitatively.
Our data are consistent with a model in which sites, to
which glass and polystyrene strongly adsorb, move within
the fluid outer cell membrane along tracks fixed to the
rigid peptidoglycan framework (Lapidus and Berg, 1982).
The coupling between the sites and the tracks is rigid, not
viscous. We do not know how the sites are propelled.
Reference: Lapidus, I. R. and Berg, H. C. (1982) J. Bacteriol., in
press.
107. THE PHOTOTACTIC RESPONSE OF CHLAMYDOMONAS
Investigator: Robert D. Smyth
The microscopic green alga Chlamydomonas swims by
doing a breast stroke with its two nagella. The motile
cells are phototactic, that is, they can swim toward or
away from a source of light. The photoreceptor for this
response is associated with the eyespot, a small red
organelle located on the side of the cell. Light absorbed
by the receptor somehow alters the flagellar beat. By
using a mutant that has only one flagellum and spins in
place on a microscope slide, we found that either an
increase or decrease in light intensity can cause a
decrease in the frequency of the fiagellar beat without
altering its form (Smyth and Berg, 1982). We are
currently using high-speed cinematography to record the
effect of light on the flagellar beat of normal biflagellate
cells held in a micropipette. These experiments are being
done in collaboration with Professor Charles Brokaw. We
find, as expected, that the two flagella respond to light
differently.
Reference: Smyth, R. D. and Berg, H. C. (1982) Cell Motility, Suppl.
1, 211-215.
80
108. CHIASMA INTERFERENCE IN EUKARYOTIC ORGANISMS
Investigator: Robert D. Smyth
Chiasma interference is one of the oldest problems in
genetics. Although known for over 60 years, the physical
basis of the phenomenon remains a mystery. Chiasma
interference refers to the fact that when homologous
chromosomes of eukaryotic organisms pair at meiosis, the
crossovers that occur between the paired chromosomes do
not occur at random, but are more evenly spaced.
Following a suggestion of Cobbs (1978), Kenneth Foster
(Department of Pharmacology, Mt. Sinai School of
Medicine, New York, New York) and I have developed
computer programs for the statistical analysis of genetic
data based on the assumption that crossing over is a
stationary renewal process, and that the distances
between crossovers follow a gamma probability distribu
tion. A single parameter, K, provides a measure of the
intensity of chiasma interference in different organisms.
The theory adequately predicts the frequency of all
genetic products recovered in crosses involving four to
seven linked markers in Aspergillus, N eurospora,
Drosophila, and Chlamydomonas. The theory suggests a
model of crossing over in which homologous chromosomes
pair at randomly selected sites along the chromosome, but
in which approximately K paired sites adjacent to a
crQssover are excluded from crossing over.
Reference: Cobbs, G. (1978) Genetics 89, 563-581.
109. THE AVOIDANCE RESPONSE IN PHYCOMYCES
Investigator: Paul W. Meyer
A Phycomyces sporangiophore (spph) bends away from
any object placed within a few millimeters of its growing
Professor: Charles J. Brokaw Researeh Fellows: Charlotte K. Omoto, Lee K. Opresko Graduate Student: David A. Myers Research Staff: Pui Ho, Sandra M. Nakada
Support: The work described in the following research reports has been supported by the National Institutes of Health, USPHS.
Summary: Evidence has accumulated that indicates that
the bending movements of cilia and flagella are generated
by a sliding microtubule process, similar to the sliding
zone. What mechanism does the spph use to detect the
object (or barrier)? We believe that an inert, diffusible
substance, gas X, is emitted by the spph. Gas X is
converted on the surface of the object to a growth
promoting, diffusible substance, gas Y. The concentration
of Y is greater on the side of the spph proximal to the
object than on the distal side, causing the spph to grow
faster on its proximal side and, thus, to avoid the object.
In order to study the avoidance response in the
diffusion limit, we have constructed a convection-free
chamber. Convection is suppressed by heating the top of
the chamber to a temperature of 0.05°C higher than that
at the bottom. The resulting density is stable:
magnesium oxide smoke particles suspended in the air
inside the chamber move with velocities less than
5 µm/sec, which is the measurement error.
Experiments in this chamber with barriers of different
chemical and gas-adsorbing properties are in progress.
They should tell us whether or not an spph really uses a
gas X to detect barriers, and, if so, what X might be.
PUBLICATIONS
Berg, H. C., Manson, M. D. and Conley, M. P. (1981) Dynamics and energetics of flagellar rotation in bacteria. Symp. Soc. Exptl. Biol. 35, in press.
Lapidus, 1. R. and Berg, H. c. (1982) Gliding motility of Cytophaga sp. strain U67. J. Bacteriol., in press.
Segall, J. E., Manson, M. D. and Berg, H. c. (1982) Signal processing times in bacterial chemotaxis. Nature, in press.
Smyth, R. D. and Berg, H. C. (1982) Change in flagellar beat frequency of Chlamydomonas in response to light. Cell Motility, Suppl.1, 211-215.
filament process responsible for muscle contraction.
Control mechanisms are needed to cause oscillatory
bending, to maintain the phase differences between
bending in different regions that are required for
propagated bending waves, and to determine the
parameters of particular bending patterns.
Our work makes particular use of ATP-reactivated
movements of demembranated sperm flagella as a source
of experimental data, and of computer programs that
simulate the movements of model flagella to relate
theoretical mechanisms to experimental data. We hope to
identify the types of control mechanisms that actually
exist in flagella and cilia, to understand how parameters
of movement such as frequency, wave length, and bend
angle are controlled, and to use this understanding to
enable detailed study of the active process that generates
sliding in flagella and muscle.
110. BENDING PAITERNS OF CHLAMYDOMONASFLAGELLA
Investigators: Charles J. Brokaw, David J. L. Luck*
The use of a mutant, uni-1, has enabled us to obtain
cells of Chlamydomonas that develop only one flagellum.
The normal asymmetric bending pattern of this flagellum
causes the cells to spin around a point on the microscope
slide and allows us to obtain good photographic records of
the flagellar bending pattern. We have used these
techniques to photograph a largeo-number of mutant strains
with altered bending patterns.
As previously reported, a mutant that is a combination
of a paralyzed mutant lacking radial spoke heads and a
suppressor mutation that restores motility without
restoring the ultrastructural defect or its associated
polypeptide deficiencies has an altered flagellar bending
pattern. In this pattern, large amplitude bends are
generated in both directions, in contrast to the normal
asymmetric bending pattern that has large bends in only
one direction.
This pattern has also now been found to be
characteristic of mutants lacking the central pair micro
tubules, also constructed by combining a paralyzed mutant
and a suppressor mutation. This pattern has also been
found to occur at a very low frequency in wild-tyi;>e cells
that are switching between the normal asymmetric
pattern and the Ca-dependent "reversal" pattern that
produces a symmetric, low amplitude, bending wave. The
reversal pattern appears to be identical in the two
mutants and in wild-type, but a detailed analysis of these
photographs has not yet been completed.
These observations suggest that the defects in the
radial-spoke/central pair system, which are correlated
with an inability to generate the normal asymmetric
bending pattern, may implicate a higher-level control
mechanism that is responsible for selecting from a
repertoire of possible bending patterns, rather than a
fundamental defect in the mechanism required to
81
generate asymmetric bending patterns.
Further analysis of asymmetric bending patterns on
wild-type cells and in other mutants confirms our first
impression that no "synchronous sliding" is associated with
generation of these asymmetric bending patterns. This
contrasts with the conclusion reached from earlier studies
on asymmetric bending patterns of demembranated sea
urchin sperm flagella. It now appears that the
synchronous sliding detected in those studies with sea
urchin sperm flagella may be an artifact resulting from
the erroneous assumption that the orientation of the
sperm head is a valid indicator of the orientation of the
basal end of the flagellum.
*The Rockefeller University.
111. ACTIVATION OF NON-MOTILE FLAGELLA
Investigators: Lee K. Opresko, Charles J. Brokaw
We previously reported that spermatozoa of the
tunicate, Ciona, when diluted directly into a Triton
demembranation solution and subsequently exposed to
MgATP, did not show reactivated motility. Reactivated
motility was only obtained when the spermatozoa were
activated to become motile in seawater before
demembranation. This activation can be induced by
theophylline, but not by permeable cAMP analogs such as
8-bromo-eAMP. We have now found conditions where a
partial activation of demembranated flagella can be
obtained with cAMP.
We would like to know why non-activated axonemes
are unable to generate spontaneous bending movements,
even though the activity of their active sliding mechanism
can be demonstrated after brief exposure to trypsin. An
obvious direction is to look for a cAMP-dependent
phosphorylation of axonemal components correlated with
the activation of motility. We find that under conditions
where cAMP gives partial activation of motility, there is
a large (5-10 fold) enhancement of phosphorylation of
flagellar proteins, in both the Triton-solubilized fraction
and the insoluble flagellar fraction. Autoradiograms of
one-dimensional gels show many phosphorylated bands in
both fractions. However, we do not yet have good
evidence for a correlation between the ability to generate
spontaneous movement in the presence of ATP and the
phosphorylation of any specific axonemal components.
Although the use of Ciona spermatozoa for these
experiments is attractive because of the clear-cut
82
distinction between activated and non-activated
axonemes, theY may turn out to be a poor choice for
biochemical study, because the sperm heads are very
small in this species, and very difficult to separate from
the flagellar axonemes.
112. MOVEMENT OF SPERM FLAGELLA WITH AND WITHOUT A TERMINAL FILAMENT
Investigators: Charlotte K. Omoto, Charles J. Brokaw
Light and electron microscope observations of Ciona
intestinalis and Lytechinus pictus spermatozoa show a thin
terminal filament at the distal end. The internal
structure of this terminal filament is composed of two
central tubules and a small number of A subfiber
extensions of the peripheral doublets. Photographs of the
movement of beating spermatozoa do not show any
obvious discontinuity in curvature at the transition region
between the 9+2 axoneme and the thinner terminal
filament. However, spermatozoa in which the terminal
filament has been removed show a clear "end effect."
This end effect involves a rapid unbending of bends that
have reached the distal end of the flagellum. Computer
simulations of flagellar models lacking a terminal
filament show a similar end effect.
When a terminal filament is present at the end of a
flagellum, bends propagate smoothly off the end of the
fiagellum, with no decrease in curvature. Addition of a
tapered terminal filament to the end of the computer
model can eliminate the end effect and give realistic
bending behavior. These computations give an estimate
for the bending resistance of the major portion of the
terminal filament of 0.03 x 109 pN nm2. This leads to
estimates of 0.01 x 109 pN nm2 for the elastic bending
resistance of an individual microtubule, and 0.2 x 109
2 pN nm for the elastic bending resistance of the 9+2
portion of the flagellum. This estimate for the value of
the bending resistance has a simpler basis than previous
estimates.
113. SULFATE INHIBlTION OF FLAGELLAR MOTILITY
Investigators: David A. Myers, Charles J. Brokaw
A slight inhibition of the beat frequency of reactivated
fie.gella by sulfate was noticed several years ago, in
comparing reactivation in solutions in which Mg +2 was
supplied by MgC12 or by MgS04• We have now found that
the inhibitory effect is caused by the MgSO 4 species
-2 rather than by SO 4 , and that MgSO 4 is a competitive
inhibitor of beat frequency. This is interesting because
MgSO 4 is an uncharged species, while the normal sub
strate, MgATP-2, and other competitive inhibitors such as - -3 -4 MgADP , ADP , or ATP , are all charged species.
114. MONOCWNAL ANTIBODIES TO ALPHA TUBULIN
Investigators: David J. Asai*, Charles J. Brokaw
Four monoclonal anti-tubulins-Abl, Ab2, Ab3, and
Ab4, all reactive for alpha tubulin and not reactive for
beta tubulin-have been characterized in terms of their
relative binding to tubulins from different sources in a
solid-phase assay and in an immunoautoradiographic
analysis. These methods revealed several differences in
relative reactivities: Ab2 bound better than Abl to
tubulins from bovine brain, chick brain, and sea urchin
sperm flagella. Although Ab2 bound better than Abl to
flagellar tubulin, this relationship was reversed when the
antibodies were applied to sea urchin egg tubulin; Abl
bound better than Ab2 to egg tubulin. Both Abl and Ab2
recognized bovine brain tubulin; however, only Ab2 also
bound bull sperm tubulin. Ab3 gave a similar pattern to
Ab2. Ab4 was relatively unreactive. Indirect immuno
fluorescence further revealed differences between Abl
and Ab2: both antibodies stained microtubules in chick
primary fibroblasts but only Abl was capable of staining
PtK2 microtubules. These results demonstrate two things:
tubulins isolated from various sources, and even from
different tissues within the same species are not identical;
and at least two of these monoclonal anti-alpha tubulins,
Abl and Ab2, are not identical. In reactivation experi
ments, both Abl and Ab2 selectively and potently
inhibited bend amplitude, without affecting beat
frequency of reactivated sea urchin spermatozoa; this
amplitude inhibition rapidly progressed until the axonemes
were completely paralyzed. Monoclonal anti-alpha
tubulin, at concentrations in excess of those necessary to
paralyze the reactivated flagella, did not inhibit outer
doublet microtubule sliding in elastase-digested axonemes.
Anti-tubulin antibodies may be preventing normal
flagellar bending by binding to the surfaces of outer
doublet microtubules and preventing tubulin subunit
rearrangements that are an essential component of the
mechanism controlling flagellar bending.
*Department of Biological Sciences, University of California, Santa Barbara.
PUBLICATIONS
Asai, D. J., Brokaw, C. J., Harmon, R. C. and Wilson, L. (1982) Monoclonal antibodies to tubulin and their effects on the movement of reactivated sea urchin spermatozoa. Cell Motility, Suppl. 1, 175-180.
Asai, D. J., Brokaw, C. J. and Wilson, L. (1981) Monoclonal antibodies to tubulin and their effects on the reactivated movement of sea urchin spermatozoa. J. Cell Biol. 91, 45a.
Brokaw, C. J. (1982) Activation and reactivation of Ciona spermatozoa. Cell Motility, Suppl. 1, 185-189.
Brokaw, C. J. (1982) Generation of the bending cycle in cilia and fiagella. Cell Motility, Suppl. 1, 137-141.
Brokaw, c. J. (1982) Models for oscillation and bend propagation by flagella. Symp. Soc. Exptl. Biol. 35, in press.
Brokaw, C. J., Luck, D. J. L. and Huang, B. (1982) Analysis of the movement of Chlamydomonas nagella: The function of the radial spoke system is revealed by a comparison of wild-type and mutant nagella. J. Cell Biol. 92, 722-732.
Brokaw, C. J. and Omoto, C. (1982) The terminal piece of Ciona sperm fiagella. Biophys. J. 37, 284a.
Professor: John J. Hopfield Weizm8Jlll Research Pellow: Noam Agmon* Research Pellow: George Geller*
SUpport: The work described in the following research reports has been supported by:
National Science Foundation Weizmann Fellowship
*Division of Chemistry and Chemical Engineering, California Institute of Technology.
summary: The general area of interest is the chemical
physics of how biological processes take place. We
attempt to abstract from biology important chemical and
physical properties; to understand, through theory and
experiments, how these properties may come about in
terms of structure, dynamics and context; and to then
take these results back into biological systems to test our
findings. Areas in which studies have been made include:
the molecular mechanisms of cooperative ligand binding in
heme proteins; the mechanism of biological electron
transfer processes; the problem of accuracy in the
synthesis of biological molecules-its limits, mechanisms
to enhance accuracy, and its origin; and emergent
properties of interacting neurons.
115. EMERGENT PROPERTIES OP NEURAL NETWORKS
Investigator: John J. Hopfield
Emergent or collective properties of systems are
83
Brokaw, C. J. and Verdugo, P. (Eds.) (1982) Mechanism and Control of Ciliary Movement. Alan R. Liss, Inc., New York.
Luck, D. J. L., Huang, B. and Brokaw, C. J. (1982) A regulatory mechanism for nagellar function is revealed by suppressor analysis in Chlamydomonas. Cell Motility, Suppl. 1, 159-164.
Okuno, M., Asai, D. J., Ogawa, K. and Brokaw, c. J. (1982) Effects of antibodies against dynein and tubulin on the stiffness of flagellar axonemes. J. Cell Biol. 91, 689-694.
Okuno, M. and Brokaw, c. J. (1982) Calcium-induced change in form of demembranated sea urchin sperm fiagella immobilized by vanadate. Cell Motility 1, 349-362.
Okuno, M. and Brokaw, c. J. (1981) Effects of Tritonextracted conditions on beat symmetry of sea urchin sperm fiagella. Cell Motility 1, 363-370.
Okuno, M. and Brokaw, c. J. (1981) Effects of AMPPNP and vanadate on the mechanochemical crossbridge cycle in fiagella. J. Muscle Res. and Cell Motility 2, 131-140.
behaviors which come about through the interactions of
many similar objects, and which are not displayed or
directly anticipated from the properties of very small
systems. Large neural systems may display such emergent
properties. The sense of the now of time, the intactness
of memories, and attention are plausibly emergent
properties. We model systems of 30-1000 neurons to
examine what biologically useful collective properties
spontaneously arise. Several such properties do occur of
which the most obvious is the interaction between
different aspects of a single memory. In the model
system, this results in the ability of the network to
reconstruct all of a particular memory from any sub-part
sufficiently large to distinguish that memory from other
stored memories. The storage of individual memories is
broadly distributed in many synapSes, and the destruction
of a small number of synapses or neurons has little effect
on memories. An extension of these ideas to memories of
time sequences and problems with spatial extent is being
pursued.
116. THE DYNAMICS OP CO BINDING TO HEME PROTEINS
Investigators: Noam Agmon, John J. Hopfield
The kinetics of ligand binding to heme proteins
depends on the structure of the protein. While at high
84
temperatures, the protein structure fluctuates rapidly and
the rate of binding is averaged over these fluctuations; at
low temperature the fluctuations are very slow, and
binding takes place for a distribution of different struc
tures. We have constructed a detailed energy surface
model on which to calculate the dynamics of ligand
binding, and have used it to describe some of the
experiments of Hans Frauenfelder and coworkers at the
University of Illinois, Urbana. The theory unifies a
considerable body of experiments in a simple structure
based model, and has predicted results for a new class of
experiments which have yet to be tried on the basis of the
old results.
117. ELECTRON TRANSFER PROCESSES
Investigators: David N. Beratan•, George Geller, Alvin D. Joran•, Peter Dervan•, Jolm J. Hoptield
A major limitation to the understanding of the
electron transfer processes control to bioenergetics has
been the absence of detailed structure for any biological
system of known electron transfer properties. Feeling
that we understand the general constructs which must be
Associate Professor: Elias Lazarides Visiting Associate: Yves Denis Plancke Senior Research Pellow: Ignacio V. Sandoval Gosney Research Pellow: Yassemi Capetanaki Research Fellows: Ingrid Blikstad, Lars Carlsson, Camilo
A. L. S. Colaco, David L. Gard, Maureen G. Price, Elizabeth A. Repasky
Graduate Students: Richard H. Gomer, Bruce L. Granger, John J. Ngai, Chung Wang
Research Staff: Adriana Cortenbach, Ilga Lielausis Laboratory Staff: Margaret M. Griffith
Support: The work described in the following research reports has been supported by:
American Heart Association Biomedical Research Support Grant (NIH) British Heart Foundation Centre National de la Recherche Scientifique The Camille and Henry Dreyfus Foundation, Inc. European Molecular Biology Organization E. S. Gosney Fund Muscular Dystrophy Association of America National Institutes of Health, USPHS National Science Foundation Sweden's National Science Foundation
Summary: Understanding of the structural complexity of
the cell's cytoplasm has taken a new turn with the
essential to the occurrence and control of electron
transfers in bacterial photosynthesis, we have embarked
on the synthesis and physical study of a series of electron
transfer molecules. The design of those molecules
incorporates the ideas believed essential to the charge
separation process. The first target molecule is
0
0
The electron transfer rate for this particular molecule is
also being calculated theoretically.
*Division of Chemistry and Chemical Engineering, California Institute of Technology.
PUBLICATIONS
Hopfield, J. J. (1982) Neural networks and physical systems with emergent collective computational abilities. Proc. Nat. Acad. Sci. USA '19, 2560-2564.
Redi, M. H., Gerstman, B. S. and Hopfield, J. J. (1981) Hemoglobin-CO binding rate. Biophys. J. 35, 471-484.
Yamane, T., Miller, D. L. and Hopfield, J. J. (1981) Discrimination between D- and L-tyrosyl tRNA in peptide chain elongation. Biochemistry 20, 7059-7064.
realization that in addition to actin filaments and micro
tubules, it contains a class of filaments, known es
intermediate filaments. Recent studies in our laboratory
have indicated that what is uniquely different about
intermediate filaments is that their structure, polypeptide
composition and cytoplasmic associations change during
differentiation and are different in various differentiated
tissues. The subunits of intermediate filaments in various
tissues are structurally related. In order to understand
their structural homology and evolutionary divergence, as
well as to study their expression in different tissues, we
have begun preparing cDNA probes for the subunits
expressed specifically in muscle, glial cells, erythrocytes
and neurons with the ultimate goal of isolating their
corresponding genes.
We have developed two model cell systems to under
stand the function and expression of the subunits of
intermediate .filaments: chicken erythrocytes and chicken
skeletal muscle. New techniques for the visualization of
the cytoplasmic distribution of these filaments in these
cell types have revealed that in the avian erythrocyte
these filaments form a three-dimensional network inter
linking the nucleus and the plasma membrane. Similarly,
in skeletal muscle these filaments interact with the
myofibril Z discs at their peripheries interlinking them to
each other and to the plasma membrane. These studies
have provided a clear example of the existence of a
transcytoplasmic integrating matrix in differentiated
higher eukaryotic cells. Both chicken erythrocytes and
skeletal muscle provide ideal systems where we can
investigate the regulation of the assembly of this matrix.
One approach is to investigate the function of proteins
that are associated with these filaments. We have
characterized two such high molecular weight proteins,
synemin and paranemin. What is intriguing about these
two proteins is that their expression varies in different
cell types and in different stages of differentiation. Thus,
apparently their expression is linked to the physiological
function of the filaments. One of them, synemin, appears
to function to crosslink intermediate filaments to each
other in avian erythrocytes. The degree of crosslinking of
the filaments as mediated by synemin appears to change
with differentiation and we are currently trying to
understand what regulates the crosslinking of the
filaments during differentiation and if such physiological
roles of these high molecular weight proteins determine
their expression. Response of those filaments to physio
logical stimuli has come from our discovery that two of
their major subunits, desmin and vimentin, are phos
phorylated in muscle cells by the cAMP-dependent protein
kinases and that the cytoplasmic levels of cAMP may
regulate their associations with various cytoplasmic
structures. Chemical analysis of the phosphorylation sites
on these two molecules has shown that they contain both
common and distinct sites which are differentially
modulated by cAMP; these observations have suggested
for the first time that these two filament subunits can
respond differentially to physiological stimuli and that
such a differential response may be one of the deter
mining factors behind their expression in different cell
types.
In trying to understand how intermediate filaments
interact with the plasma membrane, biochemical
characterization of this interaction with the erythrocyte
plasma membrane has indicated that this interaction is
most likely indirect via some component of the spectrin
actin network that underlies the erythrocyte membrane.
85
We have sought to find analogues of spectrin in non
erythroid cells since thus far this protein has been thought
of as being specific to the erythrocyte. We have
succeeded in showing that at least one form of spectrin,
a-spectrin, is expressed in non-erythroid cells. The
biochemistry behind its interaction with intermediate
filaments is currently being worked out.
Finally, we are developing monoclonal antibodies to
probe structural changes in the filaments during differ
entiation as well as to discover other proteins that may
modulate their function. In particular we would like to
understand further what regulates their association with
the Z disc during muscle differentiation.
118. STRUCTURAL ANALYSIS OF DESMIN AND VIMENTIN GENES
Investigator: Yassemi C&petanakl
Studies on the intermediate filaments of many higher
eukaryotic cells have shown that their subunits share a
number of properties including similar morphological
characteristics and polymerization properties in vitro.
However, on the basis of a number of biochemical and
immunological criteria, we can distinguish five major
classes of subunits within this class of filaments, each of
which is preferentially or exclusively expressed in various
differentiated cell types.
The two major subunits expressed in various types of
muscle are desmin and vimentin; some types of muscle
cells express predominantly desmin, others express
predominantly vimentin, and still others express
simultaneously varying ratios of the two polypeptides. To
explain these observations, previous work from this
laboratory has led to the conclusion that desmin and
vimentin share regions of amino acid sequence homology
and regions of amino acid sequence divergence
responsible, respectively, for the conserved properties of
the molecules and their presumed unique functions in a
given muscle cell type. In addition, the regulation of the
expression of these two proteins differentially in various
muscle types and their evolutionary conservation are also
unknown. As a major step in beginning to obtain an
answer to these questions,_ it is obviously necessary to
investigate the organization of these proteins at the DNA
level. For this purpose, a chicken cDNA library was
constructed from 10-day-old gizzard poly(A)+ RNA using
pBR322 as a vehicle. This library was screened for desmin
using two different 32P-cDNA probes reverse-transcribed
86
from gizzard poly(At RNA fractions obtained by
formamide-sucrose gradients. The one fraction was
enriched in desmin mRNA sequences and the other was
desmin-depleted. Further screening will be done using
desmin-specific and vimentin-specific synthetic oligo
nucleotides and the positive clones will be verified by
selection-hybridization assay and nucleotide sequencing.
The identified clones will be further used for studies of
desmin and vimentin expression during myogenesis, as well
as to isolate genomic clones and study their structure and
evolution.
119. llOLATION OP NEUROPILAMENT PROTEIN AND GLIAL PlBRlLLARY ACIDIC PROTEIN eDNAs
Investigator: Jolm J. Ngai
The relationships between the intermediate filament
subunits pose a fascinating biological problem. By many
biochemical and immunological criteria, these proteins
exhibit clear homologies, yet they show diversity as well.
It is possible that the similar polymerization properties
and subunit organization manifested by these proteins are
attributable to conserved regions of these proteins,
whereas presumed tissue- or cell-specific functions are
effected ·by the regions of divergence. In this study, I am
concentrating on two intermediate filament proteins
found in the chicken central nervous system: the 70
kilodalton (kd) neurofilament core po!Ypeptide, and the
major subunit of glial filaments, glial fibrillary acidic
protein (GFAP).
Protein sequencing data of others (Geisler and Weber,
1981; Geisler et al., 1982) have demonstrated .r70%
sequence homology between the carboxy-terminal one
third of desmin and vimentin, and a lesser degree of
similarity (.r40%) between small regions (42 amino acids)
of vimentin (or desmin) and the 70 kd neurofilament
protein. In order to better understand the relationships of
the intermediate filament proteins to one another, it is
necessary to examine the structures of their respective
genes and mRNAs. To this end, I have started by
constructing a cDNA library from chicken spinal cord
poly(A)+ RNA. Fractionation of this RNA by preparative
methylmercury hydroxide agarose gel electrophoresis and
analysis by rabbit reticulocyte lysate in vitro translations
reveal size classes of RNA enriched in either GFAP or
70 kd neurofilament protein messenger activity.
Fractions enriched for the appropriate mRNA activities
are being used as substrates for reverse transcriptase to
generate 32P-labeled cDNA probes. Candidate clones
identified by screening the cDN A library with these
probes will be further analyzed by positive hybrid-selected
translation. The resulting clones for neurofilament 70 kd
protein and GFAP will be sequenced and used to elucidate
the genomic organization of these genes. These data will
be compared to those obtained for desmin and vimentin
(see Abstract No. 118), ultimately to give a broader
understanding of the diversity and evolution of the
intermediate filament protein family.
References: Geisler, N. and Weber, K. (1981) Proc. Nat. Acad. Sci.
USA 78, 4120-4123. Geisler, N., Plessemann, U. and Weber, K. (1982) Nature
296, 448-450.
120. CYCLIC AMP-MODULATED PHOSPHORYLATION OF INTERMEDIATE FILAMENT PROTEINS IN CULTURED AVIAN MYOGENIC CELLS
Investigator: David L. Gard
The intermediate filament proteins, desmin and
vimentin, and the muscle tropomyosins are the major
protein phosphate acceptors in 8-day-old myotubes
incubated 4 hours in medium containing radiolabeled
phosphate. Addition of isoproterenol or 8-bromo-cyclic
AMP (BrcAMP) results in a two- to threefold increase in
incorporation of 32PO 4 into both desmin and vim en tin,
while no changes in incorporation of 32eo 4 into tropo
myosin or other cellular proteins are observed. The
BrcAMP-induced or hormonally-induced increase in 32Po4 incorporation into desmin and vimentin is independent of
protein synthesis, and is not due to stimulation of protein
phosphate turnover. In addition, BrcAMP does not induce
significant changes in the specific activity of the cellular
ATP pool. These data suggest that the observed increase
in 32PO 4 incorporation represents an actual increase in
phosphorylation of the intermediate filament proteins,
desmin and vimentin. Two-dimensional tryptic analysis of
desmin from 8-day myotubes reveals five phosphopeptides,
of which two show a seven- to tenfold increase in 32PO 4 incorporation in BrcAMP-treated myotubes. Four of the
phosphopeptides identified in desmin labeled in vivo are
also observed in desmin phosphorylated in vitro by bovine
heart cAMP-dependent protein kinase. Although phos
phorylation of desmin and vimentin is apparent in
myogenic cells at all stages of differentiation, BrcAMP
and isoproterenol-induced increases in phosphorylation of
these proteins are restricted to mature myotubes. These
data strongly suggest that in vivo phosphorylation of the
intermediate filament proteins desmin and vimentin is
catalyzed by the cAMP-dependent protein kinases and
that such phosphorylation may be regulated during muscle
differentiation.
121. A POSSIBLE ROLE FOR SYNEMIN REVEALED BY IMMUNOELECTRON MICROSCOPY
Investigator: Bruce L. Granger
Synemin is a 230,000 dalton polypeptide associated
with desmin filaments in smooth muscle, with desmin and
vimentin filaments in skeletal muscle, and with vimentin
filaments in avian erythrocytes (Granger and Lazarides,
1980; Granger et al., 1982). The avian erythrocyte has
been adopted as a model system for the study of
intermediate filaments because of its relative simplicity.
A combination of techniques has recently allowed ultra
structural visualization of previously unrecognized aspects
of the erythrocyte cytoskeleton (Granger et al., 1982):
cells are attached to a cationized substrate, hypotonically
lysed, broken open with a sonicator, fixed, dehydrated,
and shadowed at a low angle with platinum. The resulting
platinum replicas are stabilized with a layer of carbon,
removed from the substrate, and examined in a trans
mission electron microscope. If the disrupted cells are
incubated with specific antibodies prior to fixation or
dehydration, corresponding structures are "decorated."
This approach has revealed that vimentin, the
predominant component of the intermediate filaments in
these cells (Granger et al., 1982), indeed forms the core
polymer. Synemin, on the other hand, is present at
regularly spaced intervals along the filament axis.
Measurement of this synemin periodicity under a specified
set of sample preparation conditions has given average
values of 180 ± 40 nm (mean ± S.D.; n = 1220) for adult
erythrocytes and 230 nm ± 50 nm (n = 811) for 10-day
embryonic erythroid cells, suggesting some fundamental
change in the structure of the filaments during erythro
poiesis. Since synemin itself is not identifiable on the
filaments, the observed focal anti-synemin decoration
pattern suggests that synemin may normally exist in an
extended rod conformation along the axis of the vimentin
core, yet be either antigenically masked or non
immunogenic along most of its length. Registration of the
decorated synemin foci in laterally associated filaments,
and decoration of bridges between slightly separated
filaments, suggest that synemin mediates crosslinking of
87
intermediate filaments through self-interaction. Synemin
may play a similar role in muscle cells, where it is present
in similar relative amounts. The importance of this
crosslinking activity to intermediate filament structure
and function, and the mechanism by which this activity
might be regulated are currently under investigation.
References: Granger, B. L. and Lazarides, E. (1980) Cell 22, 727-738. Granger, B. L., Repasky, E. A. and Lazarides, E. (1982) J.
Cell Biol. 92, 299-312.
122. BIOCBEMICAL CHARACTERIZATION OF TBE INTERMEDIATE FILAMENT ASSOCIATED PROTEIN, SYNEMlN
Investigators: Camilo A. L. S. Colaco, Ignacio V. Sandoval
One approach to the elucidation of the molecular and
functional nature of intermediate filaments is the
development of an in vitro system in which their assembly
and disassembly can be studied. This in turn requires the
purification and characterization of their molecular
constituents that can then be used in reconstitution
studies to probe their functional characteristics. With
this aim in mind, we have purified and characterized
biochemically a protein component, synemin, associated
with desmin- and vimentin-containing filaments. Synemin
is a protein of molecular weight 230,000 and migrates in
analytical ultracentrifugation and column chromatography
as a globular tetramer. The amino acid content of
synemin is rather acidic and the protein has a pl of 5.34.
Synemin has a high content of serine and also incorporates
phosphate into the serine residues in a cAMP-dependent
manner. The purified synemin has no effect on the rate or
extent of desmin polymerization, although it does bind
desmin and inhibits its immunoprecipitation. At present,
studies are being undertaken to elucidate further the
binding of synemin to desmin and a possible effect on the
type of polymers of desmin formed and the effect of
synemin on vimentin polymerization. The effect of the
phosphorylation on the interactions of synemin with
desmin and its effect on . the dynamics of the in vitro
assembled filaments are also being investigated.
123. ISOLATION OF A NEW ffiGH MOLECULAR wmGHT PROTEIN ASSOCIATED WITH DESMIN AND VlMENTIN FILAMENTS FROM AVIAN EMBRYONIC SKELETAL MUSCLE
Investigators: Jennifer llreckler, Elias Lazarides
In order to investigate developmental changes in the
structure and composition of intermediate filaments, we
88
have developed techniques for their isolation from
embryonic muscle and have compared their composition
with filaments isolated from adult muscle tissue.
Filaments with a diameter of 80-120 X have been pre
pared from 14-<lay-old chick embryonic skeletal muscle,
using a physiological salt solution and gel , filtration
chromatography. The filaments obtained are composed of
the two known muscle intermediate-filament proteins,
vimentin and desmin, as well as the vimentin- and desmin
associated high molecular weight protein, synemin
(230,000 mo! wt). In addition, they contain a previously
unidentified high molecular weight protein (280,000
mol wt) which differs from synemin by isoelectric point,
molecular weight, and immunological reactivity.
Immunofluorescence on cultured myogenic cells, using
antisera to the 280,000-dalton polypeptide, has revealed
that this protein has the same spatial distribution as
desmin, vimentin, and synemin in both early myotubes,
where it associates with cytoplasmic filaments, and in
late myotubes, where it is associated with myofibril
Z lines. Examination by immunofluorescence of frozen
sections of developing embryonic skeletal muscle reveals
a gradual diminution in the presence of the 280,000-dalton
protein. The 280,000-dalton protein is undetectable in
adult skeletal and smooth muscle, as shown by immuno
n1:1orescence and immunoautoradiography. In chick
embryonic fibroblasts grown in tissue culture, only a
subpopulation of the cells is reactive with antibodies to
the 280,000-dalton protein even though all these cells
contain vimentin. In the reactive cells, vimentin and the
280,00D-dalton polypeptide exhibit an indistinguishable
cytoplasmic filamentous network, which aggregates into
filamentous bundles when the cells are exposed to
colcemid. These results suggest that this newly identified
high molecular weight protein is closely associated with
intermediate filaments containing either vimentin alone
or vimentin, desmin and synemin. The expression of this
protein appears to be developmentally regulated and does
not appear to parallel the expression of any of the other
three intermediate-filament proteins. The absence of the
280,000-dalton polypeptide in adult muscle cells and its
gradual reduction during development implies that it is
probably not required for the maintenance of Z-disc
structure after the assembly of the sarcomere.
124. CHANGES IN THE COMPOSITION OF INTERMEDIATE FILAMENTS DURING MUSCLE DEVELOPMENT
Investigator: Maureen G. Price
It has been established in this laboratory (Gard and
Lazarides, 1980) that the intermediate filaments are
redistributed during skeletal and cardiac muscle develop
ment. In early myogenesis, antibodies to desmin and
vimentin stain cytoplasmic filaments, while the same
antibodies stain the periphery of the Z discs in adult
muscle (Granger and Lazarides, 1979). Recently, we have
determined that the ratio of desmin to vimentin changes
during muscle development. Breckler and Lazarides
(1982) found that intermediate filaments isolated from
embryonic skeletal muscle are composed of desmin and
vimentin in a ratio of 2:3, whereas the salt-insoluble
residue of adult skeletal muscle contains those proteins in
a ratio of 2.5:1. Another major difference in the
composition of the embryonic and adult intermediate
filaments is the association of a high molecular weight
protein with the embryonic filaments. Antibodies to this
protein, called paranemin, fail to stain adult skeletal
muscle (Breckler and Lazarides, 1982).
The present study was designed to determine if the
pattern of expression of paranemin in early muscle
development, with its gradual loss, was requisite in all
muscle development. To this end, the intermediate
filament composition of developing smooth and cardiac
muscle was examined by immunofiuorescent staining of
frozen sections and immunoautoradiography of proteins
separated by gel electrophoresis, using antibodies to
desmin, vimentin, and paranemin. Another goal of this
study was to determine if the expression of paranemin is
linked t.o the expression of either desmin or vimentin, or
both. Therefore cells which synthesize only desmin
(gizzard smooth muscle) or only vimentin (Schwann cells
of peripheral nerve) were stained. The results are that
paranemin is expressed in embryonic visceral smooth and
cardiac muscle, but not in adult visceral muscle.
Paranemin persists in adult cardiac tissue, where it is
localized with desmin and vimentin at the Z discs.
Schwann cells and endothelial cells contain vimentin
filaments and paranemin throughout development. The
pattern that emerges is that paranemin is synthesized in
detectable amounts only in cells capable of synthesizing
either desmin or vimentin, or both proteins. Neurons and
epithelial cells do not contain paranemin, or desmin or
vimentin. The capacity to synthesize desmin or vimentin
does not guarantee synthesis of paranemin; synthesis of
paranemin ceases in desmin-containing adult visceral
smooth muscle and skeletal muscle. We propose that the
presence of paranemin indicates particular physiological
requirements of the cell. Continued research on the
vascular system, in which there is a gradient of expression
of paranemin and the intermediate-filament proteins, may
provide insight into the determinants of the expression of
paranemin.
References: Breckler, J. and Lazarides, E. (1982) J, Cell Biol. 92,
795-806. Gard, D. L. and Lazarides, E. (1980) Cell 19, 263-275. Granger, B. G. and Lazarides, E. (1979) Cell 18, 1053-
1063.
125. PLANAR ANISOTROPY IN THE AVIAN ERYTHROCYTE PLASMA MEMBRANE
Investigator: Bruce L. Granger
Avian erythrocytes are nucleated, biconvex, elliptical
discs. A coil of microtubu!es, known as the marginal
band, is present just inside the plasma membrane at the
cell's greatest circumference. Intermediate filaments
span from the nucleus to the plasma membrane. How
these cells (or any other cells) develop and maintain their
characteristic shapes and structural anisotropies is an
enigma. Studies of. the intermediate filaments of avian
erythrocytes (see Abstract No. 121) have revealed several
striking aspects of plasma membrane topography in these
cells, and have indicated that nucleated erythrocytes
might be an ideal system for the study of cellular
anisotropy.
The cytoplasmic surface of the avian erythrocyte
plasma membrane is lined with a cytoskeletal network of
proteins composed predominantly of spectrin, much as in
the anucleate mammalian erythrocyte (see Abstract
No. 126). Microtubules of the marginal band are closely
associated with this network. If the microtubules are
removed by depolymerization or sonication, ridges or
"tracks" remain on the membrane. There is apparently
one track for each microtubule in close apposition to the
membrane. These tracks were found to be positionally
stable, as if they were anchored in a non-dynamic spectrin
network. The tracks are absent from the membrane at
the poles of the cell, existing only along the blunt sides of
the elliptical disc.
89
The marginal band conceptually divides the erythro
cyte membrane into two symmetric, elliptical bowls. The
intermediate filaments do not attach uniformly to the
irlner surfaces of these bowls, but only to the region that
is more than about 1.5 micrometers from the rim. Thus,
there is a 3-micrometer-wide belt of plasma membrane
centered on the marginal band that is devoid of firmly
attached intermediate filaments.
Of interest here are the anisotropy of the microtubule
tracks and the intermediate filament anchorage points,
and their relationship to the dynamics of the plasma
membrane lipid bilayer and spectrin network. Determi
nation of how these anisotropies are established during
erythropoiesis, and how these systems interact to give the
red cell its characteristic shape, will have far-reaching
implications for the understanding of the regulation of
cell structure.
126. WIDESPREAD OCCURRENCE OF AVIAN SPECTRIN IN NON-ERYTHROID CELLS
Investigators: Elizabeth A. Repasky, Bruce L. Granger
Avian erythrocyte spectrin is composed of two high
molecular weight polypeptides designated " and a' with
solubility properties similar to mammalian erythrocyte
spectrin. We have prepared an antibody against chicken
a-spectrin, using as immunogen protein purified by two
dimensional polyacrylamide gel electrophoresis. One- and
two-dimensional immunoautoradiography shows that this
antiserum reacts only with a-spectrin in chicken erythro
cytes and crossreacts with a.-spectrin in erythrocytes from
various mammals. Immunofluorescence reveals that this
antiserum reacts with a plasma membrane component in
erythrocytes as well as in most non-erythroid avian and
mammalian cells. Frozen sections of nerve tissue and lens
tissue show intense staining. Positive staining is also seen
in endothelial cells and epithelial cells of the gastro
intestinal and respiratory tracts. Skeletal and cardiac
muscle as well as skeletal myotubes grown in tissue
culture show a similar staining at or near the plasma
membrane. Immunoautoradiography indicates that the
crossreactive antigen in these non-erythroid tissues has
the same molecular weight and isoelectric point as the
chicken erythrocyte antigen. Smooth muscle, tracheal
cilia, myelin and mature sperm appear to stain weakly or
not at all. These results suggest that the distribution of
spectrin is
Since a
more extensive than previously recognized.
cytoskeletal protein network composed
90
predominantly of spectrin is thought to be responsible for
maintaining the shape of and conferring structural
integrity to mature erythrocyte membranes, these obser
vations further suggest that the functions of spectrin thus
far elucidated for erythrocytes may be applicable to other
cell types as well.
127. CHARACTERIZATION OF SKELETAL MUSCLE FlLAMlN
Investigator: Richard H. Gomer
We have previously shown (Gomer and Lazarides, 1981)
using immunofluorescence and immunoautoradiography on
SD&-[>olyacrylamide gels that filamin exists on the stress
fibers of myoblasts and early fused myotubes, and then
disappears from these cells approximately one day after
cell fusion and before the appearance of Z-line striations
containing a-actinin. Several days later, filamin
reappears in the cells at the Z lines, shortly before desmin
and vimentin transit to the Z line. Using metabolic pulse
labeling with 35s-methionine, we were able to show that
the disappearance of filamin is in part a result of a
cessation of its synthesis.
We have used Staphylococcus aureus mediated
immunoprecipitation to isolate the filamins present in the
early and late stages of myogenesis and two-dimensional
peptide mll(>ping to compare them. Fi!amin purified from
chicken gizzard and filamins immunoprecipitated from
chick embryo fibroblasts and myoblasts all have the same
molecular weight while filamin immunoprecipitated from
cultured skeletal myotubes or purified adult skeletal
myofibrils has a molecular weight approximately 5000
daltons less than this. Immunoprecipitation of filamin
from myoblast and myotube cultures metabolically labeled
for 5 minutes with 35s-methionine show that myotube
filamin is synthesized as the lower molecular weight
variant. Two-dimensional peptide maps of filamins
labeled in vitro with 1251 and digested with three
different proteases, show that purified chicken gizzard
and chick embryo fibroblast filamins are virtually
identical, and very similar to myoblast filamin. Filamins
from cultured skeletal myotubes and skeletal myofibrils
are very similar to each other but are very different from
gizzard, fibroblast or myoblast filamins. The similarity of
fibroblast and myoblast filamins and their differences
from myotube filamin can also be seen using filamins
metabolically labeled in vivo with 35s-methionine. We
therefore conclude that myoblast and myotube filamins
are distinct gene products and that during myogenesis in
vitro, one class of filamin polypeptides is replaced by a
new class of filamin polypeptides and that the latter is
maintained into adulthood. We are currently purifying
skeletal muscle filamin so as to be able to compare its
biochemical properties, such as ability to bind actin, with
those of purified smooth muscle filamin.
Reference: Gomer, R. and Lazarides, E. (1981) Cell 23, 524-532.
128. THE METHYLATION OF HEAT SHOCK PROTEINS AT LYSYL AND ARGINYL RJ!SIDUES
Investigators: Chwig Wang
As described previously, certain heat shock proteins of
cultured chicken embryonic fibroblasts and cultured
mammalian cells are methylated (Biology 1981, No. 152).
Here we report the identification of the methylated amino
acids and some of the biosynthetic properties of this
methylation.
Using a combination of column chromatography and
thin-layer chromatography for amino acid analysis, we
found that lysyl residues are the predominant methylation
site(s) of 83,000 polypeptides and that both lysyl and
arginyl residues are methylated in the 68,000 [>Olypeptides
A and B. The majority of the methyl lysines has been
identified as E-N-trimethyHysine. E-N-mono- and E-N
dimethyl-lysines were also found as minor variants. The
major methylated arginine species was N°-monomethylarginine.
The stoichiometry of the methylation of these proteins
was determined by isolating the [>Olypeptides from cell
cultures which were grown in 3H-leucine and [methyl-3H]
methionine. It was estimated that each of the three
[>Olypeptides contains one to three methylated lysines. In
addition, there is one methyl arginine per molecule of
68 K polypeptides A and B. Nevertheless, in the presence
of protein synthesis inhibitors, the methyl groups incor
porated are substoichiometric; we estimated that less
than one methyl group is incor[>Orated per 100 molecules
under these conditions. The results suggest that the basic
amino acid methylation on these heat shock proteins
occurs during or soon after translation and there is little
turnover of the methyl groups. Indeed, the half-life of
methyl groups was estimated to be greater than 65 hours
with double labeling pulse-chase experiments.
The possible functional significance of the methylation
of these heat shock proteins is currently under
investigation.
129. THE EFFECT OF SODIUM ARSENITE ON TROPOMYOSIN PHOSPHORYLATION
Investigator: Chwig Wmig
Tropomyosin is one of the key proteins for regulating
muscle contraction. In skeletal muscle, it is composed of
two forms, a.- and a-tropomyosin, both of which are
phosphorylated. The functional significance of the two
forms and their phosphorylation are presently unknown.
We found that upon the addition of sodium arsenite to
tissue culture myotubes, the phosphorylation of tropo
myosin is specifically reduced. Upon reversal, it takes
two days for the cells to fully recover; thus the drug
effect is best characterized as slowly reversible. In
addition, we discovered that the sodium arsenite actually
facilitates the removal of phosphate from tropomyosin.
For example, the half-life of 32P phosphate in cx
tropomyosin is about 10 hours in control cells and is
reduced to less than four hours in sodium arsenite treated
cells. We have therefore concluded that the reduction of
tropomyosin phosphorylation is due to the fact that
sodium arsenite somehow activates a tropomyosin-specific
phosphatase system instead of inactivating a tropomyosin
specific phosphotransferase.
The possibility of using sodium arsenite treatment as a
tool to elucidate the function of phosphorylation of
tropomyosin is under investigation.
PUBLICATIONS
Breckler, J. and Lazarides, E. (1982) Isolation of a new high molecular weight protein associated with desmin and vimentin filaments from avian embryonic skeletal muscle. J. Cell Biol. 92, 795-806.
Professor: Jean-Paul Revel Visiting Associates: Daniel Gros, David J. Meyer Senior Research Fellow: S. Barbara Yancey Research Fellow: Cheryl M. Corsaro Graduate Student: Bruce J. Nicholson Research Staff: Jean Edens, Les B. Grim, Patrick F.
Koen
Support: The work described in the following research reports has been sup(X>rted by:
European Molecular Biology Organization National Institutes of Health, USPHS Gordon Ross Medical Foundation Albert Billings Ruddock Fund
91
Gard, D. L. and Lazarides, E. (1982) Cyclic AMPmodulated phosphorylation of intermediate filament proteins during myogenesis in vitro. Mol. Cell. Biol., submitted for publication.
Granger, B. L. and Lazarides, E. (1982) Structural associations of synemin and vimentin filaments in avian erythrocytes revealed by immunoelectron microscopy. Cell, submitted for publication.
Granger, B. L., Repasky, E. A. and Lazarides, E. (1982) Synemin and vimentin are components of intermediate filaments in avian erythrocytes. J. Cell Biol. 92, 299-312.
Lazarides, E. (1982) Intermediate filaments: a chemically heterogeneous, developmentally regulated class of proteins. Ann. Rev. Biochem. 51, in press.
Lazarides, E., Gard, D. L., Granger, B. L., O'Connor, C. M., Breckler, J. and Danto, S. 1. (1982) Regulation of the assembly of the Z disc in muscle cells. In: Proceedings of the International Congress of Developmental Biology. Basel, in press.
Lazarides, E. and Granger, B. L. (1981) The preparation and assay of desmin. In: Methods in Enzymology, Contractile Apparatus and Cytoskeleton, Vol. 85, L. W. Cunningham and D. R. Frederiksen (Eds.), pp. 488-508. Academic Press, New York.
Lazarides, E., Granger, B. L., Gard, D. L., O'Connor, c. M., Breckler, J., Price, M. and Danto, S. I. (1982) Desmin and vimentin containing filaments and their role in the assembly of the Z disc in muscle cells. Cold Spring Harbor Symp. Quant. Biol. 46, in press.
Repasky, E. A., Granger, B. L. and Lazarides, E. (1982) Widespread occurrence of spectrin in non-erythroid cells. Cell 29, 821-833.
Sandoval, I. V ., Colaco, C. A. L. S. and Lazarides, E. (1982) Purification of the intermediate filament associated protein synemin from chicken smooth muscle: Studies on its physicochemical properties, interaction with desmin and phosphorylation in vivo. J. Biol. Chem., submitted for publication.
Wang, C. W., Gomer, R. H. and Lazarides, E. (1981) Heat shock proteins are methylated in avian and mammalian cells. Proc. Nat. Acad. Sci. USA 78, 3531-3535.
Wang, c., Lazarides, E., O'Connor, C. M. and Clarke, S. (1982) Methylation of chicken fibroblast heat shock proteins at lysyl and arginyl residues. J. Biol. Chem., in press.
summary: My laboratory's continuing concern has been
the investigation of the structure of gap junctions. These
are membrane specializations through which neighboring
cells exchange low molecular weight substances, ions,
metabolites, or signaling molecules. In the central
nervous system they are called electrical synapses and in
the heart they play a major role in synchronizing the beat
of muscle cells. They are also found between many
unexcitable cells: while much remains to be learned about
their functions there, they allow metabolic cooperation
between cells, thus minimizing the effect of deleterious
92
mutations, and have been implicated in the control of
differentiation.
In the electron microscope, gap junctions are seen as
patches of "eonnexons," a single pair of which forms the
smallest possible transcellular channel. Each connexon is
probably a hexamer and contains one major protein
species in association with lipid. We have studied this
protein extensively in liver. We know that it turns over
very rapidly, and have obtained the amino acid sequence
of about 20% of the molecule. On the basis of these data,
we have been able to specify oligonucleotide sequences
that B. Yancey will use in isolating the genes which
specify for the gap junction protein(s). Partial sequence
for the protein extracted from a gap junction-like
structure in the lens of the eye, as well as comparison of
two-dimensional peptide fingerprints, suggests that gap
junctions in different tissues contain very different
proteins. Support for this idea comes from work by D.
Gros, a visitor from France. With B. Nicholson he isolated
a gap junction fraction from rat hearts in purer form than
had been done heretofore. To our surprise the two
dimensional peptide fingerprints suggest that heart
junctions are as different from liver as they are from lens.
It would thus seem that we must at present imagine that
each tissue has a characteristic gap junction protein, a
rather unexpected finding considering the fact that all of
these junctions are reasonably similar in appearance in
widely separated species and tissues. Gap junction
proteins appear to be reasonably well conserved however,
when the junctional protein in a given organ is compared
in different species.
We are now beginning to assemble a picture of how the
gap junction protein traverses the membrane, on the basis
of the amino acid sequences as well as other experiments
that suggest to us where various portions of the poly
peptide chain are located. We hope that, by combining a
molecular approach and genetic approach (C. Corsaro)
with morphological analysis, we will in the years to come
get an understanding of the organization of the connexon
and of the mechanisms by which its function is modulated
(D. Meyer).
130. TISSUE SPECIFICITY OP THE GAP JUNCTION PROTIDN
Investigators: Bruce J. Nicholson, Daniel Gros, Jean-Paul Revel
Although gap junctions have been identified in
virtually every metazoan phylum and in a wide variety of
different tissues, the characterization of their com
ponents (protein and lipid) has only been achieved in two
systems where the gap junctions are reasonably abundant
(i.e., liver and lens). As reported previously (Biology 1980,
No. 137), the junctional proteins from these two tissues
differ markedly, as judged from two-dimensional peptide
"fingerprints11 and partial, N-terminal sequencing. This,
and other morphological considerations (see Abstract
No. 136), have led to the claim that lens junctions may not
be gap junctions. It therefore was of importance to
characterize biochemically gap junctions in other tissues.
We have now identified and partially characterized a
single major protein of Mr 28,000 in gap junction fractions
isolated from rat heart (see Abstract No. 133).
In some ways, the heart and liver junctional proteins
appear similar and distinct from that of lens, thereby
apparently supporting the proposal that lens junctions are
not related to gap junctions elsewhere. The liver and
heart proteins comigrate in SDS-PAGE while the lens
junctional protein migrates slightly faster. When intact
junctional plaques are treated with trypsin (see Biology
1981, No. 153 for a detailed discussion), the junctional
proteins of heart and liver are reduced to polypeptides of
Mr 10,000, while the lens protein is only reduced to a
polypeptide of Mr 21,000. However, when the three
proteins were compared by two-dimensional peptide
"fingerprints" after tryptic or a-chymotryp~ic digestion,
the· liver, lens and heart proteins all showed very distinct
patterns. Any homology which might exist between the
proteins of these three tissues is certainly at the limits of
detection of the two-dimensional mapping system and is
only likely to be defined by more detailed knowledge of
the primary structures of the proteins. When junction
proteins from lens or liver from different mammalian
species were compared they showed very similar "finger
prints" (Biology 1981, No. 154).
These results shed no new light on the relatedness of
lens junctions with gap junctions, but they do raise the
previously unsuspected issue of diversity among the
proteins of gap junctions in different tissues. Currently,
research is being directed towards detecting any
homologies which may exist between these proteins and
defining their nature. An understanding of these
differences and similarities at the structural level could
be useful in determining structure-function correlations,
the evolutionary history of the gap junction gene and the
significance of this diversity with respect to tissue
differentiation.
131. STUDil!S TO IDENTIFY THE GENE CODING FOR THE GAP JUNCTION PROTEIN
Investigators: S. Barbara Yancey, Ellen B. Kraig, Les B. Grim, Jean-Paul Revel
The gap junction from rat liver is comprised of a single
major protein which has been isolated and characterized
biochemically and sequenced for 52 consecutive amino
acids beginning at the NH2-terminus (Nicholson et al.,
1981). From in vivo studies of its rate of turnover
(Yancey et al., 1981), the gap junction protein appears to
have a half-life of only a few hours. This suggests that
the mRNA that codes for the protein could be abundant
enough to be detected by the use of recombinant DNA
techniques. By constructing a cDNA library by cloning
ds-cDNA corresponding to total poly(A) RNA isolated
from rat liver and screening with synthetic oligo
nucleotides whose sequences have been deduced from the
amino acid sequence of the gap junction protein, we hope
to be able to characterize the mRNA for the protein, to
gain information about any post-translational modification
of the protein, and ultimately to be able to study the
organization, expression, and evolution of the gene that
codes for the protein.
Two mixtures of 14-base-long oligonucleotides repre
senting all the possible coding sequences predicted for
amino acids 1 to 5 and 44 to 48, respectively, have been
synthesized by Dr. Suzanna Horvath. We are now in the
process of constructing the cDN A library using the
plasmid pBR322 as vector and random priming with calf
thymus DNA. Each of the two synthetic oligonucleotide
mixtures will be used to screen the library for clones
whose cDNA codes for the known amino acid sequence of
the gap junction protein. To identify the gene, cDNA will
be used as well as the synthetic oligonucleotides as
hybridization probes to screen a rat liver genomic library
constructed at Caltech by James Bonner and his
associates.
References: Nicholson, B. J., Hunkapiller, M. w., Grim, L.B., Hood, L.
E. and Revel, J.-P. (1981) Proc. Nat. Acad. Sci. USA 78, 7594-7598.
Yancey, S. B., Nicholson, B. J. and Revel, J.-P. (1981) J. Supramolec. Struct. &: Cell. Biochem. 16, 221-231; Cellular Recognition, 215-226.
132. GENETIC ANALYSIS OP GAP JUNCTIONS IN CULTURED MAMMALIAN CELLS
Investigators: Cheryl M. Corsaro, Jean-Paul Revel
The isolation and analysis of gap junction-deficient
93
mutants (gap-) in differentiated cell lines in culture would
facilitate our understanding of the role of gap junctions in
growth and differentiation. We are isolating gap - mutants
in a liver epithelial cell line (BRL cells) because gap
junctions from liver have been characterized extensively
at both the morphological and biochemical levels.
The selective system involves the coculture of
tk -hprt- BRL cells (thymidine kinase--<leficient, hypo
xanthine guanine phosphoribosyl transferase-deficient)
with tk+hprt+ BRL cells in medium containing bromo
deoxyuridine and 6-thioguanine. The wild-type cells
metabolize the pyrimidine and purine analogues into toxic
nucleotides and transfer them through gap junctions to the
tk -hprt- cells. Any tk -hprt - cell which is gap - will be
resistant to this junctional transfer and survive. Two -7 clones have been isolated at a frequency of 2 x 10 after
mutagenesis with EMS. They are being characterized by
metabolic assays for nucleotide and ion transfer and by
freeze-fracture electron microscopy. The gap mutants
will be analyzed further by cell hybridization to determine + dominance or recessivity of the mutation, and gap
revertants will be isolated by culturing cells under
conditions where they must transfer nucleotides and ions
to survive.
133. THE ISOLATION OP GAP JUNCTIONS PROM RAT HEART
Investigators: Daniel Gros, Bruce J. Nicholson, Jean-Paul Revel
The isolation of gap junctions in sufficient quantities
and purity for biochemical analysis has until now been
restricted to liver and lens. However, by modifying the
previously published procedure of Kensler and Goodenough
(1980), we have simplified the isolation of gap junctions
from heart and produced fractions containing much
smaller amounts of nonjunctional material (e.g., desmo
somes). Furthermore, by using the highly sensitive
techniques of micro-polyacrylamide gels and two
dimensional peptide mapping, the protein components of
the fractions can be examined individually.
As described by Kensler and Goodenough (1980), 20 rat
hearts were homogenized and the myofilaments extracted
overnight in 0.6 M KI. The plasma membrane-enriched
fraction obtained from the KI-insoluble material by
separation on a discontinuous sucrose gradient was treated
with 0.3% N-lauryl sarcosine (Sarkosyl NL-97). This
material was loaded on a discontinuous sucrose gradient
94
containing 1 M urea and a trace of Sarkosyl, as described
for the isolation of liver gap junctions (Nicholson et al.,
1981). The final gap junction fraction, collected at the
40/54% (w/v) sucrose interface, contained between 2 and
15 µg of gap junctional protein.
Examination of this fraction by electron microscopy in
either thin-sectioned or negatively-stained samples
revealed gap junctions indistinguishable from those seen in
gap junction fractions from liver as the predominant
component. Some contamination by single membrane
vesicles, clumps of amorphous, fibrous material and
occasional strands of collagen was detected, although
there was no evidence for contamination by desmosomes
as reported by Kensler and Goodenough (1980). Analysis
of the fractions by SDS-PAGE revealed a mueh simpler
profile of polypeptides than that reported by the previous
investigators. A single major protein of molecular weight
(Mr) 28,000 was detected, although more variable com
ponents of Mr 50,000, 45,000 (a dimer), 34,000, 32,000 and
26,000 could be detected when gels were more heavily
loaded. All of these polypeptides, including that of Mr
28,000, show closely related patterns when examined by
two-dimensional peptide mapping, a technique which
provides a unique "fingerprint" for any protein. The
relationship between the components has yet to be
determined, although aggregation and proteolysis explain
some of the variability (cf. liver gap junction fractions,
Biology 1980, No. 136). However, it does appear that, as
is the case for liver and lens junctions, the heart gap
junctions are comprised of a single major protein.
References: Kensler, R. W. and Goodenough, D. A. (1980) J. Cell Biol.
86, 755-764. Nicholson, B. J., Hunkapiller, M. W., Grim, L.B., Hood, L.
E. and Revel, J.-P. (1981) Proc. Nat. Acad. Sci. USA 78, 7594-7598.
134. MODULATION OP GAP JUNCTION PERMEABILITY
Investigator: David J. Meyer
A large body of data suggests that gap junctions are
aggregates of intercellular channels (Meyer et al., 1981)
that can change their conductance in response to voltage
gradients and to changes in intracellular pH. We have
studied the modulation of junction permeability in the
mammalian liver, a tissue of special interest because it is
possible to isolate gap junctions from it in quantities large
enough for detailed biochemical and structural study.
In order to monitor conductance of the junctional
channels, we took advantage of the theoretical results
reported by Peskoff (1981). Hepatocytes separated by
about 50 µm were impaled with microelectrodes and the
voltage response resulting from a square wave stimulus
was recorded. The transient was fit with equation 21
from Peskoff (1981) using a nonlinear least square curve
fitting program. The program returned values of mem
brane resistivity (Rm), intercellular resistivity (Ri) and
membrane capacity (Cm) which yielded the best fit to the
data points. In liver superfu$ed with saline gassed with
95% oxygen and 5% co2, the average values in seven
experiments were Rm::;; 13,096 ncm2, and Ri::;; 3190 Qcm.
When the saline was switched to one gassed with 100%
co2 (a maneuver known to change intracellular pH in
other tissues), both membrane resistance and junctional
resistance increased in a time-dependent fashion. The
maximal increase in Ri was about sevenfold. All changes
were completely reversible on return to saline
equilibrated with 95% oxygen. Changes in extracellular
pH alone produced no effect on junctional or membrane
resistivity. We found that treatment with co2
also
reversibly impeded the spread of the fluorescent tracer
molecule, 6-carboxyfluorescein.
Further work will be necessary to demonstrate changes
in intracellular pH and to reveal the relationship between
intracellular pH and junctional conductance.
References: Meyer, D. J., Yancey, S. B. and Revel, J.-P. with an
Appendix by Peskoff, A. (1981) J. Cell Biol. 91, 505-523.
Peskoff, A. (1981) J. Cell Biol. 91, 519-523.
135. ORIENTATION OF THE LENS JUNCTIONAL PROTfilN IN THE MEMBRANE
Investigators: Bruce J. Nicholson, Michael W. Hunkapiller, Jean-Paul Revel
Exhaustive digestion of isolated gap junction sheets
with trypsin yields two polypeptides of Mr 10,000 in the
case of liver, but a single polypeptide of Mr 21,000 in the
case of lens junctions. NH2-terminal sequence analysis of
the Mr 10,000 polypeptides from liver has shown that the
NH2-terminus of the original Mr 28,000 protein is pro
tected from trypsin attack (Biology 1981, No. 153). As
previously argued (Biology 1981, No. 153), the size
(minimum diameter 50 !) and water solubility of trypsin
make it unlikely that any proteolysis would occur inside
the membrane, within the putative 15 1 diameter pore, or
in the 20-40 X gap between membranes. The stability of
the double membrane structure of gap junctions following
trypsin treatment would seem to confirm that proteolysis
is restricted to the cytoplasmic faces of the junction.
We have recently sequenced the trypsin-resistant Mr
21,000 polypeptide of lens junctions. As is the case for
the liver gap junction protein, trypsin largely removes
residues from the C-terminal end of the original Mr
26,000 protein. However, the lens Mr 21,000 protein has
also lost the five NH2-terminal residues of the original Mr
26,000 protein, although a second tryptic cleavage site 11
residues from the NH2-terminus of the Mr 26,000 protein
remains untouched. These results would suggest that both
the c- and NH2-terminal ends of the lens junctional
protein are exposed at the cytoplasmic surface of the
junction. Furthermore, we may have identified the point
(to within five residues) where the protein enters the lipid
bilayer at the NH2-terminus of the molecule. Although it
is clear that the liver gap junction protein also has its-C
terminus exposed at the cytoplasmic face of the junction,
the orientation of the NH2-terminus in the liver cannot be
determined with certainty until the effect of enzymes
other than trypsin are tested, since there is no tryptic
cleavage site very close to the NH2-terminus.
136. A NEUTRON DIFFRACTION STUDY OF LENS JUNCTIONS
Investigators: Bruce J. Nicholson, Shahid M. M. Khan, Edward Gogol*, Les B. Grim, David J. Meyer, Donald Engelman•, Jean-Paul Revel
Gap junctions can be identified between two cells as
an area of closely packed (frequently in a hexagonal
lattice) intramembrane particles (connexons) in regions
where the plasma membranes of adjacent cells come into
close apposition yet remain separated by a uniform gap of
2-4 nm. They have been strongly implicated as the
mediators of electrical coupling and the transfer of low
molecular weight compounds (Mr <1000) directly between
cells. Structural and electrophysiological studies and the
determination of molecular exclusion limits have sug
gested that each connexon of the gap junction forms an
aqueous pore 1-2 nm in diameter which ·connects the
cytoplasms of adjacent cells.
As a result of the ease and efficiency of preparation,
considerable interest has recently arisen in the junctions
between lens fiber cells. While showing many of the
attributes of gap junctions in other tissues, they also have
95
some unique properties such as a narrower extracellular
gap, a more dispersed array of connexons (or, in some
instances, a tetragonal crystalline packing), and a major
protein of Mr 26,000 markedly different from that of liver
gap junctions (Biology 1980, No. 137; see Abstract No.
130). These differences have led to the suggestion that
these junctions may not be related to gap junctions. In an
attempt to resolve this issue, we are in the process of
analyzing the structure of isolated lens junctions by
neutron diffraction. The advantage of this technique is
that by replacing water with heavy water, the regions of
hydration can be mapped directly without the addition of
extraneous labels. This should provide a direct method of
visualizing any aqueous channels, thus demonstrating
whether or not the lens junctions isolated for biochemical
studies do form channels of dimensions consistent with
that determined for gap junctions in other tissues and
sufficient to account for the obs~rved coupling of eye lens
fiber cells.
To interpret such neutron diffraction data, one must
be able to choose a unique, or certainly limited, number of
possible phase sets. The determination of this requires an
analysis of the lattice present in purified lens junctions to
establish whether or not it is centrosymmetric. Towards
this end, X-ray and electron diffraction studies are
currently being carried out on lens junctions isolated
under a variety of conditions. These approaches should
not only enable us to determine whether or not lens
junctions form intercellular channels of appropriate
dimensions, but may also ultimately enable us to study the
secondary and tertiary structure of the protein in the
membrane.
*Department_ of Molecular Biophysics and Biochemistry, Yale University.
137. CELL JUNCTIONS IN THE DEVELOPMENT OF FEATHER GERMS
Investigators: Jean Edens, Jean-Paul Revel
Feathers are derived from the ectoderm but their
development is closely controlled by the underlying
mesenchymal cells. In the past year we have used diverse
morphological techniques to define this interaction. We
have obtained evidence based on serial thin sectioning and
observation of thick sections in the 1000 kV microscope in
Boulder, Colorado, that processes of epithelial cells pass
through the basement lamina and make contact with the
mesenchyme. Conversely, processes of the mesenchymal
96
cells seem to pass through the basement lamina to contact
the epithelium. We believe that these cell processes may
form gap junctions allowing for direct communication
between the two interacting tissues. Physiological experi
ments aimed at testing this hypothesis are presently under
way.
PUBLICATIONS
Meyer, D. J., Yaneey, S. B. and Revel, J.-P. (1981) lntercellular communication in normal and regenerating rat liver: a quantitative analysis. J. Cell Biol. 91, 505-523.
Miller, M. M., Strader, C. D., Raftery, M. A. and Revel, J.-P. (1981) Hemoeyanin linked to protein A as an immunochemical labeling reagent for electron microscopy. J. Histochern. and Cytochem. 29, 1322-1327.
Nicholson, B. J., Hunkapiller, M. w., Grim, L.B., Hood, L. E. and Revel, J.-P. (1981) The rat liver gap junetion protein: properties and partial sequence. Proc. Nat. Aead. Sei. USA 78, 7594-7598.
Nieholson, B. N. and Revel, J.-P. (1982) Eleetron microscopy and subcellular fractionation of gap junctions in liver. Methods in Enzymology, in press.
Nicholson, B. J., Takemoto, L. J., Hunkapiller, M. w., Hood, L. E. and Revel, J.-P. (1982) The gap junetion proteins of liver and eye lens. Cell, submitted for publication.
Revel, J.-P. (1981) lntereellular eommunieation. Jn: The Paraeellular Pathway. S. E. Bradley and E. F. Pureell (Eds.), pp. 57-78. Josiah Maey Jr. Foundation.
Revel, J.-P., Yancey, S. B., Meyer, D. J. and Nicholson, B. (1981) Cell junctions and intercellular communication. In Vitro 16, 1010-1017.
Yancey, S. B., Edens, J. E., Trisho, J. E., Chang, C.-c. and Revel, J.-P. (1982) Deereased ineidenee of gap junetions between Chinese hamster V-79 cells upon exposure to the tumor promoter 12-0-tetradecanoylphorbol-13-aeetate. Exp. Cell Res., in press.
Yancey, S. B., Nicholson, B. J., Grim, L. and Revel, J.-P. (1981) The dynamie state of gap junetions. J. Supramolee. Struet. 16, 221-232.
CELLULAR NEUROBIOLOGY
Jeremy P. Brockes
A. James Hudspeth
Mary B. Kennedy
Henry A. Lester
Felix Strumwasser
Associate Professor: Jeremy P. Brockes Del E. Webb Research Fellow: Christopher R. Kintner Research Fellows: Lawrence C. Fritz, Katherine A.
Stygall Graduate Students: Karl J. Fryxell, Greg Erwin Lemke Research Staff: Arthur W. DeJohn, Wanetta Harrington,
Gil F. Richards, Teresa M. Stevens Laboratory Staff: Hortensia Zepeda
Support: The work described in the following research reports has been supported by:
The Kroc Foundation The McKnight Foundation Muscular Dystrophy Association of America National Institutes of Health, USPHS National Multiple Sclerosis Society Pew Memorial Trust Gustavus and Louise Pfeiffer Research Foundation The Del E. Webb Foundation Weingart Foundation
summary: Research in cellular and molecular neuro
biology has centered around several topics in the 'Rell
biology of nerve and glial cells and their various inter
actions. Much of our work is done on purified cell
populations maintained under the defined conditions of
culture.
Peripheral myelinated axons have a very high density
of electrically excitable sodium channels at the node of
Ranvier-the area of nerve cell plasma membrane
between successive myelinating Schwann cells. The
density in the interno-dal membrane underlying the glial
cell is undetectable. Our efforts are directed to analyzing
in vitro the factors responsible for this remarkable
clustering. During the last year we have collaborated
with Dr. M. A. Raftery's laboratory in the derivation of
monoclonal and polyclonal antibodies to the channel.
These reagents have contributed to our understanding of
the protein components of the channel and promise to be
useful for cytochemical studies.
We are also interested in the signals that pass from the
axon to the Schwann cell and instruct it to make myelin.
Our previous work has provided quantitative estimates for
the extent of this induction. A current emphasis in the
laboratory is the study of the mutant mouse Trembler,
which has a lesion in its Schwann cells leading to a
defective interaction with the myelinated class of axon.
In order to maximize the defect, the homozygote has been
bred and its mutant Schwann cells are being studied in
culture. These animals may also be valuable for studying
the nodal segregation of the sodium channel.
For the last five years we have been studying a new
growth factor, a protein component of the brain and
99
pituitary which is mitogenic for Schwann cells, astrocytes
and fibroblasts. This component, called glial growth
factor (GGF), has been extensively purified from large
quantities of bovine pituitary gland and a variety of
structural and functional studies are in progress. Our
interest in GGF and its possible role in controlling
proliferation in vivo has led to initial investigations of
limb regeneration in Urodeles-an interesting problem
which may become a major focus of future effort.
138. ANTIBODIES TO THE VOLTAGE-SENSrnYB SODWM CHANNEL
Investigators: Lawrence c. Fritz, Hsiao-Ping H. Moore*, Michael A. Raftery*, Jeremy P. Brockes
The voltage-sensitive sodium channel is the membrane
component responsible for the rising phase of the action
potential in many excitable cells. A body of experimental
evidence suggests that sodium channels are not always
uniformly distributed over a cell's surface, but can be
clustered. One striking example of this clustering occurs
at the node of Ranvier in myelinated axons. Our work has
centered on the production of antibodies against the
sodium channel from the electroplaques of the eel
Electrophorus electricus. Our intention is to use these
antibodies as cytochemical markers for studying the
process of sodium channel localization in neurons.
We have isolated and characterized a monoclonal
antibody (named VDlO) that recognizes the eel sodium
channel. This antibody can precipitate the saxitoxin
binding component from solubilized preparations of
electroplaque membranes in a dose-dependent manner.
Since it is known that saxitoxin binds specifically to the
sodium channel, the immunoprecipitation data demon
strate that VDlO recognizes some portion of the sodium
channel complex. We have further shown that VD10
reacts with a single protein band of molecular weight
250,000 (p250). This was demonstrated by separating
electroplaque membrane proteins on SDS-po!yacrylamide
gels and reacting either the gels or nitrocellulose
transfers of the gels with VDlO. This provides the
clearest evidence to date that p250 comprises part of the
eel sodium channel. VD10 does not cross-react with
sodium channel from rat brain or frog brain.
We have purified p250 by electroelution from prepara
tive SDS-po!yacrylamide gels and produced a rabbit
antiserum against it. Immunonuorescent staining of
sections of the electroplaques has demonstrated that the
100
sodium channel is restricted to the innervated caudal face
of the electrocyte, in accord with earlier physiological
results. We are currently examining the cross-reactivity
of this antiserum.
*Division of Chemistry and Chemical Engineering, California Institute of Technology.
139. CHARACTERIZATION OF TREMBLER MOUSE SCHWANN CELLS IN VIVO AND IN VITRO
Investigators: Karl J. Fryxell, Jeremy P. Brookes
The Trembler (Tr/+) mouse mutant has a pronounced
reduction in peripheral myelin, while both central myelin
and peripheral unmyelinated nerves appear normal.
Experiments (by A. J. Aguayo and collaborators) in which
axons of one genotype are confronted by Schwann cells of
another genotype produce a clear answer-the Trembler
deficit is expressed in the Schwann cell and not in the
axon. The myelin proteins P 0 and P 1 are apparently
reduced to the same extent in Trembler nerve. We would
like to understand the nature of this Schwann cell-specifie
function, which is required for the accumulation of myelin
proteins, but is not required for Schwann eell survival or
normal wrapping of unmyelinated axons.
In the original description of Trembler {Falconer,
1951), no difference was found between Tr/+ and Tr/Tr
mice. We have found, however, that homozygotes (of at
least one of the Tr alleles) do not survive to adulthood,
while Tr/+ mice do. Although these homozygotes cannot
be proven to be such by breeding experiments, they do
have a clearly recognizable phenotype in young animals.
Tr/Tr Schwann cells show significant heterogeneity in
their response to competent axons in vivo-only a fraction
of these Schwann cells make recognizable myelin and/or
are well-stained by antibodies against myelin proteins.
The cause of this heterogeneity is not yet clear, but is
almost certainly not differences in axonal diameter.
We have found that mouse Schwann cells may be
immunologically purified by methods similar to those used
for rat Schwann cells (Brockes et al., 1979), enabling us to
grow ''genetically pure" cultures of Tr/Tr Schwann cells.
Preliminary experiments indicate that cultured Tr/Tr
Schwann cells respond normally to glial growth factor;
additional experiments are planned to further charac
terize Tr/Tr Schwann cells in culture.
References: Brockes, J. P., Fields, K. L. and Raff, M. c. (1979) Brain
Res. 165, 105-118. Falconer, D.S. (1951) J. Genet. 50, 192-201.
140. LATENT INFECTION OF NERVE CELLS BY HSV-1
Investigator: Christopher R. Kintner
Herpes simplex virus type 1 (HSV-1), a human herpes
virus, latently infects the trigeminal ganglion of its
natural host. Although it is possible to produce a similar
infection in rodents, it has not been possible to latently
infect sensory ganglion cells taken from rodents and
grown in cell culture. One explanation for this apparent
failure is that the ganglion is made up of many cell types
and the lytic infection of some cell types (for example,
fibroblasts) may obscure latent infections of other ones
(for example, neurons). To obtain a HSV-1 latent
infection in vitro, it may be necessary to isolate the
appropriate cells from sensory ganglion and to define
culture conditions that permit latent infections of these
ceijs. The ability to latently infect ganglion cells in vitro
with HSV-1 would be an important tool for studying
cellular and molecular aspects of HSV-1 latency. To this
end, dorsal root ganglion neurons, Schwann cells, and
fibroblasts are being isolated from neonatal rats, cultured
in vitro as separate, pure populations and infected with
HSV-1. The infected cultures are being scored for cells
that survive the infection. Cells that are resistant to the
cytopathic effects of the virus will be tested for the
presence of latent virus.
141. GLIAL GROWTH FACTOR
Investigator: Greg Erwin Lemke
We have previously reported on the properties of glial
growth factor (GGF), a protein present in the brain and
pituitary which triggers DNA synthesis and cell division in
cultured rat Schwann cells, astrocytes, and muscle
fibroblasts (Biology 1981, No. 164). Our biochemical
characterization of this molecule has progressed along the
following lines:
(1) We have developed methods for recovering GGF
activity following SDS-polyacrylamide electrophoresis
under non-reducing conditions. These methods have
allowed us to demonstrate that all growth factor activity
resides in a molecule of molecular weight 31,000, and to
identify this protein unambiguously.
(2) By using immunoautoradiographic methods in
conjunction with a panel of monoclonal antibodies to GGF
(Lemke and Brockes, 1981), we have shown that all
antibodies which specifically precipitate GGF activity
also bind exclusively to the 31K protein.
(3) We have investigated the possible relation of GGF
to the platelet-derived growth factor (PDGF), a potent
fibroblast mitogen isolated from platelet lysates and
believed to be the principal mitogenic activity in serum
(Heldin et al., 1981). Like PDGF, GGF is a very basic
protein of molecular weight 31,000 whose activity is
destroyed by reducing agents, but which is relatively
resistant to high temperature and low pH. (Active PDGF
can also be recovered from SDS gels.) While purified
PDGF (a gift from Dr. R. Ross, University of Washington,
Seattle) shows little mitogenic activity against Schwann
cells (our results), we hypothesize that both proteins are
members of the same family of growth factors, some of
whose target cells (e.g., fibroblasts) overlap.
(4) In conjunction with Michael Hunkapiller and Leroy
Hood, we are attempting to determine the amino terminal
sequence of the GGF protein.
References: Heldin, C.-H., Westermark, B. and Wasteson, A. (1981)
Biochem. J. 193, 907-913. Lemke, G. E. and Brockes, J. P. (1981) In: Monoclonal
Antibodies to Neural Antigens. R. McKay, M. c. Raff and L. Reichardt (Eds.), pp. 133-140. Cold Spring Harbor Press, New York.
142. A CHARACTERIZATION OF MONOCLONAL ANTIBODil!S GENERATED AGAINST RAT DORSAL ROOT CELLS
Investigator: Katherine A. Stygall
The generation and initial characterization of several
monoclonal antibodies generated againSt cultured newborn
rat dorsal root ganglion cells has already been described
(Biology 1981, No. 166). As described in the previous
report, two of the antibodies bind to unfixed cultured
dorsal root ganglion cells and to rat primary muscle cells
with a very distinct pattern. These antibodies have since
been shown by competition experiments to bind to the
same or adjacent determinants.
To overcome experimental problems associated with
the use of unfixed cells, several fixation techniques were
tried in an endeavor to find one which did not severely
reduce antibody binding. Benzoquinone-fixed cells
retained antibody binding and also a good morphological
appearance under light microscopy. The density of
labeling on all cell types studied was greater when the
cells were prefixed. It has yet to be determined whether
this is due to the presence of the antigenic site inside the
cell or to a greater sensitivity of the new technique.
101
Clustered antibody binding at process branch points
and thickenings on dorsal root ganglion cells was still
apparent when the cells were pre-fixed but was less
frequent and Jess pronounced. This suggests the
possibility that in the absence of fixation some
redistribution of the antigen-antibody complex occurs.
Antibody binding had not been demonstrated on
unfixed cultured rat fibroblasts but was apparent on
benzoquinone-fixed fibroblasts. Binding to both rat
muscle cells and fibroblasts was not uniform; some cells
showed quite a high degree of labeling, others none at all.
(This Wlexplained phenomena has been observed with
several other antibodies; M. Raff, personal commWli-
cation.) My results indicate that the non-uniform
distribution of the antigenic site on the muscle cells and
fibroblasts may be related to a stage in the cell cycle and
to spreading out of the cell on the substrate. I shall do
further experiments to test this theory, and also to
determine the molecular weight of the antigen.
143. STUDil!S ON BLASTBMAL CELLS FROM REGENERATING LIMBS OF URODELES
Investigators: Christopher R. Kintner, Katherine A. Stygall, Jeremy P. Brockes
Limb regeneration in Urodeles (newts and axolotls)
begins with an accumulation of mesenchymal-like
blastemal cells. During the early stages of regeneration,
the blastemal cells divide, migrate and in later stages
differentiate into cells that make up the tissue in the
mature limb. Because of the accessibility of blastemal
cells and their similarity to embryonic stem cells, we have
begun the study of their developmental biology by doing
two things. First, we are developing methods to dis
sociate blastemal cells from regenerating newt limbs
using enzymatic digestion and defining culture conditions
that support growth in vitro of these dissociated cells. In
view of the role of nerves in stimulating division of these
cells in vivo, we are particularly interested in the effect
of GGF preparations, from both mammalian and
amphibian sources, on their proliferation in vitro.
Secondly, we are looking for antigenic markers that are
specific for blastemal cells and for each of their differen-
tiated derivatives. To do this, we are generating
hybridomas directed against blastema from early and late
stages of limb regeneration. These hybridomas are being
screened by immunohistochemistry for antibodies that
bind to specific cells of regenerating limbs.
102
PUBLICATIONS
Brockes, J, P. (1982) Identification cultured Schwann cells, and controlling their proliferation. Reconstruction. c. C. Kao and Raven Press, New York, in press.
and purification of a purified factor
In: Spinal Cord R. P. Bunge (Eds.),
Brockes, J, P. (1982) Glial growth factor-a new component of the brain and pituitary. In: Nervous System Regeneration. A. M. Giuffrida-Stella, B. Haber, G. Hashim and J, R. Perez-Polo (Eds.), Alan R. Liss, Inc., New York, in press.
Brockes, J. P. (1982) Nerve, myelin and multiple sclerosis. Engineering & Science, Vol. XLV, pp. 9-14.
Brockes, J. P. (Ed.) (1982) Neuroimmunology. Plenum Press; New York, in press.
Brockes, J, P., Fryxell, K. and Lemke, G. E. (1981) Studies on cultured Schwann cells-the induction of myelin synthesis, and the control of their proliferation by a new growth factor. J. Exp. Biol. 95, 215-230.
Professor: A. James Hudspeth Del E. Webb Research Fellow: Thomas Holton Graduate Students: Ruth Anne Eatock, Richard S. Lewis Research staff: Richard A. Jacobs
Support: The work described in the following research reports has been supported by:
National Institutes of Health, USPHS Ann Peppers Foundation Pew Memorial Trust Gustavus and Louise Pfeiffer Research Foundation Gordon Ross Medical Foundation The Del E. Webb Foundation
SUmmary: Hair cells are specialized epithelial cells that
are the sensory receptors of the vertebrate inner ear and
lateral-line organ. Each cell is a mechanoreceptor that
produces electrical signals in response to movement of its
hair bundle, a cluster of large microvilli (stereocilia) and a
single true cilium (kinocilium) projecting from the cellular
apex. The nature of the stimulus that evokes hair-bundle
displacement determines the modality to which a given
hair cell is sensitive: sound, vibration, angular accelera
tion, linear acceleration, or water movement. The
electrical response produced in the cell by appropriate
stimuli modulates the release of a chemical transmitter
from synaptic sites on the basal surface of the hair cell,
and thereby controls the firing rate of the postsynaptic
nerve fibers that convey the signal into the central
nervous system.
We are interested in the transduction process by which
mechanical stimulation evokes an electrical response, the
receptor potential, from a hair cell. We have developed in
Fryxell, K. J., Balzer, D. R. Jr. and Brockes, J. P. (1982) Development and applications of a solid phase radioimmunoassay for the Po protein of peripheral myelin. J. Neurochem., submitted for publication.
Lemke, G. E. and Brockes, J. P. (1981) An immunochemical approach to the purification and characterization of glial growth factor. In: Monoclonal Antibodies to Neural Antigens. R. McKay, M. c. Raff and L. Reichardt (Eds.), pp. 133-140. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.
Moore, H.-P. H., Fritz, L. C., Raftery, M.A. and Brockes, J, P. (1982) Isolation and characterization of a monoclonal antibody against the saxitoxin-binding component from the electric organ of the eel Electrophorus electricus. Proc. Nat. Acad. Sci. USA 79, 1673-1677.
Pappenheimer, A. M. Jr., Harper, A. A., Moynihan, M. and Brockes, J, P. (1982) Diptheria toxin and related proteins: effect of route of injection on toxicity and the determination of cytotoxicity for various cultured cells. J. Infect. Diseases 145, 94-102.
vitro preparations of hair cells from the bullfrog's
sacculus and from the alligator lizard's basilar papilla with
which is it possible to record receptor potentials and to
view displacements of hair bundles while cells are
stimulated individually with a fine probe or en masse by
oscillatory pressure changes. By this means, we are able
to determine how hair cells respond to stimuli of known
amplitude, direction, velocity, and frequency. We are
presently employing these preparations not only for
electrophysiological and biophysical studies of the hair
cell's membrane, but also for investigations of the
physiological effect of treatments which produce
permanent damage to hair cells and consequent deafness
or vertigo: overstimulation, or acoustic trauma, and
ototoxicity, or poisoning by exposure to aminoglycoside
antibiotics.
Our previous investigations have established that:
(1) it is bending of the hair bundle which produces
responses; (2) the range of hair-bundle deflection over
which a cell responds is remarkably small-an angular
extent of about ±1°; (3) hair cells are correspondingly very
sensitive, giving measurable responses in vitro to stimuli
smaller than 0.5 nm; (4) the cells possess an adaptation
mechanism which keeps them highly sensitive even in the
presence of large static stimuli; (5) the stereocilia, rather
than the kinocilium, mediate the mechanosensitivity of
the hair cell; (6) the receptor potential results from an
increase in membrane conductance of the cell, implying
that mechanical stimuli open ion channels; (7) the
channels so opened are relatively non-selective in their
ionic permeability and will pass most cations with
molecular dimensions of less than 0.6 nm; (8) the channels
open following a stimulus with a latency of less than
13 µsec at 37°; and (9) the rate of channel opening
depends upon the amplitude of the stimulus in such a way
as to suggest that mechanical force exerts a direct effect
on channel configuration.
144. IN VIVO ADAPTATION IN HAIR CELLS AND PRIMARY NEURONS OF THE BULLFROG SACCULUS
lnvestigator: Ruth Anne Eatock
Sensory adaptation is a general term used to describe
any decline with time in the response of a sensory organ
or cell to a maintained stimulus. Studies on a variety of
vertebrates have shown that adaptation is a prominent
feat~re of the responses of primary neurons innervating
the hair cells of inner-ear organs. Adaptation of a
primary neuron's response may be due to any or all of a
variety of mechanisms, including: (1) mechanical
relaxation in the accessory structures that transmit the
stimulus to the hair cells; (2) adaptation of the hair cell's
receptor current and/or receptor potential; (3) adaptation
at the synapse between hair cell and primary neuron, at
either a pre- or a post-synaptic site; (4) adaptation of the
mechari.ism for generating spikes in the primary neuron;
(5) inhibitory efferent feedback onto the hair cell and/or
primary neuron.
We have observed that the responses of hair cells of
the bullfrog's sacculus adapt in vitro to maintained
deflections of their hair bundles [(2) above]. In order to
relate this adaptation to the in vivo adaptation of primary
neurons, I have been recording the spike activity of single
primary saccular neurons and the saccular microphonic
potential, the receptor potential recorded extracellularly
from the population of hair cells. The appropriate in vivo
stimulus for the bullfrog's sacculus is linear (translational)
acceleration. For these experiments, the frog is secured
to a platform on an electromagnetic shaker that delivers
steps of vertical linear acceleration. Both the micro
phonic potential and the firing of primary neurons show a
response at the onset of a step of constant acceleration,
then adapt rapidly. The decline in response in both cases
is faster than the adaptation of hair cells in vitro, even
when efferents of central origin have been cut, suggesting
that there may be additional sources of adaptation. For
103
instance, it seems likely that there is relaxation of the
mechanical input to the hair cells in vivo, so that a step of
acceleration is not equivalent to a step deflection of the
hair bundles.
However, it also seems likely that adaptation at the
hair-cell level contributes significantly to the in vivo
adaptation, since there are some common properties. For
instance, the response decline shown by a primary neuron
during constant acceleration is partly due to a shift in the
stimulus range to which the neuron is sensitive. Such a
shift was found to be the source of the in vitro hair-cell
adaptation. Also, both in vivo and in vitro microphonic
responses have thermal Q-lOs between 2 and 3. Finally,
preliminary results indicate that the rate of adaptation of
the in vivo microphonic potential shows a Ca++ depen
dence similar to that of the in vitro microphonic current.
145. MOTION OF HAIR-CELL STEREOCilJA IN THE AUDITORY RECEPTOR ORGAN OF THE ALLIGATOR LIZARD
Investigator: Thomas Holton, A. James Hudspeth
In vertebrate auditory organs, hair cells are thought to
transduce mechanical displacements of their hair bundles
into receptor potentials. In the auditory receptor organ of
the alligator lizard (Gerrhonotus multicarinatus), the
basilar papilla or cochlea, hair cells rest on a basilar
membrane that moves in response to sound. The motion
of this membrane differs from that of the mammalian
basilar membrane in that it is not tonotopically organized;
that is, the frequency selectivity of motion does not vary
with longitudinal position along the organ (Peake and Ling,
1980). However, responses of cochlear-nerve fibers
innervating the basal portion of the papilla are tono
topically organized (Weiss et al., 1978). In this region, the
hair bundles protrude into fluid unencumbered by
overlying tectorial structures and have heights that vary
monotonically with position along the organ. We have
sought to determine whether tonotopic organization exists
at the mechanical input to the hair cells; that is, whether
hair bundles in different positions along the organ move
maximally in response to stimuli of different frequencies.
We have observed the motion of individual hair bundles
in response to mechanical stimulation in an in vitro
preparation of the lizard's basilar papilla. The organ is
cemented across a perforated partition separating two
fluids, a high-K + saline bathing the top (hair-bundle)
surface of the organ and a high-Na+ saline bathing the
104
bottom (basilar-membrane) surface. Motion of the organ,
induced by driving the fluid in the bottom compartment
with a piezoelectric bimorph element, is observed under
differential-interference-contrast optics with xenon-flash
stroboscopic illumination and recorded on 35 mm film or
videotape.
The main finding is that the motions of the distal tip
of a hair bundle and of the top surface of its hair-cell
body are different, frequency-dependent functions of
longitudinal position. As a result, the relative motion
between these structures, which constitutes the effective
mechanical stimulus to the cell, is frequency-selective
and tonotopically organized: the longest hair bundles
move preferentially at low frequencies (about 1.5 kHz),
while the shortest hair bundles move preferentially at high
frequencies (about 4 kHz). Thus, tonotopic organization
of hair-bundle motion and neural response are correlated,
suggesting that receptor-organ micromechanics partly
determines such neural response properties as frequency
selectivity and tonotopic organization.
References: Peake, w. T. and Ling, A. L. (1980) J. Acoust. Soc. Am.
67, 1736-1745. Weiss, T. F., Peake, W. T., Ling, A. L. and Holton, T.
(1978) In: Evoked Electrical Activity in the Auditory Nervous System. R. F. Naunton and C. Fernandez (Eds.), pp. 91-110. Academic Press, New York.
146. CHARACTERISTICS OF VOLTAGE-, ION-, AND TIME-DEPENDENT IONIC CONDUCTANCES IN ISOLATED HAIR CELLS
Investigator: Richard S. Lewis
The receptor potential of the hair cell is a composite
of the effects of several ionic currents. The transduction
current, produced through deflection of the hair bundle,
changes the membrane potential and thereby activates
other ionic conductances. We are attempting to describe
quantitatively the role of these voltage-, ion-, and time
dependent conductances in modulating the receptor
potential, using the whole-cell voltage-clamp technique of
Neher and colleagues (Hamill et al., 1981). Our initial
goals have been to determine the number of discrete
conductances and to find conditions that allow the study
of each in isolation. To assist in the rapid collection of
voltage-clamp data from isolated hair cells, an external
perfusion system has been designed that allows placing a
single cell sequentially in each of four different solutions
during recording of ionic currents.
Thus far, three ionic conductances have been found in
hair cells using these techniques. An inward current is
carried by Ca++ and possibly by Na+, and can be blocked
with external Mg++ or Ni++. In contrast to somatal Ca++
currents in several other preparations, this one does not
inactivate within at least 100 msec; it may underlie tonic
transmitter release at the synapse onto auditory nerve
fibers. There are two outward K + currents, both of which
are blocked by internal Cs+ but which differ in their
pharmacological sensitivities and kinetics. A fast, non
inactivating K + current is blocked by exposure to external ++ N.++ ++ d + ( . ) Mg , i , Ba , an TEA tetraethylammon1um , as
well as by removal of all external Ca++; it is most likely a ++ . + .
Ca -activated K conductance. Interestingly, its
kinetics are 10 to 100 times faster than Ca++ _activated
K+ currents described in other preparations. In addition, a
slower K+ current is present that is relatively insensitive
to external divalent cations and TEA+, but is blocked by •
external 4-aminopyridine, and shows voltage- and time
dependent inactivation. Its pharmacological and kinetic
characteristics are quite similar to those of the A current,
which has been described in both vertebrates and
invertebrates (Thompson and Aldrich, 1980).
We are now quantitatively determining the voltage-,
ion-, and time-dependences of these conductances in
order to predict their modulatory effects on the trans
duction response under various conditions. These
predictions should be testable and may yield insight into
the role of these ionic currents in shaping the physio
logical response of the hair cell.
References: Hamill, o. P., Marty, A., Neher, E., Sakmann, B. and
Sigworth, F. J. (1981) PflUgers Arch. 391, 85~100. Thompson, S. H. and Aldrich, R. W. (1980) In: The Cell
Surface and Neuronal Function. C. W. Cotman, G. Poste and G. L. Nicolson (Eds.), pp. 49-85. Elsevier/North-Holland Biomedical Press, Amsterdam.
PUBLICATIONS
Corey, D. P. and Hudspeth, A. J. (1982) Analysis of the microphonic potential of the bullfrog's sacculus. J. Neurosci., submitted for publication.
Corey, D. P. and Hudspeth, A. J. (1982) Kinetics of the receptor current in bullfrog saccular hair cells. J. Neurosci., submitted for publication.
Eatock, R. A. and Hudspeth, A. J. (1981) Adaptation in hair cells: in vitro intracellular responses and in vivo microphonic potentials from a vestibular organ. Soc. Neurosci. Abstracts 7, 62.
Holton, T. and Hudspeth, A. J. (1982) Motion of hair-cell stereocilia in the auditory receptor organ of the alligator lizard. Soc. Neurosci. Abstracts 8, submitted for publication.
Hudspeth, A. J. (1982) The recovery of local transepithelial resistance following single-cell lesions. Exp. Cell Res. 138, 331-342.
Hudspeth, A. J. (1982) Extracellular current flow and the site of transduction by vertebrate hair cells. J. Neurosci. 2, 1-10.
Assistant Professor: Mary B. Kennedy Visiting Associate: Jocelyne Lecompte Graduate Students: Mark K. Bennett, Ngozi E. Erondu Research Staff: Barbara Moore Laboratory Staff: Susanna Chan, Hortensia Zepeda
Support: The work described in the following research reports has been supported by:
The Norman W. Church Fund National Institutes of Health, USPHS Pew Memorial Trust Gordon Ross Medical Foundation The Alfred P. Sloan Fund for Basic Research
Summary: We are interested in the molecular
mechanisms by which changes in the cytoplasmic levels of
calcium ion regulate neuronal functions. It has been
known for many years that certain neurotransmitters as
well as electrical activity can cause large transient
changes in intracellular calcium concentrations. Studies
with pharmacological agents that block or mimic the
changes in calcium levels suggest that many physiological
effects that are caused by these neurotransmitters or by
electrical activity are the result of their effects on
calcium concentration. However, the number and nature
of the calcium "receptors" in the cytoplasm are still
incompletely understood. Examples of specialized
neuronal functions that are or may be regulated entirely
or in part by calcium concentrations are: formation and
maintenance of synaptic contacts during development and
regeneration; choice of transmitter phenotype during
development; transmitter synthesis; transmitter release;
modulation of the amount of transmitter released per
electrical impulse; and insertion and stabilization of
postsynaptic neurotransmitter receptors.
As one approach to understanding the mechanisms of
action of calcium in neurons, our attention has been
focused on a regulatory enzyme that is highly concen
trated in brain and can be activated about 40-fold by
calcium ion (Kennedy and Greengard, 1981). It is a
protein kinase that was discovered because it phos
phorylates an abundant brain protein, originally called
105
Lewis, R. S. (1982) Characterization of voltage- and iondependent conductances in vertebrate hair cells. Soc. Neurosci. Abstracts 8, submitted for publication.
Shotwell, S. L., Jacobs, R. and Hudspeth, A. J. (1981) Directional sensitivity of individual vertebrate hair cells to controlled deflection of their hair bundles. Ann. N.Y. Acad. Sci. 374, 1-10.
protein I and recently renamed synapsin I, that is
specifically associated with synaptic vesicles (Krueger et
al., 1977; Schulman and Greengard, 1978a,b; Ueda et al.,
1979; Kennedy and Greengard, 1981; DeCamilli et al., in
preparation). (Synaptic vesicles are specialized structures
within nerve terminals that contain neurotransmitter and
release it into the extracellular space when they fuse with
the presynaptic membrane. Calcium ion, which flows into
the terminal during each impulse, is the trigger for this
fusion event and has also been postulated to be involved in
control of the number of vesicles that are "primed" for
fusion prior to each impulse.) Because protein
phosphorylation is a regulatory mechanism that is
commonly used in many different cell types, it seemed
that understanding the properties, distribution, and
various actions of this calcium-regulated protein kinase
would help to clarify the mechanisms by which calcium
exerts its effects in nervous tissue. A similar enzyme
activity is found, in lower concentration, in other tissues
(Kennedy and Greengard, 1981); thus, these studies may
also be important in understanding the regulatory role of
calcium in non-neuronal cells.
Several general properties of the synapsin I kinase
have been described. It is found roughly evenly dis
tributed between the cytosolic and particulate fractions
after tissue homogenization. The enzymes from both
sources have been partially purified and their properties
have been compared (Kennedy, McGuinness and
Greengard, manuscript in preparation). By all criteria
thus far examined, the two are indistinguishable. For
example: they are both activated by physiological
concentrations of calcium ion; the activation is mediated
by the small calcium-binding protein, calmodulin; they
both phosphorylate the same two serine residues on
synapSin I; their affinities for syilapsin I and ATP are
identical; their protein substrate specificities are the
same; they cannot be separated by various chromato
graphic procedures. We are now interested in defining
106
mor_e rigorously the structure, substrate specificity, and
tissue and cellular locations of this enzyme. Our long
term goal is to learn enough about it to develop
pharmacological tools for use in studying its functions.
Our major efforts over the past year have been in
three areas. (1) The protein substrate specificity of the
enzyme has been examined in greater detail, and evidence
that one of the enzyme's own subunits may be auto
phosphorylated has been obtained. (2) Methods have been
developed to further purify the enzyme by standard
protein biochemical techniques. (3) Hybridomas secreting
monoclonal antibodies to the partially purified enzyme
have been selected. They will be used in the purification
of the enzyme and in studies of its structure and
localization.
References: Kennedy, M. B. and Greengard, P. (1981) Proc. Nat. Acad.
Sci. USA 78, 1293-1297. Krueger, B. K., Porn, J. and Greengard, P. (1977) J. Biol.
Chem. 252, 2764-2773. Schulman, H. and Greengard, P. (1978a) Nature 271,
478-479. Schulman, H. and Greengard, P. (1978b) Proc. Nat. Acad.
Sci. USA 75, 5432-5436. Ueda, T., Greengard, P., Berzins, K., Cohen, R. s.,
Blomberg, F., Grab, D. J, and Siekevitz, P. (1979) J. Cell Biol. 83, 308-319.
147. SUBSTRATI! SPECIFICITY AND P05SIBLE "AUTOPHOSPHORYLATION" OF RAT BRAIN CALMODULIN-DEPENDENT SYNAPSIN I KlNASE
Investigators: Mary B. Kennedy, Teresa MeGuinness•, Paul Greengard*
Our initial examination of the protein substrate
specificity of calmodulin-dependent synapsin I kinase
made use of several homogeneous proteins that are
substrates for various other protein kinases. These studies
suggested that the range of substrates recognized by the
kinase was very narrow. However, Schulman and
Greengard (1978), in their initial description of
"endogenous" calmodulin-dependent protein kinase in brain
particUlate fraction, showed that phosphorylation of
several particulate protein bands was stimulated by
calcium and calmodulin. It was of interest to us to
determine whether the synapsin I kinase, given its
apparently narrow substrate specificity when tested with
soluble homogeneous proteins, could phosphorylate any of
the endogenous brain particulate substrate proteins. In
the course of experiments to test this possibility, we made
the rather surprising observation that two of the most
prominent particulate Substrates, one at 50 kilodaltons
(kd), and one at 60 kd, are present in partially purified
enzyme preparations from both the soluble and particulate
fractions. Enzyme from both sources had been purified
about 70-fold and passed over a calmodUlin-Sepharose
affinity column, as described in Abstract No. 148. The
two substrates in the enzyme preparations and the
corresponding particulate endogenous substrates
comigrate on two-dimensional polyacrylamide gels, and
have identical phosphopeptide maps following digestion
with Staph. aureus VS protease; thus it appears that they
are the same. We made the additional discovery that one
of the substrates, the 50 kd phosphoprotein, is the major
peptide band in both partially purified enzyme prepara
tions. This band comprises 10-20% of the total protein at
this stage of purification. We used two different methods
to show that the major 50 kd protein band, the 50 kd
"endogenous" substrate protein, and the synapsin I kinase
activity migrate identically during DEAE-cellulose
chromatography and calmodulin-Sepharose affinity
chromatography. These results suggest either that the
calmodulin-dependent protein kinase and its 50 kd
substrate are associated during purification or that the
50 kd protein is a subunit of the enzyme which is
autophosphorylated. Because many protein kinases are
autophosphorylated, the second possibility is not unlikely.
Further purification of the enzyme and production of
monoclonal antibodies to the 50 kd protein should help to
distinguish between the two possibilities.
It is evident from the recovery of the 50 kd protein
after only two purification steps that it is an unusually
abundant brain protein (perhaps 0.1 % of the total). Thus,
if it is a subunit of the kinase, the implication is that
there is a great deal of the kinase in brain, and it may be
one of the major brain calcium "receptors."
Refermce: Schulman, H. and Greengard, P. (1978) Nature 271,
478-479.
*Department of Pharmacology, Yale Medical School.
148. PURIPlCATION OF SYNAPSIN I KlNASE
Investigators: Mark K. Bennett, Mary B. Kennedy
Synapsin I kinase had previously been partially purified
from the cytosolic fraction using a two-step procedure.
The first step was ion exchange chromatography on
DEAE-cellulose, followed by affinity chromatography on
calmodulin-Sepharose (Kennedy and Greengard, 1981;
Kennedy et al., 1982). These steps resulted in an
approximately 70-fold purification of the enzyme with
3-10% recovery of activity. We have been working on
other conventional biochemical techniques to further
purify synapsin I kinase. We have optimized an ammonium
sulfate precipitation step yielding an additional 1.5-fold
purification with 95% recovery of activity. Gel filtration
has also been used to further purify the enzyme, resulting
in a 3.6-fold purification, with 50% of the activity
recovered. From the gel filtration data, the Stokes radius
of the kinase is estimated to be 75 X. The 50 kd substrate
band on SDS gels remains the dominant band through these
further purifications. This is consistent with the idea that
one of the subunits of synapsin I kinase is auto
phosphorylated.
We are currently working to optimize recovery and
purification of the kinase by various techniques. Once the
kinase is purified, we hope to do detailed studies of its
subunit structure, regulation, substrate specificity, and
possible autophosphorylation.
References: Kennedy, M. and Greengard, P. (1981) Proc. Nat. Acad.
Sci. USA 78, 1293-1297. Kennedy, M., McGuinness, T. and Greengard, P. (1982)
Manuscript in preparation.
149. PREPARATION AND SELECTION OF HYBRIDOMAS wmcH PRODUCE MONOCLONAL ANTIBODIES TO PARTIALLY PURIFIBD SYNAPSIN I KINASE
Investigators: Barbera Moore, Mary B. Kennedy, Ngozi E. Erondu
One of the advantages of hybridoma technology is that
specific monoclonal antibodies can, in theory, be produced
using impure antigens. Specific antibodies to synapsin I
Associate Professor: Henry A. Lester Professor: Jerome Pine* Visiting Associate: Joel Nargeot Senior Research Fellow: Jeanne M. Nerbonne Del E. Webb Research Fellow: Lee D. Chabala Research Fellows: Robert E. Sheridan Jr., Martin M.
Weinstock Graduate Student: Mauri E. Krouse Research Staff: E. Evan Shaff er lll, Roger Spencer,
Joseph E. Venti
*Division of Physics, Mathematics and Astronomy, California Institute of Technology.
107
kinase and (if they are distinct) its 50 kd substrate protein
would be of use to us in several ways. They could be used
to purify the enzyme and determine its subunit structure,
as well as to develop a radioimmunoassay for quantitation
of the enzyme in other tissues and brain regions. The
antibodies can be labeled with fluorescent or electron
dense ligands, and used to determine the locations of the
enzyme in tissue sections at the light and electron
microscope levels. Antibodies recognizing peptides in the
antigen that are not part of the kinase may be useful for
removing contaminating proteins from the enzyme.
We have used synapsin I kinase purified through the
calmodulin-Sepharose affinity column step to immunize
mice, and have fused cells from their spleens with
myeloma cells to produce antibody-secreting hybridomas.
Two screening methods have been developed fo~ use in
selecting hybridomas that are of interest to us. The first
is a modification of conventional solid phase radio
immunoassay methods, in which serum or hybridoma
supernatants can be tested for antibody recognizing any of
the peptides in the crude antigen. The second is a
precipitation assay that tests for antibody recognizing the
synapsin I kinase. Hybridoma clones have been selected
that are positive in the first screen, and we are beginning
to test them in the second screen.
PUBLICATIONS
Kennedy, M. (1982) Control of phosphorylation of a vesicle-associated protein, synapsin I. Psycho-pharmacology Bulletin, in press.
Kennedy, M. (1983) Experimental approaches to understanding the role of protein phosphorylation in the regulation of neuronal function. Ann. Rev. Neurosci. 6, in press.
Support: The work described in the following research reports has been supported by:
American Heart Association Fulbright Fellowship Muscular Dystrophy Association of America National Institutes of Health, USPHS Pew Memorial Trust The Del E. Webb Foundation
Summary: We are interested in exploring the mechanisms
that render the nerve cell a highly sophisticated electro
chemical machine, specialized to function on a time scale
of milliseconds and a distance scale of micrometers.
108
In recent years the group has studied the synapse that
transmits impulses from a nerve cell to a muscle fiber or
(in certain fishes) to an electric organ. Acetylcholine is
the transmitter at these synapses, and the group has
explored the sequence of events that allows acetylcholine
to interact with membrane-bound receptors and to open
channels for ion flow in membranes. Several drugs and
anesthetics can block this process, and the mechanisms of
blockade provide important information about membrane
function. The experiments utilize electrophysiological
measurements-voltage-clamp and the like-in combi
nation with a series of photoisomerizable molecules that
resemble acetylcholine in some cases, and blocking drugs
in other cases. Lasers and flashlamps are used to produce
pulses of molecules near membranes, on the same time
scale as the naturally occurring events; and the electrical
measurements provide a simultaneous picture of how
channels are responding in the membrane. This avenue of
investigation seems to hold continued promise because of
new techniques for recording single ionic channels in
intact cells and in isolated membrane patches. We have
adopted these "patch-clamp" procedures, using cultured
cells. In our present experiments, photochemical manipu
lations are timed, by on-line data processing, to occur
while a single channel is either open, closed or desensi
tized.
The group is also investigating the processes that
enable intracellular messengers-cyclic nucleotides,
calcium ions and protons-to control ion channels and gap
junctions in membranes. The rationale of these experi
ments is to produce photochemical "concentration jumps"
of second messengers within intact cells under electro-
physiological investigation. The work required the
development of a new photochemical technology, based
largely on photolyzable ortho-nitrobenzyl esters, and the
introduction of several new preparations in the laboratory.
We have employed photochemical pH-jumps to turn off
gap junction channels in Chironomus salivary glands.
Also, the physiologically crucial Ca conductance of heart
muscle is increased within a few seconds after an
intracellular concentration jump of cyclic AMP-but not
of cyclic GMP! We are looking forward to detailed
information about the kinetics and localization of second
messenger action.
We are also interested in the question, how is
transmitter liberated initially in response to a nerve
impUlse? This problem is being approached in biochemical
experiments designed to define the activity of certain
6acterial toxins known to block the natural release
process. The experiments involve subcellular fractions,
rich in synaptic tissue, from the electric organs of fish.
Dr. Jerome Pine, Professor of Physics at Caltech, is
affiliated with the research group. Pine's interests center
on nerves and muscles in culture, and on those factors
that influence synapse formation, receptor production,
and synaptic change. Tissue culture dishes are used that
incorporate microelect~ode arrays, for long-term stimu
lation and recording. Other experiments involve the use
of voltage-sensitive dyes to monitor electrical activity.
An ultrasoft X-ray scanning microscope is being
developed, with the goal of studying live, developing
synapses with submicron resolution.
150. AGONIST CONCENTRATION-JUMPS AT NICOTINIC RECEPTORS
Investigators: Lee D. Chllbala, Henry A. Lester, Jeanne M. Nerbonne, Robert E. Sheridan
The association of an agonist and its receptor is
characterized by both rates and steady-state responses
that are functions of agonist concentration. The
determination of these dose-response relationships is
usually complicated by a dose-dependent decrease in the
agonist sensitivity of receptors, called "desensitization."
Desensitization generally proceeds slowly compared to the
activation of receptors. Thus, if a known quantity of
agonist can be added to the receptors rapidly (within a
few microseconds), activation of receptors can be studied
under a condition that minimizes distortions caused by
desensitization.
We are now using the photoisornerizable agonist Bis-Q
to study activation of the acetylcholine receptor at
Electrophorus electroplaques. Bis-Q is added to the cell
as the cis isomer, purified by high performance liquid
chromatography. In this form, Bis-Q is inactive at the
nicotinic receptor and causes no desensitization. A one
microsecond laser flash then creates a known concen
tration of the trans isomer of Bis-Q that proceeds to
activate receptor channels. We have determined that
little desensitization occurs during the few milliseconds
required to activate the receptors. Furthermore, at low
agonist concentrations where desensitization does not
occur, both kinetic and steady-state data obtained in this
way are comparable to those obtained with conventional
bath application of trans-Bis-Q. We expect that light-
activated agonists will be most useful for the application
of large doses of agonist, when desensitization would
otherwise be more pronounced. These experiments are
currently under way using both eel electroplaques and
cultured chick muscle.
151. CHARACTERIZATION OF SINGLE IONIC CHANNEUI OPENED BY NICOTINIC AGONISTS
Investigators: Lee D. Chabela, Robert E. Sheridan
The activation of nicotinic acetylcholine receptors
leads to discrete changes in membrane conductance as ion
channels coupled to the receptors open and close.
Thousands of such stochastic events sum to produce a
cell's response to acetylcholine. We have been studying
single ionic channels in primary cultures of pectoral
muscle from embryonic chickens. Mechanically
dissociated myoblasts are allowed to fuse under conditions
where the cells cannot attach to a substrate, thus forming
"myoballs" consisting of many fused myoblasts. A
specially constructed glass pipette with a tip diameter of
about 1 micron is then placed on the surface of the
myoball, and the appli<?ation of gentle su<?tion electri<?ally
isolates the underlying section of membrane from the rest
of the cell (Neher, 1981). If the pipette contains a
nicotinic agonist and if the patch of membrane contains
acetylcholine receptors, the opening and closing of single
ionic channels can be measured.
Channels that are opened by acetylcholine at room
temperature have an average durati~n of about 4 milli
seconds at a membrane voltage of -90 millivolts. In
agreement with other measurements on nicotinic
receptors, this average lifetime is reduced when the
membrane is depolarized. The photoisomerizable
nicotinic agonist, trans-Bis-Q, opens channels with about
the same average lifetime and dependence on membrane
voltage as the channels opened by acetylcholine, even
though the affinity of the receptor for Bis-Q is about 100-
fold greater. Furthermore, channels opened by the two
agonists have about the same conductance, 25-30 pS. The
cis isomer of Bis-Q does not open channels in this
preparation. This suggests experiments in which Bis-Q
and other azobenzene derivatives are ·Photoisomerized
while interacting with a receptor in a known state (open,
closed or desensitized).
Reference: Neher, E. (1981) In: Techniques in Cellular Physiology.
P. F. Baker (Ed.), Elsevier/North-Holland, Amsterdam.
152. THE PROPERTIES OF THE BIS-Q ACTIVATED CHANNEL AND THE NATURE OF DESENSITIZATION
Investigator: Martin M. Weinstock
109
The purpose of these experiments was twofold: first,
to compare the elementary properties of the Bis-Q
activated channel to the ACh activated channel; second,
to study the nature of the onset and recovery of
cholinergic desensitization. Muscle fibers of the fish
Xenomystus nigris were bathed in a solution containing
100 nM cis-Bis-Q while being held under voltage clamp.
A flash of light photoisomerized some of the cis-Bis-Q
(non-agonist form) to trans-Bis-Q (agonist form). This
resulted in agonist-induced current.
Subsequent light flashes caused further increases in
the ratio of trans-Bis-Q to cis-Bis-Q and accompanying
increases in agonist-induced holding current. Eventually,
a plateau was reached and further light flashes caused no
permanent increases in current. This plateau represents
the attainment of a photostationary cis/trans ratio,
although individual molecules undergo cis t. trans
conversion as a result of any given light flash.
Current noise of channels activated by both trans
Bis-Q and ACh was analyzed to find the channel
conductances and open times associated with these two
agonists. Channels activated by trans-Bis-Q and ACh had
similar conductances (20-30 pS) and open times (3-4 msec)
despite the azobenzene group of Bis-Q, which is unusual in
cholinergic agonist molecules.
The process of recovery under desensitization was
explored by continuing to flash after the equilibrium
current level was reached. At this ?Qint, light flashes
cause temporary increases in holding current which decay
back to equilibrium in seconds. This result is interpreted
according to a model in which trans-Bis-Q molecules are
tightly bound to a subpopulation of desensitized receptors
and prevent recovery to native receptor. A flash of light
at this -point may convert trans-Bis-Q molecules bound to
the desensitized receptor to the cis form. This cis-Bis-Q
molecule then unbinds, allowing the desensitized receptor
to recover.
When light was flashed on fish muscles exposed to
300-600 nM cis-Bis-Q, large (100-200 nA) agonist-induced
currents were produced. These currents decayed
exponentially in several seconds as the fiber desensitized.
This result confirms that a first order process underlies
the onset of desensitization.
110
153. A STUDY OF THE NICOTINIC ACETYLCHOLINE RECEPTOR USING A PHOTOISOMERIZABLE COMPETITIVE ANTAGONIST
Investigators: Mauri E. Krouse, Norbert H. WBSSermann*, Bernard F. lll"langer*
Competitive antagonists bind to unoccupied receptors,
preventing agonist molecules from binding to the receptor
and opening the receptor channel. At equilibrium, the
effect of increasing antagonist concentration is to
increase the number of closed receptor channels, as
though the agonist concentration were reduced. In
previous studies on the kinetics of receptor inhibition by
antagonists such as d-tubocurarine, interpretations have
been complicated by desensitization and by the high rates
of action. We have developed a more appropriate method
for measuring the kinetics of antagonist-receptor
interactions.
The photoisomerizable azobenzene derivative, cis-2,2'
bis[a-(trimethylammonium)methyU azobenzene (2BQ), has
been studied with the voltage-clamped Electrophorus
electroplaque preparation. Dose-response studies were
conducted in the presence of an agonist (carbachol) and
2BQ over a voltage range of -50 mV to -150 mV. At
cis-2BQ concentrations less than 5 µM, the dose-response
curves are shifted in a parallel fashion to higher agonist
concentrations. A dose-ratio analysis reveals that
cis-2BQ is a pure competitive antagonist with a voltage
independent dissociation constant of 0.5 µM, roughly
equal to that of d-tubocurarine. Because 2BQ can exist
in two stable photoisomers, it was also of interest to
compare the effects of several 2BQ solutions with varying
ratios of the cis/trans isomers. The apparent affinity of
the receptor for 2BQ varies linearly with the mole
fraction of the cis isomer; the extrapolated dissociation
constant is >10 µM for the pure trans isomer.
These studies, on the equilibrium between receptors
and 2BQ, provide a basis for several kinetic experiments
employing light flashes:
1) Rapid changes of the cis-2BQ concentration;
2) Structural perturbations of the antagonist-receptor
complex;
3) Studies of the possible effects of 2BQ on the open
receptor channel.
*Department of Microbiology, Columbia University College of Physicians and Surgeons.
154. STUDIBS OF TISSUE-CULTURED CARDIAC MUSCLE
Investigators: Lee D. Chabala, Robert E. Sheridan
The heart muscle cell offers a unique opportunity to
study a wide variety of membrane conductances. We have
been culturing cardiac muscle from embryonic chickens
and looking at the membrane conductances that appear at
different stages of development. The cells are dissociated
by a combination of enzymatic digestion and mechanical
agitation. Placed in suspension culture, the dissociated
myocytes coalesce into heart reaggregates. Within 24
hours in culture, the reaggregates begin to contract
spontaneously. This beating continues during the first
week of culture and gradually disappears during the
second week, although the cells remain healthy throughout
this period. At present, we have just begun to investigate
the nature of the membrane conductances in cultured
heart cells. Preliminary examinations of single ionic
currents obtained with "patch" electrodes reveal several
different types of conductances that are qualitatively
distinct from any we have seen in skeletal muscles.
155. PHOTOLABILE PROTON DONORS AND pH CONTROL OF GAP JUNCTIONS IN CHlRONOMUS
Investigator: Jeanne M. Nerbonne
We have synthesized three classes of photosensitive
molecules capable of liberating protons efficiently upon
irradiation (quantum yields 0.25-0.50). Various structural
modifications have been inCorporated to optimize the
photochemical properties (absorption maxima, conversion
efficiencies, etc.), thermal stabilities, water solubilities,
and biological usefulness of these compounds.
Presently available evidence does not allow for a clear
decision on the relative roles of intracellular protons
versus calcium ions in controlling gap junction
permeability. The first class of photosensitive proton
donors, ortho-nitrobenzyl acetates, is therefore studied in
the salivary gland of the midge, Chironomus. When glands
are bathed in the parent compound and irradiated, there is
a precipitous change in junctional resistance, consistent
with a drop in intracellular pH. Neither the unphotolyzed
compound, the nitroso photoproduct, nor light alone
produce uncoupling. Uncoupling begins within 1 sec (as
fast as we can presently measure), although complete
uncoupling requires 2-3 min following a single light flash.
The dose dependence of this time course is highly variable
in our experiments. In vitro studies, in collaboration with
J. Connor (Bell Laboratories), reveal that light induces a
pH drop within 300 msec (as fast as we can measure);
preliminary data show that this change is followed by a
much slower (half-time, 1 min) phase of proton release.
We are presently studying the absolute time course of
these light-induced pH jumps and the role of molecular
structure in this process.
156. INTRACELLULAR pH AND GAP JUNCTIONAL PERMEABILITY IN EARLY EMBRYOS
Investigator: Jeanne M. Nerbonne
In collaboration with A. Harris, D. Spray, and M. V. L.
Bennett (Albert Einstein College of Medicine), we have
attempted to utilize the ortho-nitrobenzyl acetates to
study the effect of intracellular pH on gap junctional
conductance in developing blastomeres from squid,
fundulus, and axolotl embryos. In all of these prepara
tions, we find that exposure to the drugs causes a
precipitous drop in junctional conductance and a large
drop in intracellular pH in the absence of light. We
attribute this result to enzymatic cleavage of the esters,
because it can be inhibited (albeit with some variability)
by conventional esterase inhibitors and because the
thermal hydrolysis of these compounds is much slower
(half-time, 24 hr). After inhibition of the esterases, light
induces uncoupling with a half-time of about 1 min. Since
it is difficult to inhibit the hydrolysis completely and
reproducibly, we have prepared and characterized two
new classes of photosensitive pH donors which are not
esterase substrates. We are presently utilizing the new
compounds in the gap junction preparations.
157. PHOTOSENSrnYE CALCIUM CHELATES
Investigators: Jeanne M. Nerbo1U1e, Donald C.-T. Lo*
In many cells, alterations in intracellular calcium
concentration interact with intracellular pH; furthermore,
changes in internal calcium or pH have diverse physio
logical effects. To study these phenomena, we have been
developing molecules which liberate calcium ions rapidly
and efficiently upon irradiation. These compounds will be
used alone or in conjunction with the photoactivated
proton donors to examine the intracellular buffering
mechanisms for these small ions (on a wide range of time
scales) and to measure directly the physiological responses
to step changes of concentration.
111
We have taken two approaches to developing photo
sensitive calcium chelates. In the first case, we have
exploited the well-characterized azobenzene photo
chemistry to prepare molecules with appropriately
disposed carboxylate moieties. Depending on the
wavelength of irradiation, these molecules can be photo
isomerized reversibly between two states with different
binding affinities. For the second class of compounds,
irradiation produces an irreversible reaction that results
in a large drop in the ligand affinity; free calcium is
liberated. Several compounds of both classes have been
prepared and their calcium and magnesium affinities are
being determined using spectroscopic and electrochemical
techniques.
Recently, in a collaborative effort with ~· Kaplan
(University of Pennsylvania), we have begun synthetic
work on another class of photosensitive calcium chelates: ~
molecules which can bind calcium in the physiological
concentration range only after the photoreaction.
*Undergraduate, California Institute of Technology.
158. THE APPLICATION OP A PHOTOACTIVATABLE eAMP ANALOGUE TO STUDY THE MECHANISM OF AFTERDJSCHARGE IN BAG CELL NEURONS
Investigators: Jeanne M. Nerbonne, Felix Strumwasser
The bag cells in Aplysia generate an afterdischarge
(.r30 minutes at 14°C) in response to a few seconds of
electrical stimulation. The onset of afterdischarge and
the correlated enhancement of spike broadening have been
shown to be related to an increase in intracellular cAMP
and these phenomena can be initiated by cAMP analogues
in intact bag cell clusters and in isolated neurons in cell
culture. We have initiated experiments that exploit a
photoactivatable cAMP analogue (an ortho-nitrobenzyl
ester of cAMP synthesized by J. Engels, University of
Konstanz) to study the time course and concentration
dependence of cAMP-induced spike broadening and after
discharge. This analogue is membrane permeable and
liberates cAMP upon irradiation (A = 300 nm). We are
presently determining the rate of cAMP light-induced
release.
Initial experiments on intact bag cell clusters using
extracellular suction electrodes demonstrate that the
unphotolyzed cAMP analogue (up to 500 µM and exposed
for up to 1 hour) did not cause afterdischarge. Irradiation
of the preparation for times ranging from 15 sec-2 min
resulted in discharge in 12 out of 15 experiments.
112
Irradiation alone, on the other hand, elicited discharge in
1 of 30 experiments (3 min exposure to light). The
photoactivatable cGMP analogue (up to 500 µM) did not
cause afterdischarge in the absence or presence of light (8
experiments). 1n 6 of 8 experiments with the cGMP
compound, the results suggest that following irradiation,
the threshold for electrical stimulation of afterdischarge
was increased over control values.
Because these experiments involve large cAMP con
centrations, long irradiation times and, in addition, are
not amenable to kinetic analyses of the cAMP and cGMP
responses, experiments exploiting these techniques on bag
cell neurons in culture are planned.
159. PHOTOACTIVATED CYCLIC NUCLEOTIDES PROBE THE KINETICS OF CALCIUM CHANNEL REGULATION 1N HEART
Investigators: Joel Nargeot, Henry A. Lester, Jeanne M. Nerbonne
The positive inotropic and chronotropic effects of
beta-adrenergic agonists on heart arise partly from an
enhanced slow inward calcium current. Binding of
agonists to myocardial beta receptors causes an increase
in intracellular cAMP that, in turn, is thought to mediate
the slow inward current via a chain of events including
protein phosphorylation and leading to an increase either
in the number of functional calcium channels or of their
elementary conductance. The effects of adrenaline
require about 60 seconds to reach completion in frog heart
at 25"; little is known of the rates of the intermediary
biochemical events. Furthermore, the negative inotropic
effects of muscarinic agonists are based on a decrease in
this calcium current, as well as on an increased potassium
conductance. Among the postulated intracellular second
messengers for the muscarinic response are decreased
cAMP and increased cGMP.
To obtain more information on these events, we are
exploiting photoactivatable cAMP and cGMP analogues.
The ortho-nitrobenzyl esters of cAMP and cGMP, synthe
sized by J. Engels (University of Konstanz), cleave
efficiently upon irradiation and therefore allow precise
concentration jumps that are complete within at most 300
msec. We are studying the behavior of the slow calcium
current after such jumps. In voltage-clamped atrial
trabeculae from bullfrog heart, flash-induced concen
tration jumps (1-10 µM) of intracellular cAMP increase
the calcium current by at least twofold. The effect is
complete within 30 sec and proceeds along a roughly
exponential time course, with a time constant of 10-20
sec. Increases are detectable within 2 sec after the flash.
Neither the calcium current nor the muscarinic potassium
conductance is affected by similar concentration jumps of
cG MP. Further experiments are planned on the
pharmacology and kinetics of cAMP/cGMP channel
modulation in frog heart as well as in cultured myoballs
from chick heart; but it is already possible to conclude
that the response to beta agonists is significantly limited
in rate either by the activation of protein kinase or, more
likely, by the rate of protein phosphorylation.
160. TORPEDO SYNAPTOSOMES
Investigators: Henry A. Lester, E. Evan Shaffer
We are studying the process of acetylcholine release in
pinched-off cholinergic nerve terminals (synaptosomes)
from the electric organ of Torpedo, the giant electric ray.
A procedure has been adapted from the literature for the
isolation of synaptosomes. Acetylcholine is measured
with a gas chromatograph/mass spectrograph system (in
the laboratory of Dr. D. Jenden, University of California
at Los Angeles) and with a recently reported assay that
involves (a) the hydrolysis of acetylcholine to choline,
(b) the production of hydrogen peroxide by choline
oxidase, (c) the oxidation of luminol by peroxidase, and
finally, (d) measurement of luminescence from oxidized
luminol. The luminescence assay can detect tens of
pico moles.
The major present goal is to test and exploit .the
hypothesis that certain bacterial toxins exert their well
known blockade of acetylcholine release by performing a
covalent modification of a protein crucial to the release
process. A phosphorylation assay has been developed with
lysed Torpedo synaptosomes as the intended substrate of
botulinum toxin (supplied by H. Sugiyama, University of
Wisconsin) or tetanus toxin (supplied by B. Bizzini, Institut
Pasteur, Paris). The toxins themselves do not exhibit
protein kinase activity. However,. in initial experiments
we find that both toxins can be phosphorylated by lysed
synaptosomes. The heavy chains are better substrates
than the light chains; and in the case of tetanus toxin the
phosphorylation proceeds best in the presence of 0.01 %
sodium dodecyl sulfate. It is not yet known whether
phosphorylation of the toxins is important for their action.
161. PHYSIOLOGY OF NERVE AND MUSCLE CULTURF.S
Investigators: Jerome Pine, Jolm Gilbert*
Development of culture dishes which embody an array
of 61 extracellular microelectrodes in the dish bottom has
been completed. The electrical properties of the
electrodes appear to be better than those made in early
tests of the method; this should result in improved signal
to-noise. Toxicity was originally present, presumably
caused by traces of lead incorporated in the electrodes
during platinizing, but the addition of EDTA to the
medium has apparently solved the problem by chelating
the lead in solution.
We are now growing the first healthy chick myotube
cultures in the microcircuit dishes, and developing optimal
culture conditions for dissociated chick ciliary ganglion
neurons. In culture these neurons have exhibited the
ability to make cholinergic synapses on chick muscle, and
on each other, in recent experiments done in other
laboratories. We will try to study the development of
neuromuscular jWlctions in culture, and the effects of
activity on that development. We will also grow micro
cultures of a few neurons over the electrode array to
study the patterns of synapse formation and the effects of
various forms of stimulated activity.
We are also beginning to construct a facility to utilize
voltage-sensitive dyes in conjunction with the micro
circuit dishes. In tissue culture the measurement of dye
fluorescence that is sensitive to membrane voltage has
been shown to be practical by a group headed by A.
Grinvald at the Weizmann Institute. He is interested in
combining his technique with ours, and we anticipate a
fruitful joint effort during the coming year.
*Graduate Student, Division of Physics, Mathematics and Astronomy, California Institute of Technology.
162. SOFT X-RAY MICROSCOPY
Investigators: Jerome Pine, Jeanne M. Nerbonne, Sze-Kung Kwong*
We have been involved with groups at the State
University of New York at Stony Brook and at the IBM
Watson Laboratories in a collaboration to d? soft X-ray
scanning microscopy on wet, live, biological
specimens-cultured nerve and muscle in particular.
Since the last report, no further tests in an X-ray beam
have been done, but considerable development work has
taken place. The National Synchrotron Light Source, at
113
Brookhaven Laboratory on Long Island, has just begun to
operate, and our group has a beam line dedicated to soft
X-rays at that machine. The main X-ray optical
components of the beam, aside from the final focusing
zone plate, are now in place. During the coming months,
the beam will be studied and its performance charac
terized, while the synchrotron light source is being
brought from its current state of initial test running to
becoming a useful research facility.
At Caltech, we have been working on a procedure for
producing our standard microcircuit culture dishes and
then etching the glass substrate from under the central
500 micrometer diameter region where cells are to be
studied. This should leave the electrodes in place,
supported by the thin (less than 1 micrometer thick)
polyimide insulating film of the dish bottom, on which
cells can be grown. The etched dishes will permit X-rays
to penetrate the cultures, so that microscopy can be done
on specimens whose physiological properties can be
studied before, during and after irradiation.
The most critical component of the microscope is the
focusing zone plate that consists of hundreds of narrow
concentric circular openings (zones) in a gold film. The
minimum width of these annular zones determines the
limiting resolution of the mi-croscope. For initial tests a
zone plate with a minimum width of 1350 X is being fab
ricated. The procedures for making such a zone plate are
partly developed, and success is expected in the next few
months.
*Graduate Student, Division of Physics, Mathematics and Astronomy, California Institute of Technology.
PUBLICATIONS
Chabala, L. D., Sheridan, R. E. and Lester, H. A. (1982) Characterization of single ionic channels opened by nicotinic agonists. Soc. N eurosci. Abstracts, in press.
Krouse, M. E., Lester, H. A., Wassermann, N. H. and Erlanger, B. F. (1982) A study of the nicotinic acetylcholine receptor using a photoisomerizable competitive antagonist. Soc. Neurosci. Abstracts, in press.
Lester, H. A. and Nerbonne, J. M. (1982) Physiological and pharmacological manipulations with light flashes. Ann. Rev. Biophys. Bioeng.11, 151-175.
Lester, H. A., Steer, M. L. and Levitzki, A. (1982) Prostaglandin-stimulated GTP hydrolysis associated with activation of adenylate cyclase in human platelet membranes. Proc. Nat. Acad. Sci. USA 79, 719-723.
Lester, H. A., Steer, M. L. and Michaelson, D. M. (1982) ADP-ribosylation of membrane proteins in cholinergic nerve terminals. J. Neurochem. 38, 1080-1086.
114
Nargeot, J., Lester, H. A., Birdsall, N. J. M., Stockton, J., Wassermann, N. H. and Erlanger, B. F. (1982) A photoisomerizable muscarinic antagonist. Studies of binding and of conductance relaxations in frog heart. J. Gen. Physiol. 79, 657-678.
Nargeot, J., Lester, H. A., Nerbonne, J. M. and Engels, J. (1982) Photoactivated cyclic nucleotides probe the kinetics of calcium channel regulation in heart. Soc. N eurosci. Abstracts, in press.
Nerbonne, J. M. and Lester, H. A. (1982) Photolabile proton donors and pH control of gap junctions in Chironomus. Soc. Neurosci. Abstracts, in press.
Professor: Felix Strumwasser Visiting Associates: Sudarshan Malhotra, John c. Woolum Research Pellow: Karen Goldman Herman Graduate Students: Kent R. Jennings, Joanne M. Yeakley Research Staff: Susan L. Mailheau, Laurie Minamide,
Floyd Schlechte, John M. Scotese, Delilah A. Stephens, John R. Yuen
Support: The work described in the following research reports has been supported by:
Biomedical Research Support Grant (NIH) National Institutes of Health, USPHS Pew Memorial Trust Gordon Ross Medical Foundation University of Alberta, Canada
Bum~ Our studies are concerned with how the
nervous, system generates endogenous long-lasting
programs of membrane activity. The two model systems
that we use for such studies are the peptidergic bag cell
neurons, which generate a 30-minute afterdischarge in
response to a few seconds of synaptic input, . and the
isolated eye of Aplysia which generates a circadian (J'24
hour) rhythm of neural discharge in total darkness. Our
working hypothesis is that these endogenous programs of
neural activity involve an intracellular modulation of
membrane channels and pumps.
Over the last several years we have published evidence
that the 30-minute afterdischarge to brief synaptic input
in the bag cells is intimately associated with a rise in
intracellular cyclic AMP and consequent protein phos
phorylation. The two major phosphoproteins involved have
apparent molecular weights of 33,000 (BC-1) and 21,000
(BC-2) daltons. The BC-1 protein has an elevated
phosphorylation (J'8096) by 2 min into afterdischarge which
is still sustained (J'7096 elevation) by 20 min into after
discharge. In contrast, the BC-2 protein phosphorylation
state is unchanged by 2 min into afterdischarge but is
Pine, J. and Gilbert, J, (1982) Studies of cultured cells in dishes incorporating integral microcircuit electrodes. Soc. N eurosci. Abstracts, in press.
Sheridan, R. E. and Lester, H. A. (1982) Functional stoichiometry at the nicotinic receptor. The photon cross-section for phase 1 corresponds to two Bis-Q molecules per channel. J. Gen. Physiol., in press.
Weinstock, M. M. (1982) The properties of the Bis-Q activated channel and the nature of desensitization. J. Physiol., submitted for publication.
elevated by 20 min (J' 9096). The full paper has appeared in
Journal of Neuroscience (Jennings et al., 1982).
We are now examining whether there are protein
phosphorylation changes during the circadian cycle of
neural activity in the eye. Strumwasser, Stephens and
Minamide find that under diurnal conditions there is
enhanced phosphorylation (.r60%) during "day" versus
"night" when eye extracts are tested with exogenous
protein kinase catalytic subunit. These results imply that
the endogenous phosphorylation level is actually lower in
the day versus the night.
In other experiments we have examined the sensitivity
of the circadian period to perturbations of the intra
cellular environment due to pharmacological agents such
as La3+, caffeine and lithium. Woolum and Strumwasser
have shown that all three agents produce dose-dependent
increases in t~e circadian period. La 3+ is particularly
impressive in that at low concentrations, between 2 and
5 µM, the period increases from near the control value
(23.9 hr) to 28.0 hr. Caffeine at 6.5 mM increases period
to 30.4 hr while lithium at 40 mM increases period to
32.8 hr. We believe that the La3+ result, in particular, is
due to reduced cytosolic calcium uptake by mitochondria
since butacaine blocks the period-lengthening effect as
well as the known La 3+ reduction of mitochondrial
calcium uptake. We suggest that the period of the
circadian oscillator is only regulated indirectly through
homeostatic mechanisms that buffer the chemical
environment of the cell itself. In other words the
oscillator machinery itself is independent of any homeo
static controls explaining why these agents as well as
amino acids alter period.
Localization of the cell type within the eye producing 3 the circadian oscillation is being pursued by H-2-
deoxyglucose autoradiography by Herman. We have
preliminary evidence that during the 11night11 (inactive)
phase, glycogen bodies in photoreceptors and upper retinal
neurons are more prominent and abundant than during the
"day" (active) phase. Electrophysiological localization of
the cell type is also being pursued with primary cell
cultures of the eye. Strumwasser is adapting the
extracellular suction microelectrode technique, used so
successfully in other systems to record single channels,
for longer-term membrane current recording. Our best
success to date, using this approach, has been with
isolated photoreceptors.
Finally, we are interested in tracing how circadian
information is transmitted from the eyes to the rest of
the nervous system. We approach the problem in organ
culture by dissecting all the head ganglia with attached
eyes and abdominal ganglion and depend on Mailheau for
these preparations. Our assay is to record the
spontaneous discharges of nerve cells from the axons in
the nerve trunk. Elliott and Strumwasser have developed,
with a commercial microprocessor system (Apple II Plus),
a real-time waveshape recognition system that can
separate up to 5 spikes continously, print the spike counts
and automatically plot the waveshapes at intervals for
quality checks on the sorting.
Reference: Jennings, K. R., Kaczmarek, L. K., Hewick, R. M.,
Dreyer, W. J. and Strumwasser, F. (1982) J. Neurosci. 2, 158-168.
163. CALCWM DETERMINATION IN BAG CELLS
Investigators: Jolm c. Woolum, Felix Strumwasser
We are attempting to determine the role of internal
calcium on the bag cell afterdischarge and hormone
secretion. We have constructed an optical system that
detects changes in ca2+ concentration (see Ashley and
Campbell, 1979) in a single bag cell in tissue culture.
Light is passed through a cell that has been injected with
the dye Arsenazo IIL The light is then split into its
component wavelengths and monitored at 660 and 700 nm.
When ca2+ interacts with the dye, the dye absorbs more
at 660 nm but about the same at 700 nm so the ratio of
light transmitted at 660 to that at 700 will be inversely
proportional to the Ca 2+ content of the cell. Preliminary
results obtained when a train of action potentials is
evoked in a cell show that the system is capable of
detecting changes in internal ca2+ due to the action
115
potentials. We now hope to determine if there are any
changes in internal ca2+ during an afterdischarge elicited
by addition of 8-benzylthio cAMP or other active cyclic
AMP analogs.
Refermce: Ashley, C. C. and Campbell, A. K. (Eds.) (1979) Detection
and Measurement of Free Ca2+ in Cells. Elsevier/North Holland Biomedical Press, New York.
164. AGENTS THAT AFFECT THE NEURONAL CIRCADIAN RHYTHM IN THE EYE
Investigators: John c. Woolum, Felix Strumwasser
We have continued our search for agents that alter the
circadian rhythm of the intact Aplysia eye. We have
found that agents that block K + channels in nerves
(tetraethyl ammonium and 4-amino pyridine) will ilbolish
the circadian rhythm of compound action potentials
without stopping the spontaneous compound action
potentials themselves. This result is in agreement with a
number of reports that K + flux is important in expressing
the circadian rhythm in other systems. We have also
found that several mutagenic agents that are known to
have effects on nucleic acids (ethidium bromide,
acriflavin, and 5-fluoro uridine) will lengthen the period
of the circadian rhythm. Though effects of these agents
other than on nucleic acids may be important, these
results may help to support other evidence for a role of
nucleic acids in generating the circadian rhythm.
165. ENHANCED PROTEIN PHOSPHORYLATION WITH EXOGENOUS PROTEIN KINASE IN EXTRACTS OF APL YSIA EYI!S DURING DAY VERSUS NIGHT
Investigators: Felix Strumwasser, Delilah A. Stephens, Laurie Minamide
Our studies on the mechanisms of afterdischarge in
bag cells showed that cyclic AMP elevation and protein
phosphorylation were involved. In particular two proteins
(apparent molecular weights-33,000 and 21,000 daltons)
had enhanced phosphorylation during afterdischarge
(Jennings et al., 1982). The possibility that the circadian
rhythm of impulses in the eye of Aplysia is mediated by an
intracellular modulation of membrane activity due to
altered protein phosphorylation patterns seemed worth
investigating based on the results obtained for bag cell
afterdischarge.
Last year we determined that cyclic AMP levels are
increased during the day (versus the night) phase of the
circadian rhythm (ratio ..rl.6). We have examined protein
116
phosphorylation patterns in cell-free extracts of eyes
made at the two opposite phases of circadian activity.
Protein phosphorylation patterns were determined by
simultaneous addition of y-32P-ATP and beef heart
catalytic subunit of protein kinase (Sigma) to cell-free
extracts. After 1 minute of reaction, a stop buffer was
added and proteins were separated on SDS, 13.5% poly
acrylamide slab gels by electrophoresis. After Coomassie
blue staining, autoradiograms were obtained from dried
gels.
Under diurnal conditions we find that the overall
incorporation of y-32P-ATP into phosphoprotein is .rl.6
times higher during the day versus the night ( 4 paired
experiments, each experiment involving a total of 8 eyes).
When zinc ions are left out of the reaction mixture, the
ratio of day:night incorporation is reduced to .ri.1 (2
paired experiments, diurnal conditions). This finding has
been confirmed in 4 more paired experiments without zinc
in which the diurnal cycle of lighting was stopped in the
last 24 hours before the eyes were removed (mean ratio of
phosphorylation in day:night = 0.8). These results suggest
that there may be an endogenous zinc-sensitive factor
that fluctuates between day versus night extracts. Among
the possibilities explaining these results are altered
diurnal levels of phosphoprotein phosphatases for which
there is some evidence of zinc sensitivity. The results
also imply that the endogenous state of phosphorylation is
diminished in day versus night extracts of the eye.
Reference: Jennings, K. R., Kaczmarek, L. K., Hel"'lick, R. M.,
Dreyer, W. J. and Strumwasser, F. (1982) J. Neurosci. 2, 158-168.
166. ALTERED PATl'ERNS OP PROTEIN PHOSPHORYLATION IN EXTRACTS OP APLYSIA EYES AS A FUNCTION OF PHOTOPERIOD
Investigators: Felix Strumwasser, Delilah A. Stephens, Laurie Minamide
Back phosphorylation of cell-free extracts of Aplysia
eyes was performed (see Abstract No. 165) at the two
opposite phases of circadian activity. Intact animals were
maintained either under diurnal conditions (12 hours
light:12 hours dark) or continuous dim red light ("extended
darkness") for the 24 hours just before enucleation. Under
both conditions there were at least eight phosphoprotein
bands that could be consistently identified on one
dimensional SDS, 13.5% polyacrylamide gels. Quantita
tion by microdensitometry revealed that the most
prominent phosphoprotein band, apparent molecular
weight 17 ,000 daltons, was relatively stable. It accounted
for between 24% and 30% of total 32 P-incorporation by
the 8 bands with the higher value dependent on the
presence of zinc ions in the reaction mixture. When the
LD and extended darkness groups are compared, where
zinc ions are omitted in the reaction mixture, the most
prominent phosphoprotein to change has an apparent
molecular weight of 21,000 daltons. The ratios for this
phosphoprotein band of the extended darkness to LD
groups at the two circadian phases examined were 1.4
(inactive phase) and 1.3 (active phase). Interestingly, a
phosphoprotein with this apparent molecular weight is the
most abundant phosphoprotein in the bag cells and is one
of the two phosphoproteins that we found to significantly
increase phosphorylation during afterdischarge.
167. MAPPING NEURONAL ACTIVITY IN THE EYE OP APLYSIA WITH ffiGH RESOLUTION (11H)2-DEOXYGLUCOSE AUTORADIOGRAPHY
Investigators: Karen Goldman Berman, Felix Strumwasser
The eye of Aplysia contains a circadian oscillator
system that generates a rhythm in the frequency of
compound action potentials (CAPs) in the optic nerve
(Strumwasser et al., 1979). An important question that
has not been answered in this or other neuronal circadian
systems is whether the rhythm is generated in a particular
cell or cell type, or whether the interactions of many cells
are necessary to produce the rhythm (see Page, 1981). We
are examining this question by searching for circadian
variations in the activity of neurons in the Aplysia eye
using the 2-deoxyglucose (2-DG) method for mapping
neuronal activity.
In the most recent protocol, both eyes and attached
optic nerves were removed from individual Aplysia and
the CAP activity was recorded with suction electrodes in
separate chambers. Then, incubations in (3H)2-DG media
were carried out at the peak and trough of CAP activity
while recording electrically. After incubation, the eyes
were frozen in isopentane chilled with liquid nitrogen,
freeze-substituted in 2% OsO 4 in ethanol, and embedded
in Araldite. Sections were collected on slides and dipped
in nuclear track emulsion.
Autoradiograms reveal a striking accumulation of label
in clumps at the bases of the photoreceptors, and adjacent
to the pigment granules in ciliated neurons of the upper
retina. A lower density of silver grains covers other
portions of all cells, but there are few grains over the
nuclei or extracellular spaces. Preliminary observations
of periodic acid Schiff-stained material and electron
microscopic observations suggest that the dense aggre
gations of grains in photoreceptors and ciliated neurons
occur in regions of glycogen storage. A similar labeling of
glycogen by (3H)2-DG and the incorporation of (3H)2-DG
into glycogen was recently reported by Kai Kai and
Pentreath (1981).
Autoradiograms of an eye, which was demonstrated by
recording to be at the trough of CAP activity, had dense
clusters of grains in photoreceptors and ciliated neurons,
and heavy labeling of the cytoplasm of all cells. The
opposite eye, incubated at the peak of electrical activity,
was generally lightly labeled, although a few cells were
heavily labeled. The Schiff-stained sections of these eyes
showed more prominent and abundant glycogen bodies
during the trough as compared with the peak of CAP
activity. Three additional pairs of eyes have been
incubated during electrical recording to determine
whether these unexpected results, the increased uptake of
(3H)2-DG by the inactive eye and the increased Schiff
staining, occur repeatably. We will also examine these
eyes to see if heavy labeling of certain cells is consis
tently found in eyes incubated at the peak of CAP
.activity. We also hope to improve the localization of
soluble label by applying dry nuclear track emulsion to the
sections, and to study the pattern of incorporation of
(3H)2-DG into glycogen by examining glutaraldehyde
fixed material (Kai Kai and Pentreath, 1981).
References: Kai Kai, M. A. and Pentreath, V. W. (1981) J. Neurocytol.
10, 693-708. Page, T. L. (1981) In: Handbook of Behavioral Neuro
biology, Vol. 4. J. Aschoff (Ed.), pp. 145-172. Plenum Press, New York.
Strumwasser, F., Alvarez, R. B., Viele, D. P. and Woolum, J. c. (1979) In: Biological Rhythms and Their Central Mechanism. M. Suda, o. Hayaishi and H. Nakagawa (Eds.), pp. 41-56. Elsevier/North-Holland Biomedical Press.
168. PHYSIOLOGY OF CULTURED APLYSIA PHOTORECEPTORS
Investigators: Karen Goldman Herman, Felix Strumwasser
To a large degree some of the basic features of vision,
including extreme sensitivity and ability to adapt to a
wide range of light intensities, are properties of the
photoreceptors themselves. The cultured Aplysia eye cell
preparation developed by Strumwasser et al. (1979a)
117
presents an opportunity to examine the physiology of
isolated photoreceptors in vitro. In addition, the Aplysia
photoreceptors are of special interest because they
mediate the entrainment of the circadian oscillator
(Eskin, 1976).
We have begun to study with intracellular micro
electrodes the physiology of both isolated photoreceptors
and those in small clumps of cells. The photoreceptors
responded to light with graded depolarizations, which may
be followed by an undershoot at higher light intensities.
The latency of the responses of dark-adapted photo
receptors was considerable, ranging from as long as 4
seconds for intensities near threshold and decreasing to
about a second near saturation. The input resistance of
photoreceptors in clumps was considerably less than that
of isolated photoreceptors, which is consistent with
anatomical evidence for electrical connections between
photoreceptors and other cells (Strumwasser et al.,
1979b). By injecting pulses of current during the response
to light, we found that, like other invertebrate photo
receptors, the conductance of Aplysia photoreceptors
increases when illuminated.
References: Eskin, A. (1976) J. Neurobiol. 8, 273-299. Strumwasser, F., Viele, D. P. and Scotese, J. M. (1979a)
Soc. Neurosci. Abstracts 5, 809. Strumwasser, F., Alvarez, R. B., Viele, D. P. and Woolum,
J. c. (1979b) In: Biological Rhythms and Their Central Mechanism. M. Suda, O. Hayaishi and H. Nakagawa (Eds.), pp. 41-56. Elsevier/North-Holland Biomedical Press.
169. AN ECONOMICAL REAL-TIME NEURONAL SPIKE SORTER
Investigators: Felix Strumwasser, Susan L. Maillteau, Floyd Schlechte, James Elliott*, Joseph Mcintyre**
The analysis of the patterns of nerve impulses in a
population of neurons is presently limited by the short
term nature and difficulties of either multiple micro
electrode recordings or alternatively optical recording of
membrane potential activity by dye absorption or
fluorescence. The recording of nerve impulses from
several axons simultaneously is possible in molluscan
ganglia by relatively non-invasive electrode techniques
applied to the nerve trunks (Strumwasser, 1967). In
addition, intact molluscan ganglia can be organ cultured
for several weeks (Strumwasser and Bahr, 1966) and
spontaneous nerve inpulse discharges are recordable from
the nerve trunks over this prolonged period (Strumwasser,
1971).
118
The limiting factor in analyzing multiunit activities
from individual nerve trunks is the reliable recognition
and sel?fil'ation of the same unit impulse from a back
ground of several units. One recently published procedure
records such multiunit data on magnetic tape and utilizes
software on a lab minicomputer to analyze short segments
of data (Camp and Pinsker, 1979). Two important
parameters in unit identification in this system are
conduction velocity and amplitude. The main dis
advantages of this system are lack of real-time analysis,
the necessity for preliminary recording on magnetic tape
and the dedicated use of a large lab minicomputer system
(PDP 11/34).
Our real-time spike sorting system is based on a
standard Apple Il Plus computer with 48 K bytes of
memory, a standard minifioppy disk drive and an Epson
MX80FT printer equipped with Graftrax chips for high
resolution graphics. Two of the interface cards in the
Apple computer are special purpose. One card contains an
analog-to-digital converter with a 1'look-back11 feature
which allows the viewing of the action potential wave
shape before as well as after crossing a threshold level.
The second card contains a system clock to read time-of
day during the experiment.
The overall system has two modes of operation, a spike
selection mode and a continuous run mode. In the spike
selection mode, an operator selects spikes that have
exceeded threshold to be classified from a high resolution
graphics display on a TV monitor. A selected spike is
assigned to one of five unit classes after a joy-stick
controlled cursor is used to select up to five voltage,time
coordinates which are distinctive features of the unit
waveshape. High and low limits are set at each of these
coordinates. These distinctive voltage,time coordinates
and limits are stored in memory and represent a template
identifying the unit. In the continuous run mode, an
incoming spike that exceeds the initial voltage threshold
is compared to each of the four unit waveshape templates.
When a proper match occurs, a count register is upgraded
for this unit and the full waveshape is displayed in a
selected 1?81't of the TV monitor screen. Unclassified
incoming units increment the zero class register and are
also displayed. During the run mode, spike counts are
printed at intervals selected by the operator. Quality
checks of the classified unit waveshapes are an important
feature of the overall system and are plotted by the
printer. They are plotted either on manual request or
automatically at regular intervals during the continuous
run mode.
References: Camp, c. and Pinsker, H. (1979) Brain Res. 169, 455-479. Strumwasser, F. (1967) In: Invertebrate Nervous Systems:
Their Significance for Mammalian Neurophysiology. C. A. G. Wiersma (Ed.), pp. 291-319. University of Chicago Press.
Strumwasser, F. (1971) J. Psychiatr. Res. 8, 237-257. Strumwasser, F. and Bahr, R. (1966) Fed. Proc. 25, 512.
*Digital Engineering Co., Whittier, California. **Undergraduate, California Institute of Technology.
170. ffiGH POTASSIUM STIMULATION OF 35s-METHIONINI! INCORPORATION INTO ATRIAL GLAND PEPTIDE B
Investigators: Joanne M. Yeakley, Felix Strumwasser
In Biology 1981, No. 195, preliminary results of mem
brane binding experiments employing 35s-methionine
labeled peptide B were described. In order to increase the
specific activity of the ligand for further studies, the
effect of high K + medium was assayed on 35s-methionine
incorporation into TCA-insoluble total protein. An eight
hour preincubation of intact glands in 57 mM K + seawater
(a fivefold increase) caused the subsequent incorporation
of 35s-methionine in normal K + medium to increase by
280%. Further increases in specific activity can be
obtained by radioiodination (about one hundredfold, n ;;; 1).
This approach is currently being pursued.
171. ATRIAL GLAND PEPTIDE B IMMUNOHISTOCHEMISTRY
Investigators: Joanne M. Yeakley, Felix Strumwasser
An immunohistochemical approach was adopted to
localize the distribution of immunoreactive peptide B in
the atrial gland and nervous system of Aplysia. Antisera
to a conjugate of B and thyroglobulin were raised in two
rabbits and purified for IgG. These sera demonstrated
immunospecific binding to peptide B and to thyroglobulin
by enzyme-linked immunosorbant assay. However, efforts
to localize peptide B immunoreactivity in cryostat
sections of atrial gland remain unsuccessful. Some
staining of very small cells and fibrous tissue has been
observed in the abdominal ganglion. A variety of control
experiments are presently being pursued to resolve this
problem.
PUBLICATIONS
Chiu, A. Y. and Strumwasser, F. (1981) An immunohistochemical study of the neuropeptidergic bag cells of Ai;>lysia. J. Neurosci. 1, 812-826.
Jennings, K. R., Host, J. J., Kaczmarek, L. K. and Strumwasser, F. (1981) Serotonergic inhibition of afterclischarge in i;>ei;>tidergic bag cells. J. Neurobiol. 12, 579-590.
Jennings, K. R., Kaczmarek, L. K., Bewick, R. M., Dreyer, W. J. and Strumwasser, F. (1982) Protein i;>hosi;>horylation during afterdischarge in i;>ei;>tidergic neurons of Ai;>lysia. ;f. Neurosci. 2, 158-168.
Kaczmarek, L. K., Jennings, K. R. and Strumwasser, F. (1982) An early sodium and a late calcium i;>hase in the afterdischarge of peptide-secreting neurons of Aplysia. Brain Res. 238, 105-115.
Kaczmarek, L. K. and Strumwasser, F. (1981) Net outward currents of bag cell neurons are diminished by a cAMP analogue. Soc. Neurosci. Abstracts 7, 932.
119
Strumwasser, F. (1982) Introduction: Comparative neurobiology of i;>ei;>tidergic systems. In: Pei;>tidergic Neurons: Physiology and Biochemistry. Neurobiology and Behavior Thematic Symposium. Fed. Proc. 41, in press.
Strumwasser, F., Kaczmarek, L. K. and Jennings, K. R. (1982) Intracellular modulation of membrane channels by cyclic AMP-mediated protein in i;>hosphorylation in the i;>ei;>tidergic bag cell neurons of Ai;>lysia. N eurobiology and Behavior Thematic Symi;>osium. Fed. Proc. 41, in press.
Strumwasser, F., Kaczmarek, L. K., Jennings, K. R. and Chiu, A. Y. (1981) Studies of a model i;>ei;>tidergic neuronal system, the bag cells of Ai;>lysia. In: Neurosecretion: Molecules, Cells, Systems. D. S. Farner and K. Lederis (Eds.), i;>i;>. 249-268. Plenum Press, New York.
Van Harreveld, A. and Strumwasser, F. (1981) Glutamate agonistic and antagonistic activity of L-proline investigated in the hippocampal slice. Neuroscience 6, 2495-2503.
NEUROBIOLOGY AND BEHAVIORAL BIOLOGY
John M. Allman
Derek H. Fender
Masakazu Konishi
Marianne E. Olds
R. W. Sperry
David C. Van Essen
Associate Professor: John M. Allman Senior Research Associate: Takuji Kasamatsu Visiting Associates: James F. Baker, Kunio Nakai Research Fellows: EveLynn McGuinness, Kazushige Watabe Graduate Student: David W. Sivertsen Member of the Professional Staff: Francis M. Miezin Research Staff: Maria A. Clancy Laboratory Staff: A. Anthony Balber, Claude N. Boehm,
Miriam L. Rusch, Karly Wang
support: The work described in the following research reports has been supported by:
Biomedical Research Support Grant (NIH) L. S. B. Leakey Foundation National Institutes of Health, USPHS National Science Foundation Pew Memorial Trust
SUmmary: This year Dr. Takuji Klisamatsu joined our
group as a Senior Research Associate. Dr. Kasamatsu has
a very active program that is focused on the mechanisms
of neural plasticity during the development of visual
cortex (see Abstract Nos. 173-176, 178-182, 186 and 187).
We have achieved several important technical innovations
during the past year. Francis Miezin from our laboratory
and John Power and Mike Walsh of the Biology Electronics
Shop have developed a highly versatile video display
system for producing visual stimuli to be used in the
neurophysiological study of the functional organization of
the extrastriate cortical visual areas. In most
illboratories the visual stimuli used to probe the
organization of visual cortex have been limited to bars
and gratings presented against a uniform background. The
new video system has the virtue that it can present visual
patterns that much more closely approximate natural
visual scenes. The system has the capability of presenting
a variety of different depth cues based on motion and
binocular disparity as well as stimuli presented against
moving textured backgrounds. This system has enabled us
to investigate the human perception of motion depth cues
(see Abstract No. 183) and has led to the discovery of
antagonist direction-specific mechanisms in Area MT
which may relate to the mechanisms that accomplish the
perceptual stability of the visual world during eye, head
and body movements (see Abstract No. 177). During the
past year Francis Miezin and I have developed a new
training procedure that has enabled us to use owl monkeys
as both psychophysical and neurophysiological subjects in
the exploration of the functions of the extrastriate
cortical visual areas.
123
172. THE ORGANIZATION OP THE CORTICAL VISUAL AREAS JN A STREPSIRHINE PRIMATE
Investigators: John M. Allman, EveLynn MeGuinness, Kati Shepherd*
The order Primates is divided into two suborders:
haplorhines (tarsiers, monkeys, apes, man) and
strepsirhines (galagos, lorises, lemurs). In order to trace
the evolutionary history of the cortical visual areas, we
are seeking to determine which areas are present in both
suborders. To achieve this we have been mapping the
representations of the visual field with microelectrode
recordings in the visual cortex of a strepsirhine Primate,
Galago senegalensis. Our results indicate that the
primary visual area (V-I), the second visual area (V-II), the
middle temporal area (MT), the dorsolateral (DL), the
ventral posterior (VP) and the ventral anterior (VA) are all
present in Galago. Previous data indicate that these areas
are present in New World monkeys. There is also
substantial evidence from other laboratories that most if
not all of these areas also are present in Old World
monkeys. These results suggest that these areas were
present in the primitive Primates that were the common
ancestors of these living forms. The development of these
areas may be related to the acquisition of large, frontally
directed eyes and expanded posterior neocortex evidenced
in the fossil remains of the early Primates from Eocene
deposits 55 million years old.
*Rutgers University.
173. ONTOGENY OP MONOAMJNERGIC RECEPTORS
Investigators: Giista Jonsson*, Takuji Kasamatsu
In a search for biochemical correlates of changes in
neuronal plasticity in the developing kitten neocortex, we
started to examine ontogeny of both endogenous
catecholamines (CAs) and B-adrenoreceptor binding sites.
The following two assays were used: one is the measure
ment of endogenous CAs by high pressure liquid
chromatography combined with an electrochemical
recording method developed by Adams and his associates
(Keller et al., 1976; Jonsson et al., 1980), and the other is
a standard receptor binding assay for B-adrenoreceptors
using a nanomolar amount of 3H-dihydroalprenolol as a
radioligand (Bylund and Snyder, 1976; Jonsson and
Hallman, 1978). Our preliminary result is quite sugges
tive. We have consistently noted small but significant
peaks of endogenous norepinephrine (NE) at 4 and 11
weeks, superimposed on its gradual increasing trend. This
124
maturation curve for the cortical NE, at least at its early
portion, seems roughly to fit the susceptibility curve to
monocular deprivation. The correspondence between the
maturation curve in the intracortical NE system and that
of visual cortical cells became even better when we
looked at the ontogeny of a-adrenoreceptor binding sites
as an index for the development of the NE system.
A slab (about 5 mm wide x 10 mm Jong x 2 mm thick)
of cortical tissue from the frontal and the occipital areas
will be dissected out from the brain of deeply
anesthetized (45-50 mg/kg, Nembutal, i.p.) kittens and
adult cats. Tissue samples will be kept frozen at -70°C
until their shipment to Stockholm via SAS air freight. A
hidden merit of this project is that we can pile up
materials for biochemical assay whenever we obtain more
kittens than we can use for our physiological and
morphological studies.
References: Bylund, D. and Snyder, S. (1976) Molec. Pharmacol. 12,
658-680. Jonsson, G. and Hallman, H. (1978) Neurosci. Lett. 9,
27-32. Jonsson, G., Hallman, H., Mefford, I.· and Adams, R. N.
(1980) In: Central Adrenaline Neurons-Basic Aspects and Cardiovascular Function. K. Fuxe, M. Goldstein, B. Hokefelt and T. Hokefelt (Eds.), pp. 59-71. Pergamon Press, Oxford.
Keller, R., Oke, A., Mefford, I. and Adams, R. N. (1976) Life Sci. 19, 995-1004.
*Department of Histology, Karolinska Institute, Stockholm, Sweden.
174. INVOLVEMENT OF CYCLIC AMP IN VISUAL CORTICAL PLASTICITY
Investigator: Takuji Kasamatsu
A preliminary result strongly suggested that an
increase in adenosine cyclic monophosphate (cAMP) within
the cortex may be involved in enhancing neuronal
plasticity by norepinephrine (NE) (Kasamatsu, 1980). I
first created the brain region, at the corresponding site in
both hemispheres of the kitten's visual cortex (6-7 weeks
of age), which had lost plasticity by the continuous and
localized perfusion with 4 mM 6-hydroxydopamine
(6-0HDA) in 0.4% ascorbate in saline (pH 3). After one
week of this pretreatment, the left side was perfused,
using the same implanted cannula, for another week with
dibutylyl cAMP and the right side with the vehicle
solution alone. The results are summarized as follows:
. (1) Most cells recorded in both dibutylyl cAMP-perfused
and control cortices had normal receptive fields. (2) In
the visual cortex perfused with dibutylyl cAMP, a class of
monocular cells which exclusively responded to stimula
tion through the nondeprived, ipsilateral eye was
preeminent, suggesting dibutylyl cAMP in fact restored
neuronal plasticity to the visual cortex which had Jost it
due to the prior 6-0HDA treatment. (3) The control
hemisphere of the same animals contained many binocular
cells as expected from our previous results with the
6-0HDA perfusion (Kasamatsu et al., 1979). (4) At the
concentration of 10-7 M dibutylyl cAMP stored in the
osmotic minipump system, the above effect started to
become less obvious, suggesting that 10-7 M dibutylyl
cAMP is near the threshold for enhancing plasticity. As
suggested for NE, the actual threshold concentration at
the recording site has to be much lower than this value.
Next, I would like to test, using the same paradigm of
replacement, the effects of cholera toxin, which is a
potent stimulant of plasma membrane-bound cAMP
synthesizing enzyme, adenylate cyclase (Bennett and
Cuatrecasas, 1976). Choleragen (I µg/µl or 1.2 x 10-4 M,
Miller and Kelly, 1975) will be perfused into one visual
cortex of 6-0HDA-pretreated kittens and the other
cortex with the vehicle solution alone. If I am able to
obtain a sign for enhanced plasticity in the choleragen
perfused cortex, I will repeat the same study by diluting
the toxin solution tenfold each time to determine the
threshold concentration. Then, I would like to test the
effects of choleragenoid (binding subunit, molecular
weight 66,000), as a control, which has a strong binding
capacity and thus works as a true competitive antagonist
of binding but which does not by itself have a biological
action. In in vitro study, only a brief exposure of the
tissue to low concentration of toxin (lo-10-10-8 M) is
sufficient for full biological effects (Pierce et al., 1971).
Another important test to be done is to study, in a similar
replacement paradigm, the effects of guanosine triphos
phate (GTP) and its nonhydrolyzable synthetic analogue,
Gpp(NH)p that are also known to activate adenylate
cyclase activity, especially when combined with a a--7 agonist such as isoproterenol (apparent Km at 10 M,
Aurbach et al., 1975).
References: Aurbach, G. D., Spiegel, A. M. and Gardner, J. D. (1975)
Adv. Cyclic Nucleotide Res. 5, 117-132. Bennett, V. and Cuatrecasas, P. (1976) In: The Specificity
and Action of Animal Bacterial and Plant Toxins. Series B, Vol. 1: Receptors and Recognition. P . Cuatrecasas (Ed.), pp. 1-66. Chapman and Hill, London.
Kasamatsu, T. (1980) Soc. Neurosci. Abstracts 6, 494. Kasamatsu, T., Pettigrew, J. D. and Ary, M. (1979) J.
Comp. Neural. 185, 163-182. Miller, R. J. and Kelly, P.H. (1975) Nature 255, 163-166. Pierce, N. F., Greenough, W. B. III and Carpenter, C. C. J.
(1971) Bacterial. Rev. 35, 1-13.
175. KNl!RGY METABOLISM IN THE VISUAL CORTEX
Investigator: Takuji Kasamatsu
It is important to examine whether NE-enchanced
plasticity is necessarily accompanied by an elevation of
local glucose metabolism or cerebral blood flow. As an
initial trial, I studied the effects of glucose perfusion
under the replacement paradigm in combination with
monocular lid suture. First, the visual cortex of a 5-
week-old kitten was perfused bilaterally with 4 mM
6-0HDA for a week. A week later, the left hemisphere
was then perfused with 10 mg/ml glucose (about tenfold
more concentrated than the glucose level in the plasma or
cerebrospinal fluid in cats) for the second week while the
other hemisphere was perfused with the saline alone. The
kitten's right eye was closed for the second week only.
The implanted cannula in each hemisphere was kept at the
same site for these two weeks. Because of a relatively
iarge area of tissue damage near the cannula site,
probably related to uncontrolled infection, etc., the
recording microelectrodes were placed at least 3-3.5 mm
away, on the cortical surface, from the perfusion site to
register cells with normal receptive fields (except for
ocular dominance). The electrode track was long for both
sides (6-9 mm). The preliminary result was not clear-cut.
By comparing the ocular dominance distribution obtained
from the two hemispheres, however, it may be suggested
that there was no sign of a strong shift of ocular
dominance in the hemisphere perfused with glucose for a
week together with monocular lid suture. The
binocularity in the glucose hemisphere was 4796 and it
may be compared to that of 23% in the other hemisphere
which showed the usual extent of shift. The result in the
control hemisphere may imply that the distal end of this
long electrode track left the catecholamine terminal
denervated area and entered into the boundary zone in
which some amount of NE was still available. In
supporting this guess, we obtained consecutively the last
14 cells which exclusively responded to stimulation of the
nondeprived eye. In the first half of the track (N = 16),
the proportion of binocular cells was relatively high (7 of
16 cells). It may be wise to test a wide range of glucose
125
concentrations, as done in our previous studies under the
replacement paradigm with NE and dibutylyl cAMP.
I would like to extend this line of study using
neuropeptides such as vasoactive intestinal polypeptide,
which is known to stimulate the enzymatic hydrolysis of
glycogen in mouse cortical slices at Ec502s nM
(Magistretti et al., 1981), in place of glucose. When we
use a neuropeptide in our continuous perfusion system, it
is important to pre-coat the inside of the cannula
minipump system with Prosil-28 in order to reduce the
adhE;!sion of peptide to the inner wall of the cannula and
reservoir.
Reference: Magistretti, P. J., Morrison, J. H., Shoemaker, w., Sapin,
V. and Bloom, F. E. (1981) Proc. Nat. Acad. Sci. USA 78, 6535-6539.
176. QUICK CHANGl!S IN OCULAR DOMINANCE
Investigators: Takuji Kasamatsu, Paul Heggelund*
Physiological changes in ocular dominance can quickly
take place following monocular visual experience. In a
normal animal that is acutely anesthetized and paralyzed,
if the visual cortex was concurrently perfused with a high
level of exogenous norepinephrine (NE),_ we found a
significant increase in monocular cells at the expense of
binocularly driven cells which are commonly present
(.r80%) in the normal visual cortex (Heggelund and
Kasamatsu, 1981). We interpreted this striking change in
the ocular dominance distribution as due to the nsquint11
effect which was exaggerated up to one extreme by
exogenous NE.
In our preliminary study (Kasamatsu and Heggelund,
1981), we noted that this acute "squint" effect on the
normal cortex can be blocked or reversed by approxi
mately superimposing the two sets of receptive fields by
means of an appropriate prism in front of one eye during
the NE (0.5 mM) perfusion. In the ocular dominance
histogram obtained before placing a prism (14 prism
diopter) to dislocate the image on a tangent screen (57 cm
from the cat's eye} by 8°, we found a massive loss of
binocular cells. After we finished with track 1, the
anesthetized and paralyzed animal was left facing slowly
rotating black and white gratings under light overnight.
From track 2, about 0.5 mm anterior to track 1, we
obtained this time many binocular cells as expected for
the normal visual cortex. This phenomenon also seems to
be age-dependent, the extent of loss of binocular cells
126
being larger in younger animals than in adults.
In order to confirm these initial results, I shall record
three times from the same animal successively: first to
show the W-shaped distribution, next a return to the
normal distribution by setting up an appropriate prism,
and finally reappearance of the W-shaped distribution
after removing the prism. The cortex is perfused
throughout the recordings with 0.5 mM NE.
References: Heggelund, P. and Kasamatsu, T. (1981) Adv. Physiol. Sci.
36, 233-242. Kasamatsu, T. and Heggelund, P. (1981) Soc. Neurosci.
Abstracts 1, 142.
*Neurobiology Laboratory, University of Trondheim, Trondheim, Norway.
177. ANTAGONISTIC DIRECTION..,SPECIFIC MECHANISMS IN AREA MT IN THE OWL MONKEY
Investigators: Francis M. Miezin, EveLynn McGuinness, John M. Allman
It is well known that most Area MT neurons are
strongly selective for the direction of stimulus motion.
We wished to know how MT neurons behaved when stimuli
were presented against moving textured backgrounds such
as occur commonly in natural stimulus conditions. We
have explored the receptive field structure and
directionally selective mechanisms in MT using a newly
developed microprocessor-based video display that can
present simultaneously bars and random dot patterns
moving in different directions. In nearly all MT neurons
tested, when a bar moving in the cell's preferred direction
is presented against a background of random dots moving
in the same direction, the response is greatly reduced
relative to the response to the bar moving on a
background of stationary random dots. When the
background is moved in the opposite direction, the
response to bar stimulation in the preferred direction is
either less inhibited or in some cases strongly facilitated.
The effects of background stimulation generally could be
obtained by stimuli restricted to the neuron's
conventionally defined receptive field but were stronger
when background stimulation extended into the
surrounding visual field.
In a related set of experiments we sought to map the
extent and nature of the antagonistic mechanism in the
surround. By continuously driving MT neurons by stimu
lating the conventionally defined receptive field and by
simultaneously moving fields of random dots in the
surrounding visual field, we have found that MT neurons
are affected by stimuli presented at considerable
distances from the conventional receptive field. These
surround effects are often directionally selective with the
preferred direction of the center being the direction of
maximum inhibition in the surround (see Figure 1). This
antagonistic direction-selective mechanism provides an
ongoing comparison between movement occurring locally
in the visual field and more global retinal image move
ments such as would occur during eye and head movement
or bodily transport. Thus they may be related to the
mechanisms that accomplish the perceptual stability of
the visual world during eye, head and body movements as
well as to perceptual mechanisms in figure-ground
discrimination.
178. REGROWTH OF CENTRAL CATECHOLAMINERGIC FIBERS IN CAT VISUAL CORTEX FOLLOWING LOCALIZED LESIONS WITH 6-HYDROXYDOPAMINE
lnvestigators: Kunio Nakai, Glista Jonsson*, Talarji Kasamatsu
The catecholamine (CA) system is known for its
remarkable capability of regrowth of terminal fields or
sprouting after partial lesions, provided that the proximal
axons and the somatas of CA cells are saved from initial
lesions (Moore et al., 197 4; Sachs and Jonsson, 1975).
The visual cortices of 5-week-old kittens, which had
been priorly subjected to the bilateral resection of
superior cervical ganglia, were locally perfused with 4 mM
6-hydroxydopamine (6-0HDA) for a week. By varying the
survival time (0, 2 and 4 weeks) after stopping the
6-0HDA perfusion, such cortices were then studied by
either glyoxylic acid fluorescence histochemistry (ltakura
et al., 1981) or a biochemical assay for endogenous CAs
using high performance liquid chromatography (Keller
et al., 1976). Immediately after the end of the 6-0HDA
perfusion, no CA terminals were seen within a cortical
area whose radius was about 5 mm from the perfusion site
(Kasamatsu et al., 1981a). Two weeks after stopping the
6-0HDA perfusion, the CA-terminal-depleted area
seemed to have shrunk (radius, 2-3 mm). Some of the
regrowing fibers were observed even at the edge of the
gliosis caused by the palcement of a cannula. Electron
microscopic examinations of the visual cortices of kittens
that survived for 3 weeks showed the regrowing CA
terminal boutons with pleomorphic granular vesicles,
apparently different from the normal CA terminals
containing small-cored or granular vesicles. By four
weeks, CA fibers with terminals were seen virtually
everywhere in the visual cortex including the site of
previous cannulation. At the center of perfusion,
however, the intensity of CA fluorescence did not yet
return to the normal level. In an area 3-4 mm away from
the perfusion site, complexity and intensity of fluorescent
fibers were already indistinguishable from those in the
normal. In corollary with the above result in fluorescence
histochemistry, biochemical assays 8.l.so revealed the quick
recovery of CA contents in a cortical area which had been
once depleted of its CA terminals with 6-0HDA.
Furthermore, one and two weeks after stopping the
6-0HDA perfusion, S-adrenergic receptor binding sites,
as meaaured by light microscopic autoradiography with 3H-dihydroalprenolol (Palacios and Kuhar, 1980), showed
more than a 50% increaae (in layers II and llI) at an area
2-3 mm from the perfusion site (denervation super
sensitivity). Thus, both the quick regeneration of
denervated CA terminals and the supersensitivity of aadrenoreceptors may help to explain the observed
incomplete blockade of the ocular dominance shift in
monocularly-deprived and 6-0HDA-treated visual cortex
(Kaaamatsu and Pettigrew, 1979; Kaaamatsu et al., 1979)
HCXXl"Ef"."6 9. 6 .
.....n.nn..r--.---m...--'"""--~--- 3 3" .
= ___ __,__.mnnn!!!fL-~·--- 2 I" •
..... !h~ -18"·
-----.ao11o11"""'Pcn1llr.-11111-----'"- I 5" • ....,_.....,..._..m:llDladllmilfl---- I 2" • --r---~--..az:ru!!.a...mnn.1J11:n.11:11:11nn --~......., s" • ____________ __........._ 60.
3" . a.
127
and also the incomplete suppression by the 6-0HDA
perfusion of cortical recovery from the effects of prior
monocular deprivation (Kasamatsu et al., 198lb).
We also found that the rate of CA terminal regrowth
following the 6-0HDA treatment was significantly
accelerated when 3.3 mM substance P was perfused
intracisternally.
References: ltakura, T., Kaaamatsu, T. and Pettigrew, J. D. (1981)
Neuroscience 6, 159-175. Kaaamatsu, T. and Pettigrew, J. D. (1979) J. Comp.
Neurol. 185, 139-162. Kasamatsu, T., Pettigrew, J. D. and Ary, M. (1979) J.
Comp. Neurol. 185, 163-182. Kasamatsu, T., Itakura, T. and Jonsson, G. (1981a) J.
Pharm. Exp. Ther. 217, 841-850. Kasamatsu, T., Pettigrew, J. D. and Ary, M. (198lb) J.
Neurophysiol. 45, 254-266. Keller, R., Oke, A., Mefford, I. and Adams, R. (1976) Life
Sci. 19, 995-1004. Moore, R., Bjorklund, A. and Stenevi, U. (1974) The
Neurosciences. Third Study Program. pp. 961-977. The MIT Press, Cambridge, Massachusetts.
Palacios, J. M. and Kuhar, M. J. (1980) Science 208, 1378-1380.
Sachs, c. and Jonsson, G. (1975) In: Chemical Tools in Catecholamine Research. G. Jonsson, T. Malmfors and Ch. Sachs (Eds.), Vol. 1, pp. 163-171. North Holland Publishing Co., Amsterdam.
*Department of Histology, Karolinska Institute, Stockholm, Sweden.
HCXX I "El'". "4 I 8 • 6 .
nfR1tn11J1111 3 3 Ill •
n.n...nn11n1n ... 3 fJ /II • n.t1111trllmllfl.i1.1111nmll!Htn...irtf"1llllfldhf~270.
"™""""""""""m-.ma-.i..mllllll'i..f!!~240. rffll1rflmlltl...tfD,.-..... .,_,..llllllOOnnll~21". .-tllntMrlllfli....--_.JJ!!11h!l.'Jltlln I 8" •
m1Jt1Jn111.flt)iiba..--.---..rfflkllblllllmm1so. IMfl.nnfl!TtmO..---~llhllt.1111"'1 I 2 " •
9" . 60 .
30· ~.
Figure 1. Histograms illustrating the responses of a single neuron in Area MT to 12 angles of stimulus motion (0° through 330°). The left set of histograms illustrates the response of the neuron to a moving random dot pattern wtthin a stationary window that corresponded to the conventionally defined receptive field. For each direction of stimulus movement, a 2-second foreperiod is shown, followed by a 2-second stimulus presentation period, which is underscored in the histograms, followed by a 2-second afterperiod. Stimuli were presented in pseudorandom order with 10 presentations of each direction of motion. The neurons were excited by stimuli moving at angles between 90° and 240°. In the right set of histograms, the conventional receptive field of the neuron was stimulated continuously with a noise pattern moving in the preferred direction. During the middle 2-second Wlderscored period. a random dot pattern occupying the visual field surrounding the receptive field was moved in 12 different directions. The neurori was strongly inhibited by the same directions of movement in the surroWld that excited the neuron's receptive field center (90°-240°). The ongoing response to the receptive field center stimulation was facilitated by surround movement at 0°, 300°, and 330°.
128
179. MORPHOWGY OF CATECHOLAMINERGIC TERMINALS IN CAT VISUAL CORTEX
Investigators: Kunio Nakai, Takuji Kasamatsu
The normal distribution pattern and the ultrastructure
of norepinephrine-containing nerve terminals in kitten
visual cortex have been reported (Itakura et al., 1981).
We are particularly interested in studying the nature of
synaptic contacts made by catecholaminergic (CA)
terminals in cat visual cortex. In order to label individual
terminals, the cortex was briefly perfused with a CA
specific neurotoxin, 6-hydroxydopamine (6-0HDA). We
used early signs of degeneration as a marker of CA
terminals.
Electron microscopic observation in serial sections
obtained from the 6-0HDA-treated and glutaraldehyde
fixed visual cortex has revealed that only 28.2% of
identified CA terminal boutons (N : 131) made either
asymmetrical or symmetrical synaptic contacts with
postsynaptic elements, mostly dendritic shafts and spines.
This low proportion of synapse-forming CA terminal
boutons was in contrast with the much higher proportion
(57.6%) seen among non-aminergic boutons in the same
area of the visual cortex. These results are consistent
with the previous findings based on the glyoxylic acid
KMnO 4 fixation method (ltakura et al., 1981). The
proportion of synapse-forming CA terminal boutons was
highest in layers II and III (38.1 %) in which not only the
CA terminals and fibers but also the 13-adrenergic
receptors were most richly distributed. Further, most of
the synapses of CA terminals in layers II and III were the
symmetrical type that has been considered to be an
inhibitory synapse (Ribak, 1978; Peters and Proskauer,
1980). The central CA neurons seem to have dual actions
in the neocortex: one is neurohumoral in nature and the
other is mediated bY conventional synapses.
References: ltakura, T., Kasamatsu, T. and Pettigrew, J. D. (1981)
Neuroscience 6, 159-175. Peters, A. and Proskauer, C. C. (1980) J. Neurocytol. 9,
163-183. Ribak, C. E. (1978) J. Neurocytol. 7, 461-478.
180. DISTRIBUTION OF 8-ADRENORECEPTORS IN CAT VISUAL CORTEX
Investigators: Kunio Nakai, Takuji Kaswnatsu
Using a light microscopic autoradiographic method
(Palacios and Kuhar, 1980), we studied the ontogenic
changes in the laminar distribution pattern of $
adrenoreceptor binding sites in the maturing kitten visual
cortex. In 6-week-old kitten visual cortex, the receptor
density was very high in the superficial layers I-III and
moderate in the lower layers V and VI, showing a very low
value in layer IV.
The procedure is as follows: the animal is first
perfused via the aorta with 0.1 % formaldehyde in phos
phate buffered saline. The visual cortex is quickly
dissected out and frozen with a flake of liquid C02. Ten
micron-thick sections are sliced by a cryostat (American
Optical) at -15°C and picked up on the gelatin-coated
slide glass. Slide-mounted sections are stored in the
frozen condition for a few more days with desiccant.
Incubation of warmed and dried sections with 2 nM 3H
dihydroalprenolol, with and without a displacer, 10 µM
propranolol, is carried out at room temperature. About 30
minutes after, sections are rinsed in ice-cold buffer
solution for 20 minutes. This rinsing is further followed
by quick dipping in double distilled water and finally
sections are dried on a cold plate by blowing cold air. We
use both LKB Ultrofilm for the fast return of results
(exposure time about 2 weeks) and a dipping method with
Kodak NTB2 emulsion (about 10 weeks) for photo
densitometry of silver grains. Either a piece of Ultrofilm
cut the size of the slide glass or a NTB2-coated, long size
cover slip are placed together, glued at one end of the
slide, with slide-mounted cortical sections in a dark room
under safe light. The slide glass and Ultrofilm (or
coverslip) are clamped tightly at the other end, having a
thin piece of hard Teflon sheet in between. These
assemblies are finally placed in a light-tight plastic
container with desiccant and kept at 4°C.
Reference: Palacios, J. M. and Kuhar, M. J. (1980) Science 208,
1378-1380.
181. THE "CRITICAL PERIOD" FOR PLASTICITY IN DARK-REARED CATS; DEPENDENCE ON CATECHOLAMINES
Investigators: Vilayanur S. Ramachandran•, Baruch Kuppermann
If one eye of a kitten is closed earJy in life, that eye
becomes functionally disconnected from visual cortical
neurons. Normally this effect can be produced only during
a "critical period" restricted to the first 3 months of life
(Hubel and Wiesel, 1970), but if kittens are dark-reared
for 6-12 months and subsequently exposed to a normal
visual environment with one eye closed, most of their
cortical neurons are found to be dominated by the
experienced eye (Cynader and Mitchell, 1980).
There are two possible interpretations of this effect.
First, individual cortical neurons may be actually shifting
their allegiance to the experienced eye and there may be
an 11expansion11 of columns devoted to that eye.
Alternatively, the findings may simply be the result of
selective activation of one eye's connections by the visual
experience restricted to that eye.
Using anatomical and physiological techniques, we
have been doing experiments to distinguish between these
two possibilities and have also explored the limits of this
apparent plasticity in adult animals.
(1) Two cats were dark-reared from birth until 6
months, then monocularly deprived (MD) and exposed to
light for 10 days and 3.5 weeks, respectively. The first
showed a breakdown of binocularity while the second
showed a marked shift in ocular dominance toward the
open eye. This conforms to the normal sequence of
changes that one observes in MD kittens. (2) The second
cat was subsequentJy reverse-sutured, that is, the
originally experienced eye was closed, while the initially
deprived eye was opened for 3 months. We found that this
catised a strong shift back to the newly-opened eye. (3) A
cat was dark-reared for 2 years and subsequently
monocularly deprived for 6 weeks. This cat showed shifts
in ocular dominance almost as pronounced as in 6-month
old animals. (4) 6-Hydroxydopamine was used to deplete
catecholamines from one hemisphere of three dark-reared
adult cats that were then monocularly deprived for 6
weeks. Shifts in ocular dominance were seen only in the
untreated control hemisphere. This suggests that
preserving plasticity into adulthood may at least partially
involve the continued activity of ascending catecholamine
pathways. (5) A cat was dark-reared for 6 months and
then monocularly deprived and exposed to light for 3
months. Its deprived eye was injected with tritiated 3H
proline. Although the deprived eye was injected, heavy
labeling was seen in layer 4 of both hemispheres. After
long-term monocular suture of kittens, little label is
discerned in layer 4 from the deprived eye (Shatz and
Stryker, 1978); the heavy labeling observed here suggests
that "plasticity" in adult dark-reared animals probably
occurs in the supra- and infra-granular layers of the
striate cortex.
129
References: Cynader, M. and Mitchell, D. E. (1980) J. Neurophysiol.
43, 1026-1040. Hubel, D. and Wiesel, T. N. (1970) J. Physiol. 206, 419-
436. Shatz, c. J. and Stryker, M. P. (1978) J. Physiol. 281, 267-
283.
*University of California, Irvine.
182. SEGREGATION OF GENICULOCORTICAL AFFERENT TBRMINAIB IN LAYER IV OF CAT VISUAL CORTEX
Investigators: Holger Reiter•, Michael Stryker••, Takuji Kasamatsu
In layer IV of cat visual cortex, the geniculo~ortical
terminals that receive input from either the left or the
right eye physically segregate to form the left and right
eye dominance bands in the early postnatal days. This
study was designed to determine to what extent this
terminal segregation during the critical period was
influenced by visual afferents. The animals were raised
under various visual environments such as normal,
binocularly lid-sutured and dark-reared. This study is
largely based on previous work by LeVay and Stryker
(1979).
Two injections of 40% horseradish peroxidase (HRP) in
296 DMSO were made in the optic radiation beneath the
primary visual cortex. Anterograde transport of HRP (12
hr) in the visual cortex was demonstrated by the TMB
method (Mesulam, 1978). Reconstruction of HRP-filled
terminal arborizations was made by means of camera
lucida drawings from serial sections.
The following three problems were encountered:
(1) filling of axons with a sufficient amount of HRP;
(2) timing of histochemical reactions; and (3) reconstruc
tion of HRP-filled axons. The first problem was solved by
injecting HRP slowly with a motor-driven device (30-45
min). This seemed to facilitate the axonal uptake of HRP
and thus we have consistently achieved sufficient filling.
The timing of histochemical reactions, the duration of
postfixation wash with phosphate buffer in particular, was
cruciaL Any wash over 6 hr seemed to impair HRP
activity to a point where it was largely lost in the axons,
even though there was plenty of reaction product
everywhere within the visual cortex.
The last problem of reconstruction has not been fully
solved yet. Considerable distortion of the tissue sections
due to dehydration and coverslipping makes axonal tracing
across sections extremely difficult. Usually the thickness
130
of sections decreased by up to 7596 in these procedures.
We will attempt to solve this problem by preparing thicker
sections (250 micra) than the ones used before (100
micra).
References: LeVay, S. and Stryker, M. P. (1979) Soc. Neurosci. Symp.
4, 83-98. Mesulam, M. M. (1978) J. Histochem. Cytochem. 26, 106-
117.
*Undergraduate, Occidental College, Los Angeles. **Department of Physiology, School of Medicine, University of California, San Francisco.
183. ILLUSIONS OP DEPTH PRODUCED BY MOVING RANDOM DOTS
Investigators: Constance Royden*, James F. Baker, John M. Allman
We studied the perception of depth using a computer
controlled display that presented random dots moving
within a fixed rectangle surrounded by a field of
stationary random dots. When dot motion was orthogonal
to the long axis of the central rectangle, naive subjects
100
80
PERCENT 60
FAR
RESPONSES 40
20
0
20
PERCENT 40
NEAR RESPONSES 60
80
100 CD CD 0
reported that they saw the central area in depth behind
the surround This has the appearance of a sheet of
random dots appearing at one edge of the rectangle,
moving across it, and disappearing on the opposite side.
When the dot motion was parallel to the long axis of the
central rectangle, the rectangle was most often reported
as being in front of the surround. As the ratio of length to
width was decreased, the strength of the illusions
decreased (see Figure 2). Increasing velocity from 0.3° to
3.0° per second strengthened the illusion of depth
produced by orthogonal motion. The near effect produced
by parallel motion was strongest at 1° per second. 1'lis
depth illusion produced by orthogonal motion was stronger
than the near effect elicited by parallel motion; it also
was stronger than stereoscopic (binocular disparity) depth
cues. The depth illusion produced by orthogonal motion is
thus a very strong effect and corresponds to the cue of an
occluding surface. The near effect occurs when the
inferred occluding surface is relatively small or absent.
*University of California at San Francisco.
[II] UP or DOWN
~ LEFT or RIGHT
N= 10
88 Figure 2. Reports of depth by 10 naive observers for up or down and left or right motion in five shapes of central area. The circled figure below each pair of bars corresponds to the shape tested within a 5° circular viewing aperture. The bars with vertical stripes show the combined data for up or down motion, and the bars with horizontal stripes show the combined data for left or right motion. The percentage of responses that indicated that the central area appeared to be behind the surroUI1d (far responses) is displayed above the horizontal line, and the percentage of responses that indicated that the central area appeared in front of the surroUJld (near reponses) is displayed below the horizontal axis.
184. SINGLE UNIT ELECTROPHYSIOLOGY IN THE CNS OF TWO VISUALLY PREDATORY ARACHNIDS
Investigators: David W. Sivertsen, John C. Wathey*
Jumping spiders (salticids) and wolf spiders (lycosids)
are visual predators. These arachnids do not capture prey
with silk webs, but rely on visually-guided behaviors.
Brightly colored salticids wave their front legs like
semaphores as a courtship display, and stalk their prey
with relentless efficiency (Forster, 1982).
Previous work has quantified behavior (Land, 1972),
receptor properties, and anatomy (Hill, 1975). No one has
previously recorded from the central nervous system.
Visually predatory spiders have the advantage of
simplicity, characteristic of invertebrate preparations,
yet are capable of complex visual behaviors. Phidippus
johnsonii, a salticid found in the Cft.mpus gardens, is well
suited for study.
Figure 3. Scanning electron microscope photo of salticid. The two largest spheroids are the anterior medial (AM) eyes, used for complex visual analysis tasks.
Salticids have eight eyes (Figure 3). One pair is
largely vestigial, two pair are primarily concerned with
orienting towards the target, and the anterior medial pair
(AM) perform complex analysis. The AM eyes have a 2°
wide by 20" high retina which scans (the lens is fixed).
The receptors are ordered into four distinct layers. This
array takes advantage of the focal length versus wave
length aberration to extract color information from green
to ultraviolet (Yamashito, 1976).
131
During preliminary recordings in lycosids and salticids,
we found single cells in the protocerebrum with well
defined action potentials. Visual cells had distinct
receptive fields and were selective for contrast, direction,
velocity, and size of the stimulus. We also found
vibration-sensitive cells. Spiders have specialized struc
tures in their exoskeleton for receiving vibrations. The
cells were tightly tuned for specific frequencies.
Our recording site was marked with electrolytic
lesions and spiders were thin-sectioned and stained with
haemotoxylin and eosin. Further experiments will
attempt to quantify single unit properties.
References: Forster, L. (1982) Am. Scientist 70, 165-175. Hill, D. E. (1975) Unpublished Dissertation, Oregon State. Land, M. F. (1972) J. Exp. Biol. 57, 15-40. Yamashito, T. (1976) J. Comp. Physiol. 105, 29-41.
*University of California, San Diego.
185. THE STRUCTURE AND FUNCTION OF VOCALIZATIONS IN FREE RANGING OWL MONKEY
Investigators: David w. Sivertsen, Patricia C. Wright*
Aotus trivirgatus, the owl monkey, is the only
nocturnal primate in South America. We studied the
ecology and behavior of the owl monkey for over five
months in undisturbed tropical rainforest in the· Madre de
Dios region of Peru.
We recorded calls during each monthly six night focal
group sample. We tracked and recorded neighboring
groups an additional ten nights per month.
We found nine discrete, stereotyped vocalizations and
evaluated their behavioral context. The temporal
patterning and frequency structuring of these calls are a
consequence of physical and environmental parameters.
"Hoots" are the long call of this species, a low
frequency three-note ca!L The 250 Hz fundamental is
advantageous because it is relatively unattenuated by the
spectral filtering of the forest, and it avoids competition
with the calls of frogs and insects.
Series of hoots were performed only a few nights each
month, for periods of over an hour. Hoots were performed
while the group approached fruit trees that would come
into fruit in the next month. These fruit trees were
located on territorial borders, and vicious fighting
between groups would occur in them about a week later.
We found the hooting behavior correlated with ambient
noise. A wet, drippy forest has a high broadband noise
132
level. In this situation, calls were suppressed. The owl
monkeys only hoot when their neighbors can hear. We
heard hoots 500 meters away, a distance encompassing
almost all adjacent territories.
neighbor/neighbor interactions
foraging.
Groups can assess
to optimize future
Calls also correlate with altitude and phase of the
moon, resulting in a monthly cycle of behavior (see
Figure 4). We believe the need for high light levels to
Aotus trivirgqtus
COMPOSITE AGONISTIC DATA
9/80 - 8/81
5
4
3
2
conduct canopy level fights safely to be the selective
pressure for this behavior.
Nocturnality confers the advantage of reduced
competition on the owl monkey. Np other animal
displaces Aotus from resources, diurnal raptor predation is
avoided, and lack of competition in the auditory spectrum
allows simple, parsimonious calls.
*City University of New York.
MOON PHASE
• • Hoot Nights
Fights
II
Figure 4. Hooting behavior occW's predominantly during the second week of the lunar month, when the moon is above the canopy. Agonistic encounters take place around the time of the full moon, when it is directly overhead. Because of the altitude requirement, fights that occW' later in the month take place later at night.
186. PHYSIOLOGICAL PROPKRTII!S OF NOREPJNEPHRINE-CONTAINJNG CELUI JN CAT LOCUS COKRULBUS
Investigators: Kazushige Watabe, Takuji Kssamatau
Single cell activity was extracellularly recorded from
208 norepinephrine (NE)-containing cells in cat locus
coeruleus (LC). They showed almost the same physio
logical properties as those of NE cells in rat LC. These
properties included the wide duration of a full spike
{.r2 msec), a waveform composed of A-, B- and C-spikes,
a low spontaneous discharge rate and good responsiveness
to natural stimuli such as sound and pain (Nakamura, 1977;
Watabe, 1980). Antidromic responses were obtained from
NE-containing LC cells in response to stimulation of the
dorsal lateral geniculate nucleus (LGN), the superior
colliculus (SC) and the visual cortex (VC), in accordance
with the dense innervation of such visual structures by
NE-containing axons originating from the LC. The
conduction velocity was calculated in 112 cells activated
antidromically by electrical stimulation of ascending
axons in the dorsal bundle. The mean conduction velocity
was 1.2 m/sec. This was faster than that reported for NE
cells in rat LC. 1n some of the 208 NE-containing LC
cells, responses to natural visual stimuli and electrical
stimulation of central visual structures, such as the optic
chiasm (OX), LGN, SC and VC, were studied. Twelve NE
containing LC cells responded to flashing-light with a Jong
but stable latency (mean, 60 msec),' although receptive
fields could not be plotted for them. The mean latency of
orthodromic responses of LC cells elicited by OX, LGN,
SC and VC stimulation was 21, 8.1, 5.6 and 16.4 msec,
respectively. These long orthodromic latencies may
suggest that visual inputs reach the LC polysynaptically.
It seems most likely that the reticular formation inter
venes between the LC and central visual structures as a
final common path for sensory afferents. The fact that
LC cells receive sensory afferents from various sources
through polysynaptic connections is consistent with the
above proposal.
Referenees: Nakamura, S. (1977) J. Physio!. 267, 641-658. Watabe, K. (1980) Arch. Ital. Biol. 118, 303-329.
187. RESTORATION OF NEURONAL PLASTICITY IN CAT VISUAL CORTEX BY STIMULATION OF THE LOCUS COERULEUS
Investigators: Kazusbige Watabe, Takuji Kasamatsu, Erling SchO!ler•, Paul Hegge!und•
We have been studying the role of norepinephrine (NE)
in regulating neuronal plasticity in cat visual cortex. We
previously showed that NE-containing nerve terminals in
the visual cortex are necessary to maintain and enhance
neuronal plasticity that is especially evident in the
immature visual cortex of young kittens. In all previous
studies, we directly manipulated NE-terminals within the
visual cortex by means of localized perfusion of either
catecholamine-related neurotoxin, 6-hydroxydopamine or
exogenous NE (Kasamatsu and Pettigrew, 1976;
Kasamatsu et al., 1979). 1n the present study we
investigated the effect upon visual cortical plasticity of
electrical stimulation of locus coeruleus (LC), by which
NE can be released from NE-terminals within the cortex
(Tanaka et al., 1976). We used adult cats and old kittens
that had outgrown the postnatal critical period (.r13 weeks
133
of age) for effects of monocular deprivation. Electrical
stimulation of the left LC, ipsilateral to the visual cortex
studied later, was carried out only when the animal was
allowed to have monocular vision through the left eye 2 hr
a day. The monocularly lid-sut~ed animal was otherwise
kept in the dark without LC stimulation. Ocular
dominance of visual cortical cells was determined. First,
largely reduced binocularity (33-50%) was obtained in
every animal studied (N = 5). Second, in two of them the
ocular dominance distribution was shifted toward the
monocularly exposed eye after only 12 hr of monocular
experience. In these old kittens, the shift was not so
evident as that usually observed in young kittens within
the critical period. These results suggest that activation
of LC cells can restore neuronal plasticity in cat visual
cortex.
References: Kasamatsu, T. and Pettigrew, J. D. (1976) Science 194,
206-209. Kasamatsu, T., Pettigrew, J. D. and Ary, M. (1979) J.
Comp. Neurol. 185, 163-182. Tanaka, C., Inagaki, C. and Fujiwara, H. (1976) Brain Res.
106, 384-389.
*Neurobiology Laboratory, Universi.ty of Trondheim, Trondheim, Norway.
PUBLICATIONS
Allman, J. (1981) Reconstructing the evolution of the brain in primates through the use of comparative neurophysiological and neuronanatomical data. In: Primate Brain Evolution. E. Armstrong and D. Falk (Eds.), pp. 13-28. Plenum Press, New York.
Allman, J. (1982) Evolution of the brain in primates. In: Oxford Companion to the Mind. R. Gregory (Ed.). Oxford University Press, in press.
Allman, J., Baker, J., Newsome, W. and Petersen, s. (1981) Visual topography and function: Cortical visual areas in the owl monkey. In: Multiple Visual Areas. C. Woolsey (Ed.), pp. 171-186. Humana Press, Clifton, New Jersey.
Kamei, I., Shiosaka, S., Senba, E., Takagi, H., Sakanaka, M., Inagaki, S., Takatsuki, K., Nakai, K., Imai, H., ltakura, T., Komai, N. and Tohyama, M. (1981) Comparative anatomy of the distribution of catecholamines within the inferior olivary complex from teleosts to primates. J. Comp. Neurol. 202, 125-133.
Kasamatsu, T. (1982) Neuronal plasticity maintained by the central norepinephrine system in the cat visual cortex. ln: Progress in Psychobiology and Physiological Psychology, Vol. 10. A. N. Epstein and J. M. Sprague (Eds.). Academic Press, New York, in press.
Kasamatsu, T. (1982) Enhancement of neuronal plasticity by activating the norepinephrine system in the brain: A remedy for amblyopia. Human Neurobiol. 1, 49-54.
Kasamatsu, T. (1982) A role of the central norepinephrine system in regulation of neuronal plasticity in cat visual cortex. Biomedical Res. Suppl., in press.
134
Kasamatsu, T. and Heggelund, P. (1981) Norepinephrine iontophoresis in cat visual cortex: A quick change in ocular dominance. Soc. Neurosci. Abstracts 7, 142.
Kasamatsu, T. and Heggelund, P. (1982) Single cell responses in cat visual cortex to visual stimulation during iontophoresis of noradrenaline. Exp. Brain Res. 45, 317-327.
Kasamatsu, T., Watabe, K., SchOller, E. and Heggelund, P. (1982) Activation of the central noradrenaline system: A remedy for experimental amblyopia. An abstract for the first World Congress of !BRO, Lausanne, 1982, Neuroscience Suppl. 7, 8113.
McGuinness, E. (1981) Innervation of the facial muscles by the brainstem facial nucleus. Soc. Neurosci. Abstracts 7, 896.
Miezin, F., McGuinness, E. and Allman, J. (1982) Antagonistic direction-specific mechanisms in Area MT in the owl monkey. Soc. Neurosci. Abstracts 8, in press.
Professor: Masakazu Konishi Del I!. Webb Research Pellow: Terry T. Takahashi Research Pellow: Andrew Moiseff Graduate Students: Lawrence C. Katz, Daniel Margoliash*,
James s. McCasland Research Staff: Eugene Akutagawa Laboratory Staff: Cynthia Akutagawa
*Division of Engineering and Applied Science, California Institute of Technology.
Support: The work described in the following research reports has been supported by:
Bing Chair of Behavioral Biology National Institutes of Health, USP HS National Science Foundation Pew Memorial Trust The Del E. Webb Foundation Whitehall Foundation Helen Hay Whitney Foundation
Summary: The introduction of new techniques sometimes
opens an entirely new vista in our research. The methods
of making live brain slices and recording intracellularly
from them have been used mainly by people interested in
the pharmacological aspect of brain physiology. Larry
Katz found that these methods worked well with finch and
owl brains. We think that these methods combined with
intracellular tracer injection have great potentials for the
study of neuron growth, morphology and connectivity.
Our new postdoctoral fellow, Terry Takahashi, uses these
techniques to establish neuron types and connections to
and from the owl's midbrain auditory nucleus.
Another technical advance we achieved was the
Nakai, K., Iwai, H., Kamei, I., Itakura, T., Kamai, N., Kimura, H., Nagai, T. and Maeda, T. (1981) Microangioarchitecture of rat parietal cortex with special reference to vascular "sphincters." Stroke 12, 653-659.
Nakai, K., Jonsson, G. and Kasamatsu, T. (1981) Regrowth of central catecholaminergic fibers in cat visual cortex following localized lesion with 6-hydroxydopamine. Soc. Neurosci. Abstracts 7, 675.
Petersen, S., Baker, J. and Allman, J. (1982) Directionspecific adaptation in Area MT of owl monkey. Brain Res., in press.
Watabe, K. and Kasamatsu, T. (1982) Visual afferents to locus coeruleus neurons in the cat. ARVO Abstracts, Suppl. Invest. Ophth. Vis. Sci. 21, in press.
Watabe, K. Nakai, K. and Kasamatsu, T. (1982) Visual afferents to norepinephrine-containing neurons in cat locus coeruleus. Exp. Brain Res., in press.
development of a technique to record multi-unit aj:!tivity
from the hypoglossus nerves of singing canaries. Jim
McCasland and Mark Konishi found that both the left and
the right nerves showed simultaneously a burst of spike
discharge before every note of the bird's song. These
results contradict the prevailing view that a majority of
song notes are controlled by the left hypoglossus.
Dan Margoliash's analysis of complex auditory neurons
has paid off handsomely. He found in one of the song
control nuclei of the white-crowned sparrow neurons
which responded only to playback of the song of the very
same individual he was recording from. His computer
aided techniques enabled him to determine the particular
acoustic features of the song which were necessary for
stimulating the song-specific neurons.
Andy Moiseff and Mark Konishi continued their heroic
study of the brain stem neuronal connections and their
physiological properties underlying auditory receptive
fields in the owl.
188. NEURONAL CONTROL OP BIRD SONG PRODUCTION
Investigator: James S. MeCasland
During the past year we have developed a technique
for making single-unit neurophysiological recordings from
the singing mockingbird; developed a technique for
recording bilaterally from the hypoglossal motor nerve;
and shown that there is a "higher" efferent center in the
song control pathway-nucleus interface (NIF}-than the
"highest" nucleus previously implicated in song control,
HVc.
A chronically-mounted microdrive which allows move
ment of an electrode in three dimensions has been
developed for us by Herb Adams. The stability of this
device is such that single neurons can be isolated and held
for long periods in the freely moving, singing mockingbird.
Our preliminary results using this technique in HVc show
that many cells are active during production of all song
elements, while some show highly stereotyped bursts for
only a few elements. Some units show long latency
"anticipatory" activity before song. At least some of the
auditorily responsive cells in HVc ·are inhibited during
song, as predicted from our multi-unit data. We wish to
see whether some cells show both auditory responses and
pre-motor activity, thus suggesting their involvement in
the developmentally crucial interface between auditory
feedback and motor output.
The need for making bilateral motor nerve
recordings was due to our inability to demonstrate
functional hemispheric dominance at the HVc level
(Biology 1981, No. 207). Our simultaneous bilateral
recordings from the hypoglossal nerves of singing canaries
verify that the overall timing and apparent patterning of
activities are essentially identical for the two sides, thus
suggesting the syringeal musculature itself as the site of
dominance. We are currently investigating the degree of
independence of left and right efferent pathways
following unilateral lesions and other manipulations.
Recordings from NIF in the zebra finch show longer
pre-song latencies than those from HVc, consistent with
the unidirectional pathway from NIF to HVc. Preliminary
results indicate that this pathway is essential for normal
song production, and that the input nucleus to NIF does
not relay song-related activity. Thus NIF may be a source
of timing cues for song.
189. INTRACELLULAR STAINING AND MICROANATOMY OF SINGLE NEURONS IN SONG SYSTEM BRAIN SLICl!S
Investigator: Lawrence C. Katz
The neural pathways controlling birdsong form a
system particularly amenable to studies of central nervous
system plasticity. The development of birdsong requires
that a bird use auditory feedback to modify his song. The
135
brain areas specifically responsible for song production
consist of a chain of brain nuclei (Nottebohm et al., 1976),
two of which-HVc and RA-receive auditory input (Katz
and Gurney, 1981; Katz, unpublished observations). These
nuclei are likely to be involved in song development. My
work during the past year has focused on the morpho
logical alterations of single neurons within HVc and RA
during song learning. An in vitro brain slice preparation
of relevant song nuclei has been developed. The brain is
removed from a bird, and sliced into 0.5-mm-thick
sections which are placed in a balanced salt solution in a
warmed, oxygenated chamber. The neurons in this
preparation remain healthy, exhibiting normal
electrophysiological characteristics, _and the song nuclei
HVc and RA are clearly visible. Using intracellular
injection of the fluorescent dye Lucifer Yellow, neurons
within HVc and RA can be visualized in their entirety. In
the slice preparation, both the number and quality of
intracellular dye fills is far superior to that obtainable in
vivo.
I have so far catalogued the various cell types present
in HVc and RA of adult male zebra finches, with respect
to the spatial extent and patterns of their dendrites and
axons. Results from over 50 filled cells have revealed an
unexpected complexity in the extent and distribution of
axons of these cells within their respective nuclei. How
does this local circuitry change during song development?
There are at least two possibilities: (1) the internal
"wiring" in a young bird is simple-each cell contacts (i.e.,
makes synapses with) only a small number of other
neurons within the nucleus; as the bird learns song, each
neuron contacts a greater number of cells; or (2) the
neurons in a young bird each synapse with a large number
of other neurons, and, as the bird learns song, the number
of synapses between cells is "pruned" so that only useful
contacts are retained. To distinguish between these
possibilities, I have started examining the neurons in HVc
and RA of young birds that have not yet begun to sing, as
well as those of adult birds who were deafened before
they started singing.
References: Katz, L. c. and Gurney, M. E. (1981) Brain Res. 221,
192-197. Nottebohm, F., Stokes, T. M. and Leonard, C. A. (1976) J.
Comp. Neurol. 165, 457-486.
136
190. ACOUSTIC PARAM1!TfillS UNDERLYING NEURONAL RESPONSES TO SONG IN A VOCAL CONTROL NUCLEUS OF WHITE-cROWNED SPARROWS
Investigator: Daniel Margoliash
A distinct group of brain nuclei subserves song
production in oscine songbirds (Nottebohm et al., 1976).
In one of these, HVc, it has recently been shown that some
neurons respond to noise bursts (Katz and Gurney, 1981).
This finding is interesting in light of the requirement for
auditory feedback during vocal development (Konishi,
1965). Thus, an in-depth analysis of the auditory
responses of HVc units was undertaken.
White-crowned sparrows were induced to sing by
subcutaneous testosterone implants. Songs (typically
consisting of four phrases or parts: whistle, buzz, trill,
terminal buzz) were recorded, digitized (PDP 11/40), and
modeled as frequency and amplitude functions. These
functions could be used (with special electronics) to
synthesize accurate copies of the songs. Modifications of
a function (for example, frequency shift) produced the
appropriate change in the song. In this way, a complex
stimulus (song) could be systematically modified along all
parameter dimensions. For extracellular recording,
standard techniques were used with urethane-anesthetized
birds. Tone and noise bursts, and the bird's own song,
were used as search stimuli.
Four classes of responses were found, two of which (no
response, and weak response to noise bursts) have already
been described (Katz and Gurney, 1981). A third class
responded vigorously to tone bursts. Some of these units
showed excitation to most stimuli; others preferred a
limited range of frequencies. Often the best frequency
was related to the frequency of the whistle (first phrase)
of the bird's own song. On occasion the tone burst
duration necessary for excitation ( >400 msec) was
comparable to the duration of the whistle. The fourth
class included cells responding only if combinations of
phrases were presented, the individual phrases being
ineffective. Significant changes in interphrase interval
typically did not greatly affect the response. However,
frequency shifting, or modification of the .fine structure
(for example, frequency modulation) of either phrase
could diminish or abolish the response. For many of these
neurons, the specific parameters underlying the response
to song could be delineated. These parameters reflect
integrative phenomena within the auditory system.
The specificity of class IV neurons often resulted in
selectivity within an extensive song repertoire. Thus,
some inter-dialect, and on occasion intra-dialect songs
were ineffective stimuli. This selectivity may be
coincidental, or it may be related to the fact that birds
learn song from their parents. It should now be possible to
address this question directly by recording from young
birds.
References: Katz, L. C. and Gurney, M. E. (1981) Brain Res. 211,
192-197. Konishi, M. (1965) z. TierpsychoL 22, 770-783. Nottebohm, F., Stokes, T. M. and Leonard, C. A. (1976) J.
Comp. NeuroL 165, 457-486.
191. THE AUDITORY PERIPHERY: EXTRACTION OF BINAURAL CUES FOR SOUND LOCALIZATION
Investigators: Andrew Moiseff, Masakazu Konishi
The barn owl has an acute sense of hearing which can
be used for the localization and eventual capture of prey.
We have been investigating the neural mechanisms under
lying the detection and processing of sound necessary for
extracting information about its location. Behavioral and
neurophysiological experiments suggested that owls make
use of two major binaural cues for sound localization:
(1) the difference in the intensity of sound perceived at
the two ears appears to be an important cue for the
elevation of the sound, and (2) the ongoing time disparity
between the two ears appears to be important for
establishing the azimuth of the sound. To obtain a
quantitative measure of the magnitude of these binaural
cues, we measured the intensity and time delay of sounds
reaching the ears as a function of the location of the
sound.
We measured the "cochlear microphonics11 from each
ear in response to directional sound stimulation. This
technique allowed us to measure the sound reaching each
eardrum, and is sensitive to both the absolute sound
intensity and the absolute arrival time of the sound
perceived at each ear.
Our results confirmed that by combining intensity and
time cues the owl can uniquely specify the location of a
sound. The results also provided us with an indication of
the amount of time or intensity difference to which the
owl must be sensitive-better than 10 microseconds
ongoiilg time disparity, and approximately 1 dB interaural
intensity difference.
PUBLICATIONS
Gurney, M. E. (1981) Hormonal control of cell form and number in the zebra finch song system. J. Neurosci. 1, 658-673.
Gurney, M. E. (1982) Behavioral correlates of sexual differentiation in the zebra finch song system. Brain Res. 231, 153-172.
Katz, L. c. and Gurney, M. E. (1981) Auditory responses in the zebra finch's motor system for song. Brain Res. 211, 192-197.
Senior Research Associate: Marianne E. Olds Visiting Associate: Dorwin L. Birt Laboratory Staff: Jeffrey D. Carpenter, Trau Cuong,
Lawrence Humm, Mai Thieu Trinh
Support: The work described in the following research reports has been supported by the National Institutes of Health, USPHS.
Summary: We are continuing our studies of the neural
basis of reinforcement and learning. Our studies of
reinforcement are carried out in rats using as a model
self-stimulation behavior. The animals are implanted with
chronic electrodes which are aimed at specific brain sites.
To obtain reinforcement in the form of a brief electrical
st.imulus, the subject has to depress-a lever. The number
of times the lever is depressed within a given period is the
basis for classifying sites as reinforcing or neutral or
aversive. Numerous mapping studies have revealed that
reinforcing sites are often found in the trajectories of the
central catecholamine systems or in the regions where the
cell bodies lie. Three such systems have been of
particular interest: (1) the dopamine neurons in the
substantia nigra, zona compacta, which project to the
striatum; (2) the dopamine neurons in the ventral
tegmentum, which innervate forebrain limbic structures;
and (3) the norepinephrine neurons in the locus coeruleus
together with their ascending projections to the cortex
and the hippocampus. We have given our attention to this
last system this past year on the basis of findings that the
induction of noradrenergic hyperinnervation of the locus
coeruleus region leads to enhanced reinforcement in that
region. Our aim is to determine the basis of this
behavioral effect in terms of the biochemical and
morphological changes accompanying noradrenergic
hyperinnervation.
137
Konishi, M. and Akutagawa, E. (1981) Androgen increases protein synthesis within the avian brain vocal control system. Brain Res. 222, 442-446.
Konishi, M. and Gurney, E. (1982) Sexual differentiation of brain and behaviour. Trends in Neurosci. 5, 20-23.
Mccasland, J. S. and Konishi, M. (1981) Interaction between auditory and motor activities in an avian song control nucleus. Proc. Nat. Acad. Sci. USA 78, 7815-7819.
Moiseff, A. and Konishi, M. (1981) The owl's interaural pathway is not involved in sound localization. J. Comp. Physiol. 144, 299-304.
Our studies of learning are carried out in rats
chronically implanted with electrodes for recording extra
cellular neuron activity in the auditory pathway, the
superior colliculus, and the dorsal tegmentum while the
animal learns to discriminate between two auditory
stimuli, one that is followed by a food reward, the other
not. We have found that under such conditions, the
neurons respond differently to the tones when they are
followed by the food reward than. when they are presented
not in association with the reward. Our concern in these
experiments is to determine the anatomical basis and the
function of these "learned11 changes in cellular
responsiveness. Our immediate aim is to dissociate these
changes in terms of their latencies or their appearance in
the trial sequence from the "learned" behavior of the
animal that is used as a marker to show that the
association between tone and food is being learned.
192. STUDY OF THE EFFECTS PRODUCED BY THE NEUROTOXIN 6-HYDROXYDOPAMINE INJECTED NEONATALLY IN THE RAT
Investigators: Marianne E. Olds,. Mamoni Umemoto•, Toru Itakura**, S. Kurumiya*
When the neurotoxin 6-hydroxydopamine (6-0HDA) is
injected neonatally in the ventricular system of the rat, it
has long-term consequences on the anatomy, the bio
chemistry and the function of the central catecholamine
systems. We have been using this approach to study the
effects of inducing a permanent alteration in central
aminergic transmission on reinforcement produced by
brain stimulation at sites localized in these systems. We
have obtained evidence that in the adult rat treated with
this substance at birth, the effects of brain stimulation in
the dorsal pons, in the vicinity of the locus coeruleus, are
138
greatly enhanced. We have also obtained evidence that in
these animals the levels of the catecholamines-dopamine
and norepinephrine--are reduced in cortex, hippocampus,
and to a lesser extent, in hypothalamus. In the region of
the locus coeruleus, the levels of norepinephrine are
elevated. Morphological studies based on visualization of
catecholamine-containing terminals, fibers, and cell
bodies have shown that the number of neurons in the locus
coeruleus are reduced in number or are absent in the
treated animals but the number of catecholamine fibers in
that same region is enhanced. It would seem therefore
that the catecholamine neurons of the locus coeruleus are
not responsible for the reward produced in the dorsal pons.
We are proceeding with an investigation of the source for
the increase in the norepinephrine fibers in this region.
*Psychology Department, Osaka City University, Osaka, Japan. **Department of Neurosurgery, Wakayama Medical College, Wakayama, Japan.
193. EFFECTS OF PERMANENT DEPLETION OF DOPAMINE IN THE BRAIN OF THE RAT
Investigators: Mamoru Umemoto*, Marianne E. Olds
When 6-hydroxydopamine (6-0HDA) is given to rats, it
produces effects on the catecholamines dopamine and
norepinephrine. If selective action on dopamine is
required, it is possible to use the tricyclic antidepressant
desmethylimipramine (DMJ) as a pretreating agent before
6-0HDA to achieve this effect because DMI inhibits the
uptake of the neurotoxin into the terminals of
norepinephrine neurons. We have used this approach to
study the effects of the selective depletion of dopamine
on reinforcement obtained in the substantia nigra, on the
regional concentrations of dopamine and norepinephrine,
and on the morphology of the substantia nigra, with a view
to determining whether enhanced reinforcement can be
obtained in the substantia nigra.
Our findings indicate that reinforcement can be
obtained in these animals but it is more difficult to
demonstrate because reward sites are sparser. The
biochemical findings show effects in one direction only, a
depletion of dopamine. This effect is, however, graded,
being more marked in the forebrain than in the
mesencephalon. The levels of norepinephrine in the test
animals are the same as in controls. We are in the process
of investigating the morphological changes induced in the
substantia nigra zona compacta by the neonatal
treatment. The aim of these studies is to establish on a
firm footing the reduction of rewarding sites in the
substantia nigra and to determine whether this behavioral
finding and the biochemical results correlate with
morphological changes in this region.
*Psychology Department, Osaka City University, Osaka, Japan.
194. BEHAVlORAL AND BIOCHEMICAL EFFECTS OF DEAFFERENTING THE HIPPOCAMPUS NEONATALLY OF ITS NORADRENERGIC INPUT
Investigators: Marianne B. Olds, James Smith*, John Lane*, Jeffrey D. Carpenter
The anatomical findings in the brains of adult rats
treated neonatally with 6-hydroxydopamine (6-0HDA)
suggest that one of the principal targets of the toxic
substance is the hippocampus. This is a region that
receives noradrenergic input principally from the
adrenergic neurons of the locus coeruleus, and it receives
serotonergic input from the raphe nuclei. In animals
treated with 6-0HDA by the intraventricular route, the
histological material shows selective degeneration of the
rostral hippocampus-septum complex, in particular the
axon-dendrite network linking the several types of cell
bodies found in this structure. Inasmuch as this structure
is also implicated in the regulation of emotionality and
has been postulated to exert an inhibitory role in the
activity of brainstem structures, we have reasoned that
this particular projection of the ascending branch of the
coeruleus noradrenergic system may be responsible for the
behavioral effects of animals treated at birth in the
lateral ventricles with 6-0HDA. Two behavioral effects
are especially noteworthy. One is the hyperreward in the
dorsal pons and the hyporeward in the hypothalamus; the
other is a hyperactive reaction to stimuli, either new or
part of the animal's environment. We are using a
stabilimeter to measure the startling variety of move
ments executed by the animal when it has been removed
from its home cage. The treated animals perform more
movements of the type counted by our apparatus and for a
longer time than controls. This syndrome develops over
time and becomes slightly attenuated when the animal
reaches old age, but it remains a permanent feature of its
behavioral repertory.
In the biochemical studies carried out with the
collaboration of Dr. James Smith and Mr. John Lane of
the University of Louisiana Medical School, the levels of
catecholamines are measured in three subdivisions of the
hippocampus, the Cal and Ca3 regions and the dentate
gyrus, because anatomical findings from other
laboratories have indicated that the distribution of the
adrenergic input to this structure is uneven, its greater
density being in the dentate. In addition, levels are
measured in the pons, ventral, and dorsal regions for
effects in the A6-A4 and Al-A3 group cells, and in the
hypothalamus and caudate for effects on the dopamine
systems, since 6-0HDA used without pretreatment with
desmethylimipramine may have preferential effects on
the noradrenergic systems. We have tested these animals
in the stabilimeter and have not replicated the hyper
reactivity seen when 6-0HDA is injected into the lateral
ventricles. It is possible that the concentration of 6-
0HDA was not high enough to produce the damage in the
hippocampus seen with the neonatal intraventricular
injections or, alternatively, that the damage was too
selective to induce the changes in adrenergic innervation
seen with the ventricular route. We are at present testing
identically treated animals for reward in the diencephalon
and the dorsal pons, in the locus coeruleus region.
*University of Louisiana Medical School.
195. ANATOMICAL BASIS AND FUNCTION OF LEARNED ENBANCED NEURAL RESPONSIVENESS 1N THE AUDITORY SYSTEM OF THE RAT
Investigators: Marianne E. Olds, Dorwin L. Birt, Jeffrey D. Carpenter
In previous studies we have obtained evidence of
associative neural changes in the medial portion of the
medial geniculate bodies and in the deep layers of the
superior colliculus in the rat. Inasmuch as the latencies of
these changes suggested that they might be related to the
movements of the animal during the presentation of
auditory stimuli that signal a food reward, the present set
of experiments is designed to dissociate the neural
changes from the "learned" behavior both in terms of the
latencies of their occurrence with respect to signal
presentation, and in terms of the trial sequence.
we have selected three strategies to demonstrate this
dissociation. In the first, we have altered the paradigm
used hitherto to include three food magazines instead of
one. The appropriate magazine in a trial is signaled by a
139
click given 200 msec after the onset of one of the tones,
which can be either paired or not pa.ired with a food
reward. The absence of information during the first 200
msec about which of the three food magazines will be the
"hot" magazine during a given trial is intended to
eliminate the directional movements toward the food
magazine in the one-food magazine situation.
In the second strategy, we introduce a delay in the
one-food magazine paradigm. It is also intended to
eliminate movements during the first 200 msec of the
stimulus presentation. In this situation, if the animal
moves during this interval, the trial is aborted. The aim is
to obtain an interval free of movements, under the control
of the experimenter, for analysis of neural changes during
that period.
In the third strategy, we use the one-food magazine
situation but we substitute brain reward for the food
reward. As a rule the brain reward induces movements
but it does not have directionality or have as a component
an orienting response toward the food magazine. We have
evidence that the nonspecific movements induced by the
brain reward can be conditioned. We are hoping to use
this method to show that the associative neural changes
we have uncovered remain, even though the movements
associated with the food-task have been eliminated.
PUBLICATIONS
Birt, D. and Olds, M. E. (1981) Associative response changes in lateral midbrain tegmentum and medial geniculate during differential appetitive conditioning. J. Neurophysiol. 46, 1039-1055.
Olds, M. E. (1981) Reinforcing effects of morphine in the nucleus accumbens. Brain Res. 237, 429-440.
Olds, M. E. (1982) Developmental aspects of pontine selfstimulation in the rat. Devel. Brain Res., submitted for publication.
Umemoto, M., Kurumiya, S., Itakura, T. and Olds, M. E. (1982) Catecholaminergic hyperinnervation but Joss of cell bodi~ in the locus coeruleus (AS) in rats treated neonatally with 6-hydroxydopamine: histochemical fluorescence study of self-stimulation. Brain Res., submitted for publication.
Umemoto, M., Kurumiya, S., Itakura, T. and Olds, M. E. (1982) Enhanced pontine self-stimu1ation, noradrenergic hyperinnervation in LC, but loss of AS neurons after neonatal 6-0HDA. Science, submitted for publication.
Umemoto, M. and Olds, M. E. (1981) Presynaptic alphaadrenergic mediation of self-stimulation in locus coeruleus in rats pretreated neonatally with 6-hydroxydopamine. Brain Res. 219, 107-119.
140
Professor: Roger W. Sperry Senior Research Associate: Charles R. Hamilton Visiting Associates: Evelyn L. Teng, Eran Zaidel Graduate Students: Alice M. Cronin-Golomb, Jay J. Myers Research Staff: Amy M. Canada, Eef Goedemans, Lois E.
MacBird, Betty A. Vermeire
Support: The work described in the following research reports has been supported by:
Biomedical Research Support Grant (NIH) Frank P. Hixon Fund National Institutes of Health, USPHS Pew Memorial Trust University of California, Los Angeles University of Southern California
summary: Our research has continued to focus on
hemispheric specialization and left-right cross integration
in human subjects who have undergone surgical
disconnection of the cerebral hemispheres. Correlated
investigations, carried out in split-brain monkeys by
Dr. Charles Hamilton, have attempted to carry further
the analysis of basic hemispheric interrelations with
combined surgical and testing techniques not applicable in
human subjects.
196. HEMISPHERIC DIFFERENCES IN ABILlTY TO RECOGNIZE FIGURE AND BACKGROUND
Investigator: Alice M. Cronin-Golomb
Four complete commissurotomy subjects, LB, NG, RY
and AA, were tested for the ability to recognize
components of pictures presented in central vision to right
or left visual field. A test picture, exposed tachisto
scopically for 150 msec, consisted of one of four solid
figures of amorphous shape, centrally placed against one
of four Gibson gradients as background. When required to
identify either the test figure or the background from a
four-choice array in free vision, the subjects demon
strated a left/right difference not observed in control
subjects: the left hemisphere performed very well in
identifying the figure, but only at near-chance level in
recognizing the background, especially in early blocks of
trials. In contrast, the right hemisphere was equally adept
at identifying the figures and grounds. A hierarchy of
performance within and between hemispheres was
observed, such that the subjects performed best on the
figure task with the left hemisphere; less successfully,
though still well above chance, on either the figure or
ground task with the right hemisphere; and at near-chance
level on the ground task with the left hemisphere. When
either the "figure" or "ground" was presented alone, both
hemispheres were essentially perfect in selecting that
"figure" or "ground" from a four-choice array.
These results support the view that the left
hemisphere tends to zero in on the "focal" component of a
picture, and that the right is better at perceiving the
"whole" situation. What is new is the finding that,
although the subjects claimed to be aware that attention
to both the figure and ground elements of the test
composite was necessary in order for them to perform
well, they nevertheless failed to obtain ground infor
mation when it was presented to the left hemisphere. The
left hemisphere was apparently "compelled" to attend
selectively to the figures. Further experiments are
planned to examine more systematically the figure-ground
variables involved.
197. BACKGROUND INFLUENCE ON PERCEPTION OF SIZE AND LOCATION IN LEFT AND RIGHT HEMISPHERES
Investigator: Alice M. CronhrGolomb
The relative abilities of right and left hemispheres to
determine the size and position of solid dots on a variety
of backgrounds were measured in commi.Ssurotomy
subjects LB, NG, RY, and AA. A dot appeared against
either a plain white background, or against a Gibson
gradient of horizontal lines, receding naturally toward the
top of the stimulus card. The stimulus card was presented
in central vision for 150 msec to either right or left visual
field. When the test dot appeared against a white
background, each of the nine choices of the answer array
in free vision (three dot sizes X three positions) was also
located on a white ground. Likewise, when the test dot
was presented against a gradient background, each of the
dots of the nine-choice array appeared against the same
gradient background.
Two subjects (NG and AA) and two normal controls
demonstrated a superior performance with the natural
gradient, relative to the plain background. In the
commissurotomy subjects, this superiority appeared within
the right hemisphere exclusively. In subsequent tests with
the same dot size and localization task with the back
ground gradient inverted, only the right hemisphere
performance was disrupted, as it was enhanced by use of
the natural gradient. The observed tendency of the right
hemisphere to be more influenced by background
information may explain reports of the right1s greater
susceptibility to illusions, and may, indeed, be the basis of
some illusory effects observed in normal subjects.
198. RIGHT/LEl'T PROCl!SSING OP PERSPECTIVE CUES FOR VISUAL DISTANCE IN COMMJSSUROTOMY SUBJECTS
Investigator: Allee M. Cronin-Golomb
In normal subjects it has been shown that an object
viewed in the top half of a field is perceived to be smaller
than it actually is, and, when viewed in the bottom half
field, to be perceived as larger than it really is. When a
solid dot of one of three sizes in one of three vertical
{top, middle, or lower) positions was flashed in central
vision to either right or left visual field for 150 msec,
commissurotomy subjects LB, NG and AA demonstrated
the above normal effect for the right hemisphere only.
The effect was enhanced when a natural gradient back
ground (Gibson's horizontal lines, receding toward the top)
was used, rather than a plain white ground, and the effect
was diminished when the gradient ground was inverted.
Further, the right hemisphere of three subjects (LB,
NG and AA) more often chose the correct answer (i.e.,
that dot of the nine-choice array which was of the same
size and position as the test dot) when the stimulus card
represented a relatively "correct" distance relationship
(e.g., small dot at the top) than when the relationship was
relatively "incorrect" (e.g., small dot at the bottom). The
opposite effect was obtained for the left hemisphere in
subjects NG and RY, in whom left hemisphere
performance was better on trials involving "incorrect"
than "correct" relationships. The latter effects were
independent of background used. These findings suggest
that the separated hemispheres may be analyzing some
visual perspective cues in different ways.
199. VISUAL FIBLD ABNORMAIJTll!S IN COMMJSSUROTOMY SUBJECTS
Investigator: Jay J. Myers
Abnormalities in the visual fields of commissurotomy
subjects were noted in the course of measurements taken
to permit the use of the lateral limits method for
lateralizing visual input. These have been further
examined using perimetric techniques with a tangent
screen at a one-meter viewing distance. Measurements
were taken for both binocular and monocular vision, with
moving and stationary targets, and under conditions
requiring verbal or manual report. Detailed measure
ments were obtained for two commissurotomy subjects
(LB and NG) while a cursory examination was made of two
others (AA and RY).
141
There was some indication of field defects in all of
these subjects. Although reliable results were not
obtained for RY because of difficulties in maintaining
fixation, the other three all showed abnormalities
restricted to, or more pronounced in, the right visual field
(RVF) projecting to the left hemisphere. As reported on
other occasions, NG showed evidence of grossly con
stricted visual fields (tunnel vision), possibly an hysterical
condition. AA showed a similar, less severe constriction
limited to the RVF. LB reported blurry vision in the RVF
interspersed with patches of clear vision and blindspots.
The defects were probably not detected in previous
testing because in all cases, the region of foveal vision is
spared.
These abnormalities of vision are difficult to explain.
Their presence with monocular viewing by either eye
indicates a central rather than retinal origin, the nature
of which remains obscure. Damage to central visual
pathways from the surgery or from earlier epileptic
activity or some sort of functional reorganization of the
substrates of visual perception are among possibilities
that need to be ruled out.
200. NAMING OF STIMULI FELT WITH THE LEl'T HAND FOLLOWING FOREBRAIN COMMJSSUROTOMY
Investigator: Jay J. Myers
In contrast to earlier observations, recent reports
increasingly suggest that long-term commissurotomy
subjects can at times verbally identify stimuli projected
to the right hemisphere (RH) through the left visual field.
This finding has been taken to imply development of RH
speech, an interpretation that has since been questioned
(Biology 1981, No. 232) from evidence that the left
hemisphere (LH) rather than RH is responsible for the
verbalizations. Whether the LH gains access to such
information through ipsilateral sensory projections or by
interhemispheric channels has not been determined. In an
effort to further explore this question in a different
modality, a series of tests was conducted to assess the
ability of subjects LB and NG to name tactual, alpha
numeric characters (5 cm plastic letters and digits)
through blind unimanual stereognosis with the left and
right hands.
The present results conform closely with prior findings
in the visual modality. LB was able to name the tactual
stimuli when presented to either hand with very few
errors. NG could name stimuli in her right hand with a
142
few errors but correctly identified very few stimuli felt
with the left hand unless the number of alternatives was
reduced to two or three and unless informed in advance of
their identities. During one block of 24 trials, NG gave
completely reversed responses to two letters presented to
the left hand. Similar reversals had also been noted
during visual testing within the left visual field.
Taken together, these findings strongly suggest that
interhemispheric channels allow the LH to sometimes
verbally identify stimuli projected to the RH. The close
similarity of performance within the visual and tactual
modalities, especially for subject NG, further argues
against the use of ipsilateral sensory projections.
201. SERIAL REVERSAL LEARNING: TWO HEMISPHERES ARE BE'ITER THAN ONE AND THE LEFT IS BETTER THAN THE RIGHT
Investigator: Evelyn L. Teng
A previous study (Biology 1981, No. 230) with four
cornmissurotomy patients has shown that the left and the
right hemispheres learned equally readily on a two-choice
discrimination problem that involved tactile inputs and
manual responses, but the right hemisphere was clearly
inferior to the left one in mastering discrimination
reversals (i.e., when the designations of correct and
incorrect responses were reversed). In addition, even the
left hemisphere of the commissurotomy patients did not
perform as well as three control patients over a series of
reversals, suggesting that learning with both hemispheres
is better than learning with a single one; however, this
between-group comparison cannot give unequivocal
conclusions because the two groups were not matched in
age, IQ, and other possibly pertinent variables.
The aim of the present study was to make within-·
subject comparisons between learning with both
hemispheres and learning with a single hemisphere. The
same two-choice serial reversal learning task was
presented to each hand of four commissurotomy patients
under two conditions: (1) behind an opaque screen, as was
the case in the previous study, so that only the hemisphere
contralateral to the working hand could participate in the
task; and (2) in free view, so that information about the
task was available to both hemispheres. Results show
that, with vision, the two hands performed at comparable
high levels. Without vision, the right-hand performance
showed minor impairments, and the left-hand performance
showed major deteriorations. Control patients consis
tently performed at high levels with either hand, with or
without vision. These results confirm that serial reversal
learning is better performed with two hemispheres than
with one hemisphere, and the right hemisphere is
particularly deficient in this task. Work is under way to
check whether or not the above findings also hold when
different discriminanda and response requirements are
involved, and to explore the reasons of the right
hemisphere deficiency.
202. LEXICAL DECISION AND SEMANTIC FACILITATION IN THE SPLIT BRAIN
Investigator: Eran Zaidel
Consider a hemifield tachistoscopic experiment in
which the subject has to decide whether a brief target
character string flashed to one or the other visual
hemifields is a word or not (lexical decision). The target
is preceded by another lateralized word (the prime) that
may or may not be semantically associated with the
target word. In central vision, it is known that when the
prime is a semantic associate of the target, the decision
about the target is faster (semantic facilitation). In a
second experiment, the printed prime is replaced by an
unlateralized spoken word. A control test was also
administered in which lateralized targets without any
primes were presented. Three or four complete, and one
or two partial, con'lmissurotomy patients participated in
the experiments.
The results are summarized in Table 1. Lexical
decision of lateralized targets alone, without primes,
disclosed generally bilateral competence (4 out of 5
patients) and no hemispheric difference in latency (3 out
of 4 patients). The partial commissurotomy patient
showed a pattern of latency and accuracy that was
completely consistent with that of the complete
comissurotomy patients.
Lexical decision of lateralized targets preceded by
lateralized primes showed bilateral competence in three
patients, and bilateral incompetence in one. There was a
trend for more accurate left hemisphere (LH) decisions
but no consistent hemispheric difference in latency. Only
one patient (LB) showed significant facilitation, and then
only in the LH.
Lexical decision of lateralized targets with free
auditory primes showed again bilateral competence and no
consistent hemispheric superiority in accuracy. Latency
data showed significant but mixed hemispheric
superiorities. Furthermore, auditory facilitation was
about equally likely to occur in either hemisphere.
Table 1
Summary of performance of commissurotomy patients
on lexical decision and semantic facilitation
Targets
Lexical Bl decision - - - -Semantic facilitation
Hem~s~heri_c LH spec1ahzat1on
alone
4/5 RH 5/5 LH
- - - - -
Visual Auditory primes primes
Bl 3/4 RH Bl 4/5 RH 3/4 LH 4/5 LH - - - - - - - - -
LH? Bl 4/5 RH 4/5 LH
·-Mixed Mixed
Bl = bilateral competence; LH = left hemisphere, RH = right hemisphere; 3/4 RH = 3 out of 4 right hemispheres, etc.
What is the relation of lexical decision to reading?
The same targets used in the lexical decision tasks were
subsequently used for a reading test in which two
complete commissurotomy patients were required to point
to multiple choice arrays of pictures in free vision, in
response to the lateralized target. Hemispheric
performance estimates for both hemispheres were
generally higher for the reading than for the decision task.
Furthermore, the tachistoscopic assessment of reading
competence in these patients actually underestimates the
hemispheric reading vocabularies as assessed with a
custom-made contact lens technique for free ocular
scanning. It would seem, therefore, that lexical decision
provides a conservative estimate of competence for
reading in the disconnected RH.
The same lexical decision was administered to normal
subjects (Radant, 1981). These subjects showed evidence
of RH competence for both decision and semantic
facilitation. Thus, the disconnected RH, in turn, appears
to underestimate the contribution of the normal RH to
language.
Reference: Radant, A. (1981) Undergraduate Honors Thesis, Depart
ment of Psychology, University of California, Los Angeles.
143
203. DISCONNECTION SYNDROME AS A MODEL FOR LATERALITY EFFECTS IN THE NORMAL BRAIN
Investigator: Eran Zaidel
Consider a dichotic listening experiment showing a
right ear advantage (REA) or a hemifield tachistoscopic
experiment showing a right visual half-field advantage
(RVFA) for some language stimuli. Both are commonly
interpreted to reflect left hemisphere (LH) specialization
in processing the stimulus material. But how are we to
interpret the observed laterality effect? Does it reflect
exclusive LH specialization for processing so that in
reading stimuli the RH must first shuttle across the
corpus callosum to the LH at some cost in latency and
stimulus quality, hence the REA or RVFA? Or can the RH
also process its own input, but not as quickly and
accurately as the LH, hence the REA or RVFA?
Let us call the first possibility the "callosal relay"
model, and the second possibility the "direct access"
model. The latter focuses on relative hemispheric
specialization, whereas the former emphasizes callosal
connectivity. Complex tasks that require inter
hemispheric interaction may involve components of both
models.
How can we tell whether a given task fits one model or
the other? Direct access tasks involve no callosal
transfer and can be done by both hemispheres so that a
small and comparable laterality effect should be observed
in the normal and in the disconnected brains. Callosal
relay tasks, on the other hand, should result in massive
laterality effects in the split brain, where callosal
transfer is impossible, but in only small effects in the
normal brain. Other behavioral criteria for direct access
and callosal relay tasks in the normal brain include
(i) response hand by hemifield interaction (direct access),
(ii) a main hand effect without a hand by hemifield
interaction (callosal relay), and (iii) a hemifield by
stimulus complexity interaction (direct access).
Conditions i and ii may not hold for choice RT tasks where
the motor response reflects a simple binary choice (e.g.,
yes/no).
Examples of tasks that fit both models actually exist.
Thus, dichotic listening to nonsense CV syllables is a
callosal relay task. It shows a massive REA in the
disconnected LH, signaling left hemisphere specialization,
but only a modest REA in the normal brain. Further, the
disconnected RH cannot do the task at all so that the left
ear signal must be transferred to the LH via the corpus
144
callosum for further processing (Zaidel, 1976). Lexical
decision of concrete, imageable words, on the other hand,
fits the direct access model (see Abstract No. 202).
The existence of direct access and callosal relay tasks
makes it possible to analyze the possible selective roles of
relative hemispheric specialization or of callosal connec
tivity in giving rise to diverse laterality effects as a
function of individual differences in sex, handedness, and
cognitive profile.
Reference: Zaidel, E. (1976) Jn: UCLA Conference on Human Brain
Function. BR!, UCLA, Los Angeles, pp. 103-110.
204. LEFT HEMISPHERE SUPERIORITY FOR PERCEPTION OF SEQUENTIALLY-PRESENTED STIMULI
Investigators: Harold W. Gordon*, Eran Zaidel
While it is generally accepted that the left cerebral
hemisphere is dominant for the processing of sequences,
the evidence is limited in the case of auditory stimuli.
There are difficulties in investigating the lateral
asymmetry of sequential processing in normal subjects
because long sequences may not be conveniently
lateralized to the left or right hemisphere by conventional
tachistoscopic or dichotic techniques. Accordingly, most
studies rely on observations of dysfunction in neurological
patients w!th verified unilateral left or right lesions
(Bentin and Gordon, 1979). This problem has been
eliminated by use of unimanual presentations of tactile
stimuli to split-brain patients showing LH superiority for
both common objects and nonsense shapes (see Biology
1973, No. 54). Jn the present study we used a specially
designed scleral contact lens with an attachment that
restricts vision to one half-field. This lens was used in the
present study with two patients who have undergone
complete forebrain commissurotomy (NG, LB) to inves
tigate the ability of each hemisphere separately to
process sequentially-presented stimuli.
Sequential perception was assessed with two tests, one
in which the sequential stimuli were presented audibly,
one in which they were presented visually. For the
auditory test, easy-to-recognize sounds such as rooster
crowing, trumpet fanfare, and bird chirping were
presented in sequences of 2, 3, 4, and 5. · The subject's
task was to point to associated pictures in the same
sequence as the sounds had been presented. The
associated pictures were confined to one visual field (one
hemisphere), assuring that assessment of each hemisphere
was made independently. The visual sequence test was
presented by a Super-8 movie. Six circles arranged in a
hexagon array were "colored in" at a rate of 4/sec in
sequences of 3, 4, and 5. The subject's task was to
indicate (by pointing) the correct sequence of the "colored
in" circles on a response card containing the same 6-circle
array. With the lens system, both the stimuli and response
card could be confined to one visual field, again assuring
the assessment of one hemisphere at a time. The scoring
for both tests gave weighted credit for longer sequences,
and partial credit for partially correct sequences (e.g., the
first 3 correct in a sequence of 5 would get some credit
even if items 4 and 5 were omitted or reversed).
The results for both the auditory and visual tests were
the same for both subjects. The score for the left
hemisphere was consistently better than the right,
reflecting the facts that the left hemisphere was more
consistently correct and was able to process longer
sequences. In order to enhance the chances of the right
hemisphere success, the test had been given first as a
practice run to the right hemisphere, followed by testing
the left hemisphere and then retesting the right hemi
sphere. For both subjects, the score of the right
hemisphere was still only about half that of the left
hemisphere. The right hemisphere correctly reported
about half of the trials with 3 in a sequence and
occasionally reported correctly 4 in a sequence. The left
hemisphere regularly reported 3 in a sequence with few
errors and often correctly reported 4 and even 5 in a
sequence. Whereas the performance for either hemi
sphere was below normal, the reduced capacity of the
right hemisphere compared to the left in the same subject
was clear. The results give direct support for left
hemisphere superiority in perception of sequences whether
they are presented by the visual or auditory modality.
Reference: Bentin, S. and Gordon, H. w. (1979) J. Neuro!. Neurosurg.
Psychiat. 42, 715-723.
*Department of Psychiatry, University of Pittsburgh, School of Medicine.
205. RIGHT HEMISPHERE SUPBRIORITY FOR PROCESSING MENTAL IMAGES OF RECTANGULARSOLJDS
Investigators: Harold W. Gordon*, Eran Zaidel
Specialized abilities of the r-ight cerebral hemisphere
have been variously described as 11spatial, 11 and
"synthetic, 11 in reference to better performance on tasks
of pattern perception, orientation, localization, and
musical perception. As with most studies in this area,
conclusions are inferred from performance deficits in
patients with verified unilateral brain lesions, or in quick
flashing (tachistoscopic) or right/left competition
techniques, dichotic (auditory) or dichhaptic (tactual)
presentation, in non-neurological subjects. Patients with
complete commissurotomy offer a unique opportunity to
explore the capabilities of each hemisphere separately and
thereby compare the performance of each in the same
individual. Accordingly, the contact lens system was used
to present a test of three-dimensional mental imaging in
each hemisphere separately in order to obtain direct
compariscn between the right and left hemisphere ability
In this domain.
The test consisted of a picture of a stack of 8-10
rectangular solids arranged randomly along each of the
three Euclidean axes. (Imagine a cube made up of several
rectangular blocks.) One block was designated a "key"
block and was shaded in the picture. The task for the
subject was to indicate which other blocks were touching
the ''key" block. Some of the touching blocks could be
directly observed, others had to be inferred or imagined
by extrapolating to the third dimension. The test was
presented to each hemisphere separately, first to the right
hemisphere, then to the left. It consisted of 30 drawings
of six different stacks of blocks.
The results showed a striking superiority of the right
hemisphere for the one subject (LB) who was able to
perform the task. The score of complete~y correct
responses was 26/30 for the right hemisphere and 16/30
for the left. Three of the four incorrect responses for the
right hemisphere were due to false positives while only
2/14 were false positives in the left hemisphere. There
were 108 blocks that could be directly observed to be
touching the "key" blocks over the 30 trials. The right
hemisphere was able to point to all of them without error;
the left pointed to 102. There were 29 touching blocks
that were hidden so that contact had to be inferred from
extrapolating to the third dimension. The right hemi
sphere pointed out 28_of the 29; the left pointed out only
9. The importance of right hemisphere superiority in this
task is most evident when contrasted with left hemisphere
superiority in sequencing tasks and, of course, in verbal
skills. These results complement earlier findings about
right hemisphere superiority in three-dimensional spatial
representation (Levy, 1969).
145
Reference: Levy, J. (1969) Unpublished Doctoral Dissertation,
California Institute of Technology.
*Department of Psychiatry, University of Pittsburgh, School of Medicine.
206. HEMISPHERIC SPECIALIZATION FOR ORIENTED LINES
Investigators: Charles R. Hamilton, Betty A. Vermelre
The split-brain preparation allows the two cerebral
hemispheres to be tested independently for speicalized
abilities. Our progress in studying hemispheric
differences in monkeys is summarized in this and the next
two abstracts. Overall, the possibility of human-like
differences in the processing abilities of the two
hemispheres remains tenable even though negative results
still outnumber the positive ones.
Last year we reported that the left hemisphere learned
to differentiate lines tilted 15° from each other about 2.5
times more readily than did the right hemisphere (Biology
1981, No. 235). This finding has now been confirmed with
eight additional monkeys. Furthermore, these 16 monkeys
showed no consistent hemispheric specialization while
discriminating symmetrical geometric patterns presented
in the same training apparatus. This controls for
unrecognized asymmetries in our testing paradigm or in
our surgical preparation. We are now determining the
thresholds for discrimination by each hemisphere and are
trying to pin down the exact cues being used by the
monkeys. For example, besides the tilt of the lines there
are correlated differences in the position of the ends of
the line as well as possible uncontrolled asymmetries in
the stimuli. If our results remain robust, we may finally
have a tool for examining the mechanisms of hemispheric
specialization with invasive techniques that are not
practical for use with human subjects.
207. HEMISPHERIC DIFFERENCES IN FACIAL DISCRIMINATION
Investigators: Betty A. Vermelre, Catherine K. Ifune*, Charles R. Hamilton
We previously reported no differences between the left
and right hemispheres of split-brain rhesus monkeys in
learning to discriminate photographs of the faces of
conspecifics (Biology 1976, No. 154). Because recognition
of emotion in human faces is more lateralized in man than
is individual recognition, we decided to test our monkeys
with photographs of monkeys making different
146
expressions. As reported last year (Biology 1981, No.
234), we made four discriminations based on differences in
expression with the individual held constant and four
based on individuals with the expression constant. To
date, eight subjects have learned these eight problems.
Overall, they have not shown consistent differences
between the left and right hemispheres although the
retention of these discriminations was considerably, but
not yet significantly, better with the right hemisphere.
When the handedness of the subjects is taken into account,
there is a significant correlation between the handedness
and dominance indices of the monkeys such that the
hemisphere opposite the preferred hand learned more
readily. Several monkeys have also been tested with each
hemisphere for the ability to recognize new photographs
of the expressions or individuals in order to assess the
generalizability of the acquired discriminations. While
noticeable hemispheric differences exist for the subjects,
these data are still too incomplete for a consistent picture
to be drawn. Final interpretation of all the above results
must wait until all 16 monkeys in the balanced design are
tested.
*Undergraduate, California Institute of Technology.
208. SEQUENTIAL PROCESSING IN THE TWO HEMISPHERES OP SPLIT-BRAIN MONKEYS
Investigators: Betty A. Vermeire, Charles R. Hamilton
Twelve split-brain rhesus monkeys were tested for
differences in the abilities of their two cerebral
hemispheres to learn discriminations based on the
comparison of sequentially-presented visual stimuli
(Biology 1980, No. 217). Across all monkeys there was no
generalized advantage for either hemisphere in learning
these discriminations. However, there was a significant
correlation between each monkey's handedness and the
hemisphere that learned more readily; the more proficient
hemisphere tended to be contralateral to the pre
operatively preferred hand. These data suggest that
monkeys' handedness may be more closely related to
cognitive processing than is usually believed. In addition,
the results show that in these monkeys, as in right-handed
human subjects, sequentially-received information is
processed better by the hemisphere contralateral to the
preferred hand.
Experiments with human subjects have usually required
reporting the order of stimulus presentation. Therefore,
to make our experiments more comparable to those with
human subjects,
sequential task.
we are testing our monkeys with a new
Up to four circular windows arranged
vertically before the monkey flash briefly in succession,
with their order different on each trial. To obtain a pellet
of food, the monkeys must reconstruct the sequence by
· pushing the Y1indows in the same order in which they were
flashed. Left and right hemispheres are being tested
separately. This experiment should extend our previous
finding by using a task known to be better latera!ized to
the left hemisphere of man.
209. INTERHEMISPHERIC COMMUNICATION IN PARTIALLY SPLIT-BRAIN MONKEYS FOR PERCEPTUAL PROCESSES
Investigators: Charles R. Hamilton, Betty A. Vermeire
An approach to studying the functions of different
regions of cortex by behavioral methods has been
developed for use with monkeys (Biology 1979, Nos. 205-
208; Hamilton, 1982). Jn brief, a restricted set of visual
areas is left interconnected with the opposite hemisphere
by a small commissural bridge and the functions of these
areas inferred from the kinds of information that can still
be conveyed interhemispherically. We have chosen two
perceptual tasks, interocular transfer of tilt aftereffects
and stereopsis, for initial study because they are thought
to depend on binocular neurons in the early stages of
visual processing and hence upon known commissural
connections.
We have recently shown with five monkeys that tilt
aftereffects can be reliably measured. The subjects
continuously gaze at a high contrast square wave grating
that is unpredictably replaced with a thin, dim test line.
If the line is vertical, a push is rewarded; if the line is
tilted, withholding a response is rewarded. When the
grating is tilted about 10° from vertical, human subjects
typically pick as vertical a test line tilted 2° or 3° in the
same direction. Furthermore, if the grating is viewed
monocularly, normal subjects show about 7096 interocular
transfer of this illusion. All five monkeys, tested
binocularly, have demonstrated a reliable tilt aftereffect
of about 1°. The smaller magnitude of the aftereffect of
monkeys compared with people probably represents less
complete adaptation to the grating caused by an average
viewing time of about 8096. Tests for interocular transfer
of the aftereffect are now being run with four monkeys
that have either the anterior commissure or the splenium
intact. If transfer occurs in the splenium intact group, as
expected, then tests with further subdivisions of the
splenium will be continued to pinpoint the visual areas
that are involved.
The same partially split-brain monkeys will be tested
for preservation of stereopsis along the vertical meridian
of the visual field. We will test local stereopsis with
traditional stereograms and global stereopsis with
dynamic random dot stereograms. Although optic chiasm
section destroys the principal source of binocularity,
cortical cells near the representation of the vertical
midline can still receive information through the commis
sures from the contralateral eye and therefore could
support stereopsis. The commissural bridges permitting
midline stereopsis should allow inferences as to which
visual areas are used.
Reference: Hamilton, c. R. (1982) In: Analysis of Visual Behavior,
MIT Press, pp. 693-717.
210. INTERHBMISPHI!RIC COMMUNICATION IN PARTIALLY SPLIT-BRAIN MONKEYS DURING LEARNING
Investigators: Charles R. Hamilton, Betty A. Vermeire
Discrimination learning is usually thought to occur
farther downstream from the primary visual cortex than
the perceptual functions discussed in the preceding
abstract. Thus interhemispheric transfer should occur via
commissural fibers connecting areas farther into the
visual hierarchy. We are attempting to determine if the
learning of discriminations based on different cues is
transferred across different commissural regions. At
present we are comparing interhemispheric transfer of
discriminations of geometrical patterns, characteristics of
monkey faces, and direction of movement in monkeys with
an intact anterior commissure or splenium. So far, all
three transfer through the splenium as expected.
Discriminations of patterns and movement also transfer
through the anterior commissure, which is consistent with
known cortical connections to inferotemporal cortex;
there is not yet enough data to decide if facial discrimi
nations also transfer through the anterior commissure.
Once the basic magnitude of transfer of these tasks is
established for these two routes, additional surgical
fractionation of the interhemispheric pathways will be
pursued to further characterize the pathways and areas
involved.
147
PUBLICATIONS
Benowitz, L. I., Bear, D. M., Rosenthal, R., Mesulam, M., Zaidel, E. and Sperry, R. W. (1982) Hemispheric specialization in nonverbal communication. Science, in press.
Hamilton, c. R. (1982) Mechanisms of interocular equivalence. In: Analysis of Visual Behavior. D. J. Ingle, M. A. Goodale and R. J. W. Mansfield (Eds.), pp. 693-717. MIT Press, Cambridge.
Hamilton, c. R. and Vermeire, B. A. (1982) Hemispheric differences in split-brain monkeys learning sequential comparisons. Neuropsychologia, in press.
MacKay, D. M. (1980) Letters to the Editors, The interdependence of mind and brain. Neuroscience 5, 1389-1391.
MacKay, D. M. and MacKay, V. (1982) Explicit dialogue between left and right half-systems of split brains. Nature 295, 690-691.
Myers, J. J. and Sperry, R. w. (1982) A simple technique for lateralized visual input that allows prolonged viewing. Behavior Research Methods and Instrumentation, in press.
Zaidel, E. (1981) Hemispheric specialization for reading: From Franz to Sperry. Brain Research Institute Bulletin, UCLA, 5, 9, 13-19.
Zaidel, E. (1981) Hemispheric intelligence: The case of the Raven Progressive Matrices. In: Intelligence and Learning. M. P. Friendman, J.P. Das and N. O'Connor (Eds.), pp. 531-552. Plenum Publishing Corp., New York.
Zaidel, E. (1982) Reading in the disconnected right hemisphere: an aphasiological perspective. In: Dyslexia: Neuronal, Cognitive and Linguistic Aspects. Y. Zotterman (Ed.), pp. 69-91. Wenner-Gren Symposium Series, Vol. 35. Proceedings of an International Symposium held at the Wenner-Gren Center, Stockholm, June 3-4, 1980. Pergamon Press, Oxford.
Zaidel, E. (1982) Advances and retreats in laterality research, Extended commentary on J. L. Bradshaw and N. S. Nettleton (1981) The nature of hemispheric specialization in man. The Brain and Behavoral Sciences, in press.
Zaidel, E. (1982) On multiple representations of the lexicon in the brain: The case of the two hemispheres. In: The Neurobiology of Language. M. StuddertKennedy and G. Adelman (Eds.), NRP Monograph. MIT Press, Cambridge, in press.
Zaidel, E. (1982) Disconnection syndrome as a model for laterality effects in the normal brain. In: Cerebral Hemisphere Asymmetry: Method, Theory and Application. J. Hellige (Ed.), Praeger, New York, in press.
Zaidel, E. and Peters, A. M. (1981) Phonological encoding and ideographic reading by the disconnected right hemisphere: Two case studies. Brain and Language 14, 205-234.
Zaidel, E., Zaidel, D. and Sperry, R. W. (1981) Left and right intelligence: Case studies of Raven's Progressive Matrices following brain bisection and hemidecortication. Cortex 17, 167-186.
148
Associate Professor: David c. Van Essen Research Pellow: Andreas Burkhalter Graduate Students: George J. Carman, Herman Gordon,
Baruch Kuppermann, John H. R. Maunsell Research Staff: Michael Connolly, Carol Shotwell
SUpport: The work described in the following research reports has been supported by:
Fogarty International Research Fellowship National Institutes of Health, USP HS National Science Foundation Pew Memorial Trust
Summary: The cerebral cortex of primates has recently
been found to contain a large number of distinct visual
areas which collectively carry out many diverse aspects of
visual perception and attention. Our laboratory uses a
combination of anatomical and physiological techniques to
identify cortical areas and study their functional
organization in the macaque monkey. Our major
apprOaches are (1) to trace the connections between
various areas, (2) to determine how the visual field is
represented within each of them, and (3) to ascertain the
functional properties of cells in different areas. This year
we can report significant progress on several fronts. Two
new visual areas have been identified, raising the total
number of known visual cortical areas in the macaque to
12. Detailed analyses have been made of the topographic
organization of several visual centers. This has led to a
better understanding of the transformations taking place
at different levels of a single sensory system. Using a
variety of anatomical tracer techniques, new pathways
between various areas have been identified. As well as
contributing to a rapidly expanding list of known intra
cortical connections in the macaque, this information has
led to an important insight concerning the hierarchical
organization of cortical visual areas. In particular, the 12
known areas can be grouped into seven hierarchical levels
on the basis of specific features of their patterns of
connectivity.
Several interesting results have come out of studies of
single unit properties in extrastriate visual cortex. A
detailed analysis of area MT has shown that this area is
specialized for processing three aspects of stimulus
motion: direction, speed, and depth in space. The way in
which motion-in-depth is analyzed differs from that
expected on the basis of previous studies, however. This
has important implications for our understanding of the
psychophysics of motion perception in humans. We have
also obtained interesting results on the functional
organization of several areas in the ventral part of the
hemisphere. The most striking result has been the finding
of a high incidence of color-selective cells in at least two
areas, V2 and VP. This implies that color analysis is not
restricted to a single extrastriate area, V 4, as had been
hypothesized in reports from other laboratories.
A separate facet of our research progr~m concerns the
development and plasticity of the mammalian neuro
muscular junction. Our interest is in the phenomenon of
synapse elimination, whereby the majority of synapses
initially formed in developing muscles are removed in a
relatively brief period after birth. During this past year
we have obtained evidence that there are two distinct
stages of synaptic reorganization during postnatal
development. Moreover, the rules governing the likeli
hood that any given synapse will survive appear to differ
during these two stages. Further analysis of these events
may provide insights relevant to the understanding of
competitive interactions elsewhere in the nervous system.
211. FUNCTIONAL SPECIALIZATION l'OR MOTION ANALYSIS IN THE MIDDLE TEMPORAL AREA OP THE MACAQUE
Investigator: Jolm H. R. Maunsell
The middle temporal visual area (MT) in the macaque
monkey is known to contain a large percentage of cells
that are selective for the direction of stimulus motion.
We were interested in learning more about the responses
of neurons in MT to this and other aspects of visual
stimuli. Computer generated stimuli were used to
examine quantitatively the responses of 168 cells in MT.
The results confirmed earlier reports on the high
degree of direction selectivity in this ~ea. On average,
the response of a cell to motion in its preferred direction
was about 10 times that to motion in the opposite
direction. Tuning for direction was sharp, with most cells
giving half-maximal responses when stimulus motion was
changed by only 30 degrees from optimal.
In addition to having direction selectivity, most
neurons in MT were shown to be sharply tuned for stimulus
speed. On average, the responses of a given cell fell to
half-maximal within a factor of four on either side of the
optimal speed; many cells were inhibited by motion at
speeds which were far from the preferred value. Although
most cells were sharply tuned for speed, individual cells
had different preferred speeds, so the range which they
collectively covered was relatively broad, running from
about 1 to 250 degrees/second.
It was possible to compare the results of these and
other tests with existing quantitative data reported by
John Allman's laboratory on the response properties in the
homologous area in another primate, the owl monkey.
Overall, the data from the two species are remarkably
similar. This similarity is striking in light of the fact that
the owl monkey is a nocturnal, New World species, while
the macaque is diurnal and from the Old World. The high
degree of conservation of response properties between
these different species may provide a clue to the role of
MT in visual function.
212. ANALYSIS OF MOTION lN THREE-DIMENSIONAL SPACE lN THE MIDDLE TEMPORAL AREA OF THE MACAQUE
Investigator: John H. R. Maunsell
The macaque monkey, like man, makes use of the
different views seen by the two eyes to judge the distance
to points in space. Objects that do not lie on the plane of
fixation have images in the eyes that fall on non
corresponding parts of the retinas. The sign and
magnitude of this retinal disparity is used in stereoscopic
vision to determine the distance of objects. Vl and V2 in
the macaque are known to contain neurons that are
selective for retinal disparity. Because most neurons in
the ·middle temporal area (MT) are sensitive to the
direction and speed of visual motion parallel to the
fixation plane, it is of interest to know if this area is
further specialized for handling information about motion
in three-dimensional space. We examined neurons in MT
with stimuli that simulated objects moving parallel to the
fixation plane at different distances from the animal, and
also stimuli that simulated motions with components
toward or away from the animal-motion-in-depth.
The majority of neurons tested (52/76) showed striking
sensitivity to the disparity of stimuli moving parallel to
the fixation plane. Most of these cells could be classified
into one of four types of response. The first group
responded well only over a narrow range of disparities
near zero. These cells could be expected to signal
information about objects on or near the fixation plane.
The seeond group had responses that were complementary
to the first, responding best to stimuii in front or behind,
but not on, the fixation plane. The final two groups also
responded well over broad ranges, but were more
selective. One responded well only to stimuli in front of
the fixation plane, the other preferred stimuli that were
beyond.
149
None of the neurons that were tested was truly
selective for motion with components toward or away
from the animal. Because cells had tuning for disparity,
they generally preferred motions in depth which moved
from a bad disparity to a preferred disparity over fronto
parallel motion at a bad disparity, but the best overall
responses were almost always to fronto-parallel motion at
the preferred disparity. We therefore do not regard such
cells as selective for motion-in-depth. Nonetheless, the
presence of a large percentage of disparity tuned cells in
MT indicates that this area is very well suited for the
analysis of motion in three-dimensional space.
213. CORTICAL AND SUBCORTICAL CONNECTIONS OF AREA MT lN THE MACAQUE
Investigator: John H. R. MaWISell
MT is a well-defined visual area ·that is highly
specialized for the analysis of visual motion. Knowledge
about which visual areas it has connections with -can
provide insight into how this aspect of visual information
is processed in the nervous system. While MT is known to
receive input from Vl and V2, little is known about its
other connections. We injected both 3H-proline and
horseradish peroxidase into MT to demonstrate its inputs
and outputs.
Three successful injections were made in MT. The
demonstrated patterns of cortical and subcortical
connections were consistent among the different
injections. Connections were found between MT and many
other eortical areas, and most of these pathways were
shown to pass information in both directions. On the basis
of the cortical layers in which projections originated and
terminated, it was possible to group almost all the
cortical areas with which MT has connections into two
categories: those at lower levels, which predominantly
supply input to MT, and those at higher levels, which
receive input from MT. The inputs to MT arose from Vl,
V2, V3, VP, and a region tentatively designated VA in
ventral extrastriate cortex. The areas receiving output
from MT include two previously unidentified areas that we
have called the medial superior temporal area (MST) and
the ventral intra-parietal area (VIP). Our laboratory has
previously recorded from the cortex in MST; we have
found that this area has many neurons that are direction
selective, like those in MT, but they have much larger
receptive fields. It is likely that MST represents a
subsequent stage in the cortical processing of information
150
about motion. One other area, V 4, had reciprocal
connections with MT whose laminar distributions were
intermediate between the input and output types.
MT also projects to many subcortical structures,
including the pulvinar (lateral and inferior subdivisions),
the pregeniculate nucleus, the thalamic reticular nucleus,
the claustrum, the putamen, the caudate nucleus, the
superior colliculus, and the pontine nuclei. Only the
pulvinar was shown to return a projection to MT. The
projection to the pontine nuclei is of particular interest,
since these nuclei project directly to the cerebellum, a
major center of the control of body movements. This
suggests that MT may supply information about move
ments in the visual field that is used for guiding body
movements.
214.. SPATIAL ORGANJZATION OP DIRECTIONALLY-SELECTIVE NEURONS IN THE MIDDLE TEMPORAL AREA OP MACAQUE
Investigators: Andreas Burkhalter, Jolm H. R. Maunsell
The large proportion of neurons selective for the
direction of stimulus motion found in the middle temporal
area (MT) of the macaque suggests that this area is
involved in motion analysis. Physiological studies have
shown clustering of cells having similar direction
preferences in MT, suggestive of a columnar organization
of preferred direction.
We have used the 2-deoxyglucose labeling technique to
study the pattern of labeling in MT after stimulation with
spots moving in a single direction (four experiments) or
alternating between movements to the left and to the
right (one eXI?eriment). Animals were exposed to the
stimulus in only one visual hemifield, which allowed
comparison of the stimulated and the control hemisphere
within the same individual. The patterns of labeling in the
stimulated and the control hemispheres were compared to
the labeling pattern in an unstimulated control animal,
which had both eyes covered during the eXI?eriment.
In both hemispheres of the control animal, labeling was
densest in layer IV and was non-uniform throughout
extrastriate cortex. No obvious asymmetries were
observed between the two sides. This picture was similar
within MT, where occasional patches and radially-oriented
columns occurred.
In the unidirectionally stimulated animals, there was a
significant asymmetry, mainly in parts of MT where fields
outside the fovea are represented and where the stimu
lation was most effective. The labeling on the stimulated
side was arranged in distinct columns that extended
radially throughout the entire thickness of cortex. Their
cross section was mostly oval with about 1.2 mm in the
long and 0.3-0.5 mm in the short axis. The center-to
center spacing between individual columns ranged from
0.6 to 1.2 mm. Occasionally two neighboring columns
merged in layer IV, but separated again in deeper layers.
In the bidirectionally stimulated animal, the pattern of
labeling in the two hemispheres was asymmetric, as in the
unidirectional cases. The individual columns, however,
were about twice as wide (0.6-0.8 mm) as in the uni
directional eXI?eriments. The center-to-center spacing
remained about equaL
From these results we conclude that stimulus direction
in MT is represented in columnar fashion. This is evidence
that an anatomically-defined functional system of
direction of stimulus motion exists in extrastriate visual
cortex. Interestingly, the direction columns in MT are not
arranged in stripes or slabs, as occurs for ocular
dominance columns in striate cortex; rather, they form
discrete, moderately elongated zones which often are
immediately adjacent to columns representing the
opposite direction of movement.
215. FUNCTIONAL PROPERTIES OP SINGLE CELLS IN VISUAL AREAS V2, VP AND VA OP VENTRAL EXTRASTRIATE CORTEX IN THE MACAQUE
Investigators: Andreas Burkhalter, Jolm H. R. Maunsell, David C. Van Essen
To learn more about the functional subdivision of
macaque extrastriate visual cortex, we have recorded
from single cells in three areas in the ventral part of the
occipital lobe: V2, VP and a less well-characterized area
tentatively designated VA. Neurons were examined for
their selectivity to stimulus shape, orientation, direction,
speed, binocular disparity and color; responses were
analyzed quantitatively.
In three monkeys 117 units were examined, of which 57
were in V2. The remaining 60 were in cortex anterior to
V2 and are considered as a single group (VP IV A) for the
present, because final assignments to VP or VA have not
been completed.
The most striking result to date has been the finding of
a high incidence of color-sensitive neurons in V2 and
VP IV A. In both regions nearly half of the cells studied
were sensitive to the wavelength of the stimulus, and
many of these responded only to a narrow portion of the
visible spectrum and far less, if at all, to white light.
These observations show that the analysis of stimulus
color is strongly emphasized in several visual areas and
not just in a single area, V4, as suggested by reports from
other laboratories.
In both regions there were many cells that were
selective for stimulus parameters other than color. A
higher percentage of cells were orientation-selective in
V2 (84%) than in VP/VA (59%). In contrast, the incidence
of direction selectivity was higher in VP/VA (41%) than in
V2 (23%), and the same was true for disparity selectivity
(56% in VP/VA, 33% in V2). Interestingly, none of the
cells in V2 was selective for more than two of the
parameters that we tested for, whereas 30% of the VP/VA
cells were selective for three parameters (e.g., color,
orientation and disparity).
These results demonstrate a considerable degree of
functional specificity in individual cortical neurons. It is
likely, however, that each visual area among the ones
under study is involved in more than one aspect of the
analysis of form, color, and motion in the visual world.
216. TRANSFORMATIONS IN THE VISUAL REPRESENTATION IN THE RETIN<>-GENICULOSTRIATE PATHWAY
Investigators: Michael Connolly, David C. Van Essen
It is well known that there is a marked emphasis of
central vision relative to the visual periphery in most
visual centers. This obviously is due in part to the
elevated density of retinal ganglion cells in the central
retina, but it has been controversial whether the
distribution of ganglion cells can account entirely for the
topographic organization of higher visual centers. The
issue is of importance for understanding the ways in which
each center is specialized for processing different aspects
of visual information.
Last year (Biology 1981, Nos. 241 and 242), we
reported on the use of two-dimensional maps of striate
cortex and the lateral geniculate nucleus (LGN) in the
macaque as a means of facilitating the accurate analysis
of visual topography of these structures. In continuing
this approach over the past year, our primary goal has
been to obtain accurate expressions of cortical and LGN
"magnification factors," i.e., distance within the cortex or
LGN per degree of visual field. A previous report by
Daniel and Whitteridge (1961) had suggested that the
cortical magnification factor is inversely proportional to
eccentricity in the visual field and that it is independent
of polar angle and of the direction in the cortex along
151
which it is measured. However, we have found that none
of these relationships is exactly correct. Magnification
declines more steeply than the inverse of eccentricity; it
depends on polar angle, as there is more cortex devoted to
the horizontal meridian than to the vertical meridian; and
the representation is anisotropic in many regions, with a
greater magnification along lines of constant polar angle
than along lines of constant eccentricity.
Similar complexities exist in the representation in the
LGN, but there are important quantitative differences.
The dependence of magnification on eccentricity is not as
steep as in the cortex. The anisotropies in the LGN differ
from those in the cortex in a manner that may be related
to the way in which alternate LGN layers project to form
interdigitating ocular dominance stripes in the cortex.
Comparisons between retina, LGN, and cortex indicate
that there are major transformations at each stage. In
particular, retinal ganglion cell density, determined in
other studies, does not change with eccentricity as rapidly
as do the magnification factors in the LGN and cortex.
Thus, the postulate of "peripheral scaling," i.e., that
central sensory representations are dictated strictly by
the density of peripheral innervation, does not hold in the
macaque visual pathway.
Reference: Daniel, P. M. and Whitteridge, D. (1961) J. Physiol. 159,
203-221.
217. THE PATTERN OF OCULAR DOMINANCE STRIPES IN MACAQUE STRIATE CORTEX
Investigators: Michael Connolly, David C. Van Essen
In order to achieve binocular fusion and stereopsis, it
is necessary to have precisely aligned inputs from the two
eyes. The first stage of the visual pathway at which there
are binocular inputs to individual neurons is in the striate
cortex. Binocular integration at this level involves a
network of ocular dominance columns, or stripes, which
receive inputs alternately from the left and right eyes.
Previous studies of the pattern of ocular dominance
stripes in the macaque monkey have been incomplete
because a large portion of striate cortex is buried within
the complex folds of calcarine sulcus. In the present
study we have used the technique of preparing two
dimensional cortical maps to obtain a complete
representation of ocular dominance stripes in a single
hemisphere. A large dose of 3H-proline was injected into
one eye of an adult macaque, and the animal was allowed
to survive long enough to permit transneuronal transport
152
of label through the lateral geniculate nucleus and up to
striate cortex. Histological sections through the cortex
were processed for autoradiography to reveal alternating
labeled and unlabeled regions within layer !Ve, the layer in
which geniculocortical fibers terminate.
A number of interesting features of the ocular
dominance pattern are evident from our preliminary
analysis of the completed cortical maps. As expected
from previous studies, the ocular dominance stripes
intersect the margins of striate cortex roughly at right
angles. Surprisingly, though, the stripes are significantly
wider near the border, where the vertical meridian is
represented, than they are near the horizontal meridian
representation. Over most of striate cortex, the stripes
run approximately orthogonal to the representation of the
horizontal meridian. Although lines of constant
eccentricity in the visual field tend to run in the same
direction on the map, the two sets of contours are not
strictly parallel to one another. In the region of central
representation {less than 5° eccentricity), the pattern of
ocular dominance stripes is even more complex, and in
places the stripes run parallel to the horizontal meridian
representation. Another significant difference between
center and ?eriphery concerns the balance between left
and right-eye inputs. For the central representation, the
inputs are equally balanced, but for eccentricities of 20°
and above the contralateral eye shows an unexpected
dominance. A final noteworthy feature is that the optic
disc, which is a nearly circular structure within the retina,
is represented as a highly elongateQ.. region in the cortex.
This supports previous physiological evidence for the
existence of significant anisotropies in the visual repre
sentation within the cortex.
This work was done in collaboration with Dr. Simon
Le Vay of Harvard Medical School.
218. TWO STAGES OF SYNAPTIC REORGANIZATION IN THE RABBIT SOLEUS MUSCLE
Investigators: Herman Gordon, David c. Van Essen
During early postnatal development of mammalian
muscles, a major reorganization occurs in the pattern of
innervation. In the rabbit soleus muscle, our experimental
system, individual muscle fibers at birth are innervated by
an average of four motoneurons, but over a period of two
weeks each muscle fiber comes to be innervated by only
one motoneuron. Last year (Biology 1981, No. 243), we
reported that concomitant with this wholesale loss of
synapses, the distribution of tensions produced by
individual motor units becOmes relatively more diverse.
On the assumption that motor unit tension accurately
reflects the number of muscle fibers innervated by a
single motoneuron, we inferred that motoneurons differ in
their abilities to retain synaptic sites within the muscle.
In particular, synapses from large motor units are
evidently at a competitive advantage and those from
small motor units at a disadvantage during the first two
weeks after birth.
In the past year, we have examined the motor unit
sizes in muscles from animals five to six weeks old and
have obtained evidence for a second stage of postnatal
synaptic reorganization. At this age the motor unit
tensions are considerably less diverse than at two weeks
or even at birth. Thus, there appears to be a protracted.
secondary period of reciprocal sprouting and elimination
of synapses favoring the growth of small units and the
shrinkage of large units. Recently, we have followed an
analogous sequence of events in the soleus muscle of the
rat as well.
We would like to know how the secondary
reorganization occurs. Are muscle fibers transiently
denervated by shrinkage of large motor units or are they
transiently polyinnervated by sprouts from expanding
small motor units? We hope to address this problem using
anatomical techniques.
219. MECHANISMS INVOLVED IN THE CONTROL OF GENICULATE CELL SIZE IN THE CAT
Investigator: Baruch Kuppermann
Intravitreal injections of tetrodotoxin (TTX), a potent
sodium channel blocker, into one eye of seven-week-old
kittens have been shown to. have a dramatic effect on cell
size in the lateral geniculate nucleus {LGN) within one
week ·after initiation of the TTX treatment {Biology 1981,
No. 222). The changes in cell size occur throughout the
LGN, but vary according to location in the LGN. In the
binocular region of the LGN, where adjacent laminae
receive input from different eyes but from the same area
of visual space, cells in laminae receiving input from the
untreated eye increase in size, and cells in laminae with
input from the TTX-treated eye shrink in size. In the
monocular segment, where cells receive input from the
visual periphery through the contralateral eye only, TTX
treated cells shrink in size, but the untreated cells remain
normal in size. Animals given monocular TTX treatment
and placed in the dark during the week-long treatment
period exhibit a uniform change in cell size throughout the
LGN: no increase in cell size is observed in the untreated
laminae, and a decrease in cell size is seen in TTX-treated
laminae. These results suggest at least two mechanisms
involved in the control of LGN cell size: (1) deprivation
effects due to inactivity per se, and (2) competitive
effects due to differential activity of the treated and
untreated layers of the LGN.
Recent work has shown that while major changes in
cell size are observed after one week of monocular TTX
treatment, there is not a correspondingly obvious
difference in the levels of cytochrome oxidase (a mito
chondrial enzyme) between TTX-treated and untreated
laminae. Kittens subjected to months of monocular lid
suture have been shown by Wong-Riley (1979) to have a
decreased level of cytochrome oxidase activity in laminae
receiving input from the sutured eye, as determined by
staining fixed LGN tissue with cytochrome CIII. In the
present situation, after a short (one week) period of TTX
induced monocular inactivity, there resulted comparable
changes in LGN cell size without the corresponding
change in cytochrome oxidase levels.
TTX was also applied to one eye of adult cats, and a
change in cell size was observed in the LGN of these
S:nimals. The cells in the inactive laminae were uniformly
smS:ller than in the untreated laminae, both in the
binocular and the monocular regions of the LGN. This
uniformity in change of cell size suggests that it is the
silencing of activity alone that affects cell size in adults.
Experiments are currently under way to determine
whether the cortex is involved in the observed changes in
LGN cell size. By locally perfusing TTX into one cortical
hemisphere, done in conjunction with monocular TTX
injection, it is possible to silence the primary visual
cortex of one hemisphere without affecting the
differential activity of LGN layers subserving the treated
and untreated eyes. If the cortex is involved in the
geniculate cell size changes, the pattern of changes in the
LGN ipsilateral to the TIX-treated cortical hemisphere
should be measurably different than in the untreated
hemisphere. Preliminary experiments indicate that the
cortex may indeed be involved in the control of LGN cell
size, but to what extent and under what conditions has yet
to be determined.
Reference: Wong-Riley, M. (1979) Brain Res. 171, 11-28.
153
PUBLICATIONS
Burkhalter, A., Van Essen, D. c. and Maunsell, J. H. R. (1981) Patterns of 2-deoxyglucose labeling in extrastriate visual cortex of unstimulated and unidirectionally stimulated macaque monkeys. Soc. Neurosci. Abstracts 7, 172.
Burkhalter, A. and Van Essen, D. C. (1982) Functional properties of neurons in the ventral posterior area (VP) of the macaque monkey. Soc. Neurosci. Abstracts 8, in press.
Connolly, M., LeVay, S. and Van Essen, D. C. (1982) The complete pattern of ocular dominance stripes in macaque striate cortex. Soc. Neurosci. Abstracts 8, in press.
Connolly, M. P. and Van Essen, D. c. (1982) The representation of the visual field in the lateral geniculate nucleus of the macaque: anisotropies, laminar differences, and individual variability. Manuscript in preparation.
Gordon, H. and Van Essen, D. c. (1981) Motor units diversify in size as synapse elimination proceeds in the neonatal rabbit soleus muscle. Soc. Neurosci. Abstracts 7, 179.
Maunsell, J. H. R. and Van Essen, D. C. (1982) Functional properties of neurons in the middle temporal visual area (MT) of the macaque monkey. I. Selectivity for stimulus direction, velocity and orientation. Manuscript in preparation.
Maunsell, J. H. R. and Van Essen, D. C. (1982) Functional properties of neurons in the middle temporal visual area (MT) of the macaque monkey. II. Binocular interactions and the sensitivity to binocular disparity. Manuscript in preparation.
Maunsell, J. H. R. and Van Essen, D. C. (1982) The connections of the middle temporal visual area (MT) in the macaque monkey. Soc. Neurosci. Abstracts 8, in press.
Maunsell, J. H. R. and Van Essen, D. c. (1982) Cortical and sub-cortical connections of the middle temporal visual area (MT) in the macaque monkey. Manuscript in preparation.
Van- Essen, D. C. (1982) Neuromuscular synapse elimination: structural, functional and mechanistic aspects. In: Neuronal Development. N. C. Spitzer (Ed.), pp. 333-376. Plenum Press, New York.
Van Essen, D. c., Maunsell, J. H. R. and Bixby, J. L. (1981) The middle temporal visual area in the macaque: Myeloarchitecture, connections, functional properties and topographic organization. J. Comp. Neurol. 199, 293-326.
Van Essen, D. C., Maunsell, J. H. R. and Bixby, J. L. (1982) The organization of extrastriate visual areas in the macaque monkey. In: Multiple Cortical Somatic Sensory-Motor, Visual and Auditory Areas and Their Connectivities. C. N. Woolsey (Ed.), pp. 157-170. Humana Press, New Jersey.
Van Essen, D. c., Newsome, W. T. and Bixby, J. L. (1982) The pattern of interhemispheric connections and its relationship to extrastriate visual areas in the macaque monkey. J. Neurosci. 2, 265-283.
Van Essen, D. c., Newsome, W. T. and Maunsell, J. H. R. (1982) The representation of the visual field in striate cortex of the macaque: anisotropies and individual variability. Manuscript in preparation.
NEUROGENETICS
Seymour Benzer
Ronald J. Konopka
Professor: Seymour Benzer Sherman Fairchild Distinguished Scholar: Obaid Siddiqi Senior Research Fellows: Lawrence M. Kauvar, Mark A.
Tanouye Research Fellows: Shinobu c. Fujita, Tadmiri R. Venkatesh,
Stephen L. Zipursky Graduate Student: Sandra L. Shotwell Research Staff: Eveline Eichenberger, John B. Reinitz,
Devra C. Spurr, Marika Szalay Laboratory Staff: Sharon W. Lee
Support: The work described in the following research reports has been supported by:
The James G. Boswell Foundation for Virus Research
Fairchild Foundation Lawrence A. Hanson Foundation National Institutes of Health, USPHS National Science Foundation Pew Memorial Trust Helen Hay Whitney Foundation
Summary: Our activities are directed at mechanisms
underlying the development and function of the nervous
system, using the fruit fly, Drosophila, in which
connections between genetics, molecular biology,
physiology and behavior can be traced.
Monoclonal antibodies offer highly selective markers
for identifying surface molecules that distinguish neurons
and other nervous system components from one another.
By injection of Drosophila brains into mice and subsequent
hybridization of the host's activated spleen cells with a
myeloma cell line, we have generated a large collection of
hybridomas that produce monoclonal antibodies. These
are being used to study the chemical architecture of the
nervous system, the developmental appearance of each
antigen, and the genes involved.
The excitability of membranes depends on ion-specific,
voltage-dependent channels whose biochemical identities
and molecular mechanisms are incompletely understood.
Since these proteins, and others that comprise essential
components of the nervous system, are encoded in genes,
they are subject to mutational changes. The adult fly
possesses a pair of "giant" axons that can be impaled by
microelectrodes, to record resting and action potentials.
Such intracellular recordings provide a method for
analyzing the membrane properties of mutants suspected
of having defective ionic channels. Our experiments
indicate that certain Shaker mutations may affect
potassium conductance. Genetic mapping of various
Shaker alleles and deficiencies indicates a complex struc
ture of the controlling chromosomal region. By using
recombinant techniques to clone the genes in question,
157
this system may be used to identify the relevant
molecular components of the membrane.
Memory presumably embodies changes in functional
strength of patterns of synaptic connections. Genetic
methods can be used to delete or modify individual steps
in the process of memory formation, leading to identifi
cation of those steps. In certain paradigms, Drosophila
can learn, and mutants deficient in learning can be
isolated. The finding that the dunce mutant is deficient in
one kind (form II) of cyclic nucleotide phosphodiesterase is
an exciting first step. Our results show that the dunce+
gene is the structural gene for the form II enzyme, rather
than a regulatory gene. This leads to other questions,
such as whether there is an identifiable molecular product
influenced by form II that is associated with memory.
220. MONOCLONAL ANTIBODIES AGAINST THE DROSOPHILA NERVOUS SYSTEM
Investigators: Shinobu c. Fujita, Sandra L. Shotwell, Seymour Benzer
We have extended our panel of antibodies with which
to dissect the fly nervous system by performing fusion
experiments using mice immunized with fly brain or fly
head homogenates. Screening was with indirect immuno
fluorescence on cryostat sections of fly heads. Eighty
five new hybridoma lines were established, bringing the
total to 115. Of these, 61 antibodies are highly specific to
or primarily directed toward the nervous system (brain,
retina, nerves) and have diverse staining patterns. Several
others are specific to tissues such as muscle (e.g., Ab3E2)
or lens (Ab3F12). Most of the remaining antibodies have
broader tissue specificities; some are highly organelle
specific, such as to nuclei (e.g., Ab8C5). The majority
(64%) of the antibodies were identified as IgGs, 28% as
IgMs. The stability of antibody production in hybridomas
was tested for 11 lines in long-term flask cultures. Ten
lines continued to secrete specific antibodies for a three
month test period.
The antibodies serve to identify architectonic regions
of the nervous system. For example, our findings indicate
that the first optic ganglion (lamina) has an antigenic
composition quite distinct from the rest of the brain.
AbDlO, specific to neuropil, stains most central neuropil
homogeneously, but in the lamina it stains a regular array
of columns. On the other hand, AbD12A, which strongly
stains the cellular cortex of the brain, also intensely
stains the entire lamina, but not those columns. Ab5D6
158
also stains the cortex, but the entire lamina only faintly.
Ab5B12 and Ab3Fll, while not specific to the nervous
system, stain the lamina more intensely than any other
CNS region. An antibody was also found that stains only
the neuropil of the lamina (Ab10Hl2).
Two antibodies (4B6A and 6Bll) stain nerves, retina
and the brain neuropil, the calyx of the mushroom body
and the subesophageal nerve being stained particularly
intensely. Moreover, a group of cells immediately
overlying the calyx is also stained in marked preference to
other cell bodies, indicating that these antibodies
recognize a subset of cells in the fly brain. An antibody
was found (Ab2E6) that intensely stains most tissues in the
head, yet the neuropil of the optic lobes is only very
weakly stained. Thus, the antibodies not only define
specific presence but also specific absence of antigens in
a particular tissue.
221. IDENTIFICATION OF POLYPEPTIDES RECOGNIZEDBYMONOCLONALANTIBODII!S DIRECTED AGAINST DROSOPHILA TISSUES
Investigators: Stephen L. Zipursky, Shinobu c. Fujita, Seymour Benzer
We are utilizing monoclonal antibodies to identify
polypeptides that are unique to cell types or organelles
within the Drosophila nervous system. Identification of
such proteins will facilitate localization of the genes that
encode them, and generation of mutations that affect
their expression will aid in determining their function. We
have employed the technique of "western blot11 analysis to
detect specific polypeptides recognized by our panel of
monoclonal antibodies directed against Drosophila tissues.
We have observed bands on western blots for 51 of the
115 antibodies tested so far. Of these, a subclass of 25
reacted with single polypeptide bands corresponding to
molecular weights from 14 to 200 kilodaltons (kd). These
antibodies vary in tissue specificity, from highly specific
to general. For instance, Ab3F2 recognizes a high
molecular weight polypeptide in the retina, whereas
AbGllB binds to a 55 kd polypeptide in all Drosophila
tissues tested. A second subclass of monoclonal anti
bodies reacted with multiple bands of the western blot.
Within this group, binding of a single antibody may occur
with from two to more than ten distinct bands. As was
observed for antibodies that recognized single bands, this
subclass of antibodies also manifests staining patterns
that range from being highly specific, e.g., lens (Ab3F12)
or muscle (Ab4E9), to general, e.g., Ab2G4A. Of· the
antibodies that bound to single bands, 92% were IgG and
4% were lgM whereas of those that recognized multiple
bands, 54% were IgG and 39% were lgM. The immuno
globulin class of the remaining antibodies has not been
determined.
These results demonstrate that monoclonal antibodies,
readily generated with Drosophila homogenates, can
identify polypeptides unique to various tissues. These
antibodies should prove useful in studying the molecular
genetics of Drosophila development.
222. MONOCLONAL ANTIBODII!S SPECIFIC TO NUCLEI
Investigators: Seymour Benzer, Shinobu c. Fujita
Several monoclonal antibodies were obtained that are
highly specific to nuclei of Drosophila tissues (Ab8C5,
Ab7C6, and 24D4; all three are lgGs). Ab8C5 was studied
in some detail. It stains nuclei at all stages of Drosophila
development, from embryo to adult. In addition, it stains
nuclei of a wide range of animal and plant species tested,
from yeast and protozoa up to human brain. The antigen
survives fixation with 2% formalin (30 min, room
temperature). It does not stain live cells. The identity of
the antigen for this antibody is being investigated. This
antibody will be highly useful as a universal fluorescent
counterstaining reagent for nuclei.
223. GENERATION OF MONOCLONAL ANTIBODII!S AGAINST DROSOPIIILA RETINA
Investigators: Shinobu c. Fujita, Stephen L. Zipursky, Tadmiri R. Venkatesh, Seymour Benzer
The visual sys tern is the best analyzed part of the
nervous system of Drosophila (Meinertzhagen, 1973;
Ready et al., 1976). A fusion experiment using a mouse
initially immunized against whole Drosophila head
homogenate, then boosted with pure retina dissected from
acetone-dried flies gave rise to many positive hybridomas
whose culture supernatants gave specific immuno
fluorescence staining of retina in cryostat sections of fly
heads. Some appear to be specific to lens, crystalline
cone, photoreceptors, rhabdomeres, pigment cells, or
basement membrane. Some stain retina intensely, but
also stain the first optic ganglion and a regular array of
fibers in the second optic ganglion (e.g., Ab26G5). About
70 such hybridoma lines are being cloned. Interestingly, in
this fusion experiment, antibodies specific to nonretinal
tissue were rare.
Ref.,.,ences: Meinertzhagen, I. A. (1973) In: Developmental Neuro
biology of Arthropods. D. Young (Ed.), pp. 51-104. Cambridge University Press, London.
Ready, D. F., Hanson, T. H. and Benzer, s. (1976) Devel. Biol. 53, 217-240.
224. MONOCLONAL ANTIBODil!S REVEAL ANTIGENIC PROFILES OF DROSOPHILA CELL LINES
Investigators: Ronald J. Konopka, Shinobu C. Fujita, Seymour Benzer
A number of cell lines have long been established in
Drosophila, but their nature has been difficult to define
with the small number of parameters available, such as
content of particular enzymes or reactions to conven
tional antisera (Moir and Roberts, 1976).
Many of our monoclonal antibodies were tested on four
established lines: Kc, Ore-R, CL-7 and shi, using indirect
immunofluorescence on live and acetone-treated cells.
For 17 antibodies with broad tissue specificity (as
determined on cryostat sections of fly heads), each of the
four cell lines expressed a high percentage (varying from
11 to 15 positives for the four lines) of the antigens. In
contrast, for 26 antibodies specific to nervous tissues, the
Kc line was positive for only four, while the shi line
reacted to 19 out of 20 tested. The other two cell lines
were intermediate. This shows that the shi line expresses
a neural antigenic profile, in contrast to the Kc line. As
our panel of monoclonal antibodies consists mostly of
those with either neural or broad tissue specificity, it
remains to be seen whether these cell lines express
antigens characteristic of other differentiated tissues.
Some antibodies stained acetone-treated but not live cells
(e.g., Ab8C5, AbD12A), while some others stained both
similarly (AbDllA, Ab2A9), indicating intra- or extra
cellular localization of the antigens, respectively.
Cultured cell lines will be useful for preparation of -large
quantities of antigen molecules.
Reference: Moir, A. and Roberts, D. B. (1976) J. Insect Physiol. 22,
299-307.
225. ANTIBODil!S DISTINGUISH CELL TYPES IN PRIMARY CULTURE OF DROSOPHILA CNS
Investigators: Shinobu C. Fujita, Nobuyuki Suzuki•, Chung-Fang Wu•
The Drosophila larval brain can be dissociated and
cultured in vitro, with many cells retaining neuronal
159
characteristics (Wu et al., 1981). Such a system allows
the identification of cell types expressing particular
neural antigens, thus distinguishing cells of different
neuronal subclasses or differentiation states. Also, a
panel of antibodies could be used to eliminate particular
cell types by immunocytotoxicity. To probe these
possibilities, selected monoclonal antibodies were tested
on live or fixed cells in such cultures. Antibodies with
broad tissue specificity (AbDllA, Ab2A8, Ab4H3)
intensely stained all cells, whether large or small, and
with or without processes. Some other antibodies stained
subsets of cells. For instance, AbD8A, which stains the
entire fly brain, stained primarily clumps of small cells
with bundles of processes. Ab2E9, which stains fly retina,
neuropil and nerves, stained only a small number of larger
cells in culture. This study is being combined with
immunohistology of the larval brain to establish the
identities of the distinct cell types.
Reference: Wu, C.-F., Suzuki, N. and Poo, M.-M. (1981) Soc. Neurosci.
Abstracts 7, 598.
*Department of Zoology, University of Iowa, Iowa City.
226. LENS-SPECIFIC ANTIBODY BINDS TO KNOWN LENS-SPECIFIC POLYPEPTIDES
Investigators: Shinobu C. Fujita, stephen L. Zipursky
Monoclonal Ab3F12 specifically stains the lenses of
the compound eyes and ocelli of Drosophila. A whole
homogenate of the fly was analyzed by two-dimensional
electrophoresis, followed by electroblotting. The antibody
was found to bind specifically to a family of spots having
pl of around 6, with the major component in the 55-60 K
region. This family of spots had been previously
identified, by dissection experiments, to be among a set of
lens-specific polypeptides (Fujita, 1980). This antibody
and others will provide sensitive histological markers and
biochemical probes in molecular approaches to the
development of the compound eye.
Reference: Fujita, S. c. (1980) Taisha 17, 489-494.
227. WHOLE MOUNT STAINING PROCEDURE
Investigator: Shinobu C. Fujita
In studying the antigen distribution within a tissue,
whole mount preparations (Zipser and McKay, 1981) have
certain advantages compared with tissue sections. The
neural lamella ensheathing the fly brain presents a barrier
160
to antibody molecules. Therefore, a procedure employing
collagenase (EC3.4.24.3) (Sigma type VII) was developed to
allow indirect immunofluorescence staining of the entire
Drosophila brain. Brains dissected from acetone freeze
dried flies (Fujita and Hotta, 1979) are soaked in Tris
buffered saline containing 1 mM EGTA and 5 mM NaN3 (TBS). The tissue is then incubated in Tris buffer, pH 7 .5,
containing 10 mM CaC12 and collagenase (150 U/ml) for
10 min. After 5 min wash in TBS, the brain is fixed with
2% formalin in phosphate buffer for 15 min, followed by a
1 hr wash in TBS plus 1 % NP-40. Incubations with the
first and second antibodies are each overnight, with half
day-washes in TBS. All steps are at room temperature.
Fresh brain became very fragile upon collagenase treat
ment. Aldehyde fixation before collagenase treatment
interfered with action of the enzyme. Elastase was not
effective. Using this procedure, AbB7 (IgG) stained
numerous fibers throughout the brain.
References: Fujita, S. C. and Hotta, Y. (1979) Protein, Nucleic Acids,
Enzymes 24, 1336-1343. Zipser, S. and McKay, R. (1981) Nature 289, 549-554.
228. CLONJNG THE Shaker GENE
Investigators: Alberto Ferrus*, Carlos V. Cabrera, Mark A. Tanouye
We have previously used chromosomal rearrangements
broken in and around Shaker (Sh) to localize the gene to a
small region between X chromosome salivary bands 16E2
and 16F8 (Tanouye et al., 1981). Further, one EMS
induced allele, ShKsi 33, was localized proximal to a
breakpoint in 16E2-4 and distal to a breakpoint in 16Fl-4.
In order to extend the genetic analysis of Sh, we are
studying its molecular genetics using recombinant DNA
techniques.
We have obtained a cDNA clone that maps by in situ
hybridization to 16F. The cloned DNA maps to a unique
site, proximal to a breakpoint in 16E2-4 and distal to a
breakpoint in 16Fl-4, the same location as ShKSl33.
Further, genomic blots with several restriction enzymes
produce a single band. We are now in the process of
walking along the chromosome in both directions in order
to obtain the entire chromosomal region originally
ascribed to Sh by genetic means.
Reference: Tanouye, M. A., Ferrus, A. and Fujita, S. C. (1981) Proc.
Nat. Acad. Sci. USA 78, 6548-6552.
*Centro de Biologia Molecular, Universidad Autonoma, Madrid.
229. SINGLE CHANNEL STUDIES IN DROSOPIULA MUTANTS
Investigators: Mark A. Tanouye, Chun-Fang Wu*
Electrophysiological studies in Drosophila have
suggested that various mutations affect fundamental
macromolecular mechanisms underlying ionic conduc
tance. For example, Shaker (Sh) mutants appear to have
altered K + channels. The temperature-sensitive paralytic
mutant, nap ts (no action potential, temperature sensitive),
appears to have alterations in the voltage-sensitive Na+
channel.
Identification of channel defects in mutants has, until
recently, relied largely on indirect means. One of the
most direct ways to demonstrate channel defects is to
record single channel currents. Current flowing through
individual ion channels can be measured in small patches
of nerve or muscle membrane (Neher et al., 1978). A
small-diameter glass pipette is used to electrically isolate
and voltage clamp the membrane patch. An advantage of
patch recording is that it is particularly adaptable to
small cells, such as Drosophila neurons.
We are employing patch recording methods for analysis
of normal and mutant Drosophila cultured neurons.
Primary tissue cultures of dissociated cells are prepared
from the isolated nervous systems of third instar larvae.
Cells cultured for 24 hr exhibit excitable membrane
properties and have one or more long neuronal processes
with branches (Wu et al., 1981). These differentiated cells
may be maintained in culture for up to one week.
Patch recording methods provide a means for
unequivocally identifying the classes of channels affected
by different mutations. It is anticipated that the
molecular details of channel activation, inactivation, ion
selectivity and channel distributions will be revealed by
using genetic mutations which alter these processes.
References: Neher, E., Sakmann, B. and Steinbach, J. H. (1978)
Pfltigers Arch. 375, 219-228. Wu, C.-F., Suzuki, N. and Poo, M.-m. (1981) Soc. Neurosci.
Abstracts 7, 598.
*Department of Zoology, University of Iowa, Iowa City.
230. GIANT FIBER ACTIVATION OF DIRECT FLIGHT MUSCLES JN DROSOPIULA
Investigators: Mark A. Tanouye, David G. King*
Escape in several Dipterans, including Drosophila, is
initiated by a complex sequence of behavioral and
physiological events (Nachtigall and Wilson, 1967; Kaplan
and Trout, 1974; Tanouye and Wyman, 1980). An escape
jump acts to get the animal into the air. The indirect
flight muscles are activated. The wings are elevated,
opened, and properly oriented. The flight motor system is
started. A large portion of escape initiation including the
escape jump, wing elevation, and indirect flight muscle
activation is driven by the cervical giant fiber (Tanouye
and Wyman, 1980). The present experiments examined
giant fiber system contributions to the control of wing
position.
Intracellular microelectrode recordings were made
from the fibers of direct flight muscles. Recordings from
the two main direct wing elevators, pa 1 and pa 2
(terminology from Zalokar, 1947), showed that although
both muscles could be activated by nerve fibers
(unidentified) descending from the brain, neither was
driven by the giant fiber. These results suggest that the
first wing elevation seen during giant fiber-driven escape
initiation is apparently due solely to activation of the
tergotrochanter muscle (Tanouye and Wyman, 1980) which
functions dually as a jump muscle and an an indirect wing
elevator.
Recordings from the two main direct wing opener
muscles, pa 3 and pa 4, showed that although both muscles
could be activated by nerve fibers descending from the
brain, only pa 3 was driven by the giant fiber. These
results suggest that wing opening during escape initiation
is mediated by giant fiber activation of pa 3.
References; Kaplan, W. D. and Trout, W. E. (1974) Genetics 77,
721-739. Nachtigall, W. and Wilson, D. M. (1967) J. Exp. Biol. 47,
77-97. Tanouye, M. A. and Wyman, R. J. (1980) J. Neurophysiol.
44, 405-421. Zalokar, M. (1947) Revue Suisse de Zoologie 54, 17-33.
*Southern Illinois University School of Medicine, Carbondale.
231. ANTENNAL PHYSIOLOGY OF DROSOPHILA
Investigators: Oba.id Siddiqi, John B. Reinitz
Behavioral experiments show that Drosophila has a
well-developed sense of smell. It can discriminate
between a variety of attractants and repellants and
memorize single odorants as well as their combinations.
The underlying peripheral and central mechanisms of odor
perception are, at present, poorly understood. We are
studying olfactory mechanisms of Drosophila with the help
of mutations that block smell responses. A number of
161
X-Iinked olfactory (olf) mutations causing specific
anosmias are available (Figure 1). Electrophysiological
experiments on normal and mutant flies aimed at
identifying neurological defects in the mutants have been
initiated.
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Figure 1. Olfactory genes on the X chromosome of Drosophila; brackets indicate the sets of chemicals to which responses are blocked. Three of the olf genes appear to be closely linked. Data by V. Rodriguez and 0. Siddiqi.
The electroantennogram (EAG) is an easily measured
response of the antenna to odor stimuli. It reflects the
receptor potential in the sensory neurons. We have
examined the EAG of Drosophila against a variety of
volatile chemicals. The amplitude of the EAG is related
to odor concentration by a power law (log response :;; k log
stimulus concentration). Most chemicals elicit a mono
phasic negative response but certain odorants produce a
positive potential. The threshold and maximal responses
vary with individual chemicals. The strongest responses in
Drosophila are to acetates and alcohols. The fly responds
also to certain aldehydes and ketones, but only weakly or
not at all to several volatiles that attract other insects
(plant hydrocarbons and amines). The EAG responses are
thus indicative of well-defined receptor groups.
We have compared the effects of simultaneous and
successive stimulation with paired odorants in order to
determine the elementary set of olfactory chemo
receptors. It is assumed that if two odorants, A and B,
act on independent receptors, the amplitude of the EAG in
response to B will not be affected by an overlapping
stimulus of A; the responses should be additive. On the
other hand, if A and B act on the same receptor, the
response to B should be competitively inhibited by the
presence of A. Experiments with over 50 chemicals show
that the elementary receptor groups of Drosophila
162
correspond, in the main, to chemical groups such as
acetates, alcohols, ketones, aldehydes and fatty acids.
Some of these groups appear to be heterogeneous, i.e., act
on more than one chemoreceptor, while others are
homogeneous. The olf mutants belonging to four different
genes, olf A, olf B, olf C and olf D, were examined. Olf C
exhibits an enhanced threshold for ethyl acetate, while
the others show an unimpaired EAG.
The EAG is a combined response of over 1000 sensory
neurons in the third antenna! segment. In order to know
the response characteristics of olfactory sensillae, it is
necessary to observe the responses of single neurons. The
absence of observable defects in the EAG in some of the
mutants might mean that these mutations affect the CNS.
On the other hand, a mutation which alters the distribu
tion of chemoreceptors on the . sensory neurons could
profoundly alter sensory coding of olfactory information
without changing the EAG. We have begun, therefore, to
make extracellular recordings from single units in the
antenna. Several different types of olfactory units in the
antenna with characteristic firing patterns have been
identified. On the lateral face of the third segment,
surrounding the sacculus, there is a cluster of units
primarily responsive to ethyl acetate. We hope to
construct a detailed map of the functional organization of
Drosophila antenna. The availability of such a map should
make it easier to identify peripheral defects in anosmic
mutants.
232. ANTENNAL BIOCHEMISTRY
Investigators: Lawrence M. Kauvar, Obeid Siddiqi
A simple method for preparing large numbers of pure
antennae from Drosophila has been established. The
antennae, which are attached to the fly by a narrow stalk,
are easily dislodged by gentle shaking of frozen rues.
After being sieved through appropriately sized nylon
mesh, the antennae are free of fly bodies and large debris.
Contamination by small leg fragments and halteres is
further reduced by a series of unit-gravity sedimentations
in dilute non-ionic detergent (Figure 2). Anatomical and
electrophysiological studies of antennae from Drosophila
and larger insects have indicated that the primary
olfactory receptors are located in the fine hairs that
cover the surface of the antenna. We have been able to
shear off these hairs from purified antennae by
mechanical abrasion. This preparation should allow a
variety of biochemical techniques to be applied to
problems of chemosensory physiology.
Figure 2. Scanning electron micrograph of mass prepared antenna! third segments (with feathery aristo attached). Sensory hairs ccwering the surface of the antennae are undamaged.
233. cAMP PHOSPHODIESTERASE IN NORMAL AND dunce FLIES
Investigator: Lawrence M. Kauvar
The purification of cyclic nucleotide phospho
diesterases (PDE) from normal Drosophila, reported last
year, has now been adapted to allow substantial purifica
tion of PDE from dunce mutant stocks. Evidence has been
obtained for dunce as the structural gene for one of the
forms of PDE found in adult flies, the PDE-II enzyme.
For one mutant allele, dunce 1, the PDE-II -is markedly
more thermally labile than normaL For a second allele,
dunce2, the Michaelis kinetic constant, Km' is tenfold
higher than normal. In each case, the PDE-II defect is
present in both crude homogenates and purified prepara
tions. Furthermore, flies heterozygous for either dunce1
or dunce2 contain PDE-II characteristic of both mutant
and normal types. Thus, the dunce mutations are
apparently not acting by causing abnormal processing of a
normal PDE-II, since a processing defect should produce a
single, uniform population of PDE-Il molecules. The
thermal !ability and Km defects of PDE-Il have both been
mapped genetically to the dunce gene. In combination
with previously retwrted data showing linear dependence
of PDE-Il activity per fly with the dosage of the dunce+
gene (Kiger and Golanty, 1979; Shotwell, 1982), this work
establishes dunce as the structural gene for PDE-Il.
Consequently, defective cAMP metabolism is the primary
lesion leading to dunce's impaired memory in the olfactory
associative conditioning paradigm of Dudai et al. (1976).
References: Dudai, Y., Jan, Y.-N., Byers, D., Quinn, w. and Benzer, S.
(1976) Proc. Nat. Acad. Sci. USA 73, 16841688. Kiger, J. A. and Golanty, E. (1979) Genetics 91, 521-535. Shotwell, S. (1982) Ph.D. Thesis, California Institute of
Technology.
234.. DOSAGE SENSlTIVITY AND REGULATION OF CYCIJC AMP PHOSPHODIESTERASE II FOR THE dtmce MEMORY MUTANT GENE OF DROSOPHILA
Investigator: Sandra L. Shotwell
The Drosophila memory mutant dunce has reduced
activity for one of the cyclic AMP phosphodiesterases
present in fruitflies, PDE II (Byers et al., 1981). Does this
enzymatic defect result from abnormal regulation of PDE
Il activity, or is dunce the structural gene for PDE Il?
The following results present evidence that dunce is a
structural gene for PDE II, indicating a role for this
enzyme in the learning ability of Drosophila.
Male flies bearing the mutant alleles dunceMll or
dunceM14 have greatly reduced PDE II activities (4.0% ±
0.9% and 1.8% ± 0. 7% of normal, respectively). The
addition of a normal copy of the dunce gene present on
the duplication chromosome Dp(1;2)w +Slb7 restored
PDE II activity to normal levels in these males (dunceMll;
Dp = 96% ± 2. 7% of normal levels and dunceM14; Dp =
88% ± 13%). This indicates that dunce flies have normal
PDE II regulation from the level of transcription to the
level of enzyme activity. The presence of a normal copy
of the dunce gene also fully restores learning ability in dunceMll and dunceMl 4 males.
In Drosophila, as a rule, the activity level of an
enzyme is proportional to the number of copies of the
_enzyme's structural gene that are present in a cell.
Homogenates of flies with gene arrangements providing
the equivalent of O, 0.5, 1.0, 1.5 and 2 times the normal
dose of the gene were assayed for PDE II and had,
respectively, 0.03 ± 0.01, 0.64 ± 0.02, I.O, 1.45 ± 0.10, and
1.67 ± 0.13 times the PDE II specific activity of normal
flies. This demonstrates a one-to-one correlation between
PDE II activity and the number of copies of the norrrial
dunce gene at five different doses.
Thus, PDE II appears to be regulated normally in dunce
mutants, and the dose dependence is consistent with the
ideas that dunce is a structural gene for the enzyme and
that altered PDE 11 represents the primary defect in dunce
flies. In addition, Davis and Kiger (1981) and Kauvar
(1982) have also shown differences in kinetic and thermo
stability properties of two mutant forms of PDE II. These
163
studies indicate that PDE II plays a role in the learning
ability of normal flies.
Refer'"1ces: Byers, D., Davis, R. and Kiger, J. A. (1981) Nature 289,
79-81. Davis, R. L. and Kiger, J. A. (1981) J. Cell Biol. 90, 101-
107. Kauvar, L. M. (1982) J. Neurosci., in press.
235. SUPPRJ!SSION OF THE FEMALE STERILITY PHENOTYPE ASSOClATED WITH THE dtmce MEMORY MUTANT OF DROSOPHILA
Investigator: Sandra L. Shotwell
Mutants at the dunce locus on the Drosophila X
chromosome show three phenotypic defects-learning
disability, female sterility, and elevated levels of cyclic
AMP due to reduced activity of one of the cAMP
phosphodiesterase activities present in normal flies,
PDE II (Byers et al., 1981). In this study, three different
genetic modifications are shown to suppress female
sterility without affecting the other two phenotypes. This
indicates that female sterility is subject to suppression at
a secondary level, and that the remaining mutant pheno
types of reduced PDE II activity and poor learning lie
closer to the primary defect in dunce mutants.
The first two genetic modifications are second
chromosome mutations isolated by Madeline Crosby
(personal communication) that suppress the sterility of
females homozygous for the mutant allele dunce2. One of
the suppressors, su(fs)C67, is recessive. Females homo
zygous for dunce2 on the X chromosome, and su(fs)C67 on
the second chromosome, are fertile, but still have reduced
PDE II activity as do sterile female dunce2 controls (27%
and 25%, respectively, of the PDE II activity of normal
females). The second suppressor, Su(fs)C119, is dominant.
Females homozygous for dunce2 on the X chromosome and
heterozygous for Su(fs)C119 on the second chromosome
are fertile, but also have similarly reduced PDE II activity
as dunce2 controls (26% and 20%, respectively, of normal
PDE II activity levels). One of the suppressors was tested
for learning, and it does not improve the learning of
dunce 2 flies. While normal flies achieve a learning score
of 0.30, dunce2 females scored 0.03 in the absence, and
0.02 in the presence, of Su(fs)C119. The su(fs)C67 has not
been tested for its effect on learning.
The third genetic modification that separates female
sterility from the other two defects is a deficiency of the 64°15 X chromosome Df(l)N l (see Kiger, 1977) that deletes
bands 3C4 to 3D3, a region just to the left of the dunce
164
gene. Females were constructed bearing this deficiency
on one X chromosome and a second deficiency
Df(l)dm 75e19, which deletes the dunce gene, on the other
X chromosome. While most of these females are fertile,
10-20% are sterile. Df(l)N64jlS /Df(l)dm 75el9 females
are poor learners (learning score = 0.04, D. Byers, personal
communication). In this study, individual sterile and
fertile females of this genotype were found to have
similarly reduced PDE II activity (23% and 25%,
respectively, of normal PDE II activity).
These results indicate separate genetic regulation of
the female sterility associated with dunce mutants. The
continued expression of poor learning and reduced PDE II
activity indicates that these mutant phenotypes lie closer
to the primary defect of dunce mutants.
References: Byers, D., Davis, R. L. and Kiger, J. A. Jr. (1981) Nature
289, 79-81. Kiger, J. A. Jr. (1977) Genetics 85, 623-628.
236. HORMONAL REGULATION OF CYCLIC NUCLEOTIDE PHOSPHODIESTERASE ACTIVITY 11!1 CLONAL DROSOPHILA CELL LINES
Investigators: Sandra L. Shotwell, Ronald J. Konopka
Drosophila contains three cyclic nucleotide phospho
diesterase activities, one that hydrolyzes both cAMP and
cGMP (PDE I), one specific for cAMP (PDE II), and one
specific for cGMP (PDE III) (see Kauvar, 1982). We have
assayed these three enzymes in three cultured clonal cell
lines (designated CL7, CL12 and CL14) and found that
each cell line contains all three enzymes at specific
activity levels within the range seen for adult tissues.
These lines had been established by Ronald Konopka and
Steven Wells from late Drosophila embryos, and cloned in
soft agar. The presence of each enzyme in all three
clonal cell lines indicates that a single cell type can
contain multiple forms of PDE.
Why would a single cell contain more than one enzyme
capable of hydrolyzing a given substance? One possibility
is that the enzymes are subject to separate regulation,
thus allowing different subcellular pathways to utilize
Assistant Professor: Ronald J. Konopka Gosney Researeh Fellow: Janine Perlman Graduate students: Steven H. Green, Dominic Orr,
Randall F. Smith Research Staff: David J. Baker, Leila Gonzalez, Thomas
E. Lee, Steven M. Wells
cAMP as an intracellular messenger. Support for this
hypothesis comes from the observation that the PDE
activities of one of the cell lines, CL14, have different
responses to ecdysone, a hormone involved in pupation.
The effect of ecdysone on PDE activity in the other cell
lines has not yet been tested. After adding 2 x 10 - 6 M
ecdysone to CL14 cells, cGMP hydrolysis by PDE 1 and
PDE III declines dramatically, while the hydrolysis of
cAMP by PDE 1 and PDE ll does not. The decline in cGMP
hydrolysis is apparent by day 1 after adding ecdysone, and
reaches a maximum by day 5, when hydrolysis is reduced
by over 85% with respect to untreated CL14 cells. These
results demonstrate that it is possible to have separate
hormonal regulation of similar enzymatic activities. The
molecular mechanism of this effect is under investigation.
Reference: Kauvar, L. M. (1982) J. Neurosci., in press.
PUBLICATIONS
Fujita, S. C., Ferrus, A., Shotwell, S. L. and Benzer, s. (1981) Monoclonal antibodies against the Drosophila nervous system. Soc. Neurosci. Abstracts 1, 120.
Kauvar, L. M. (1982) Defective cAMP phosphodiesterase in the Drosophila memory mutant dunce. J. Neurosci., in press.
Kauvar, L. M. (1982) The Dff'Ophila temperaturesensitive paralytic mutant nap shows reduced binding of 3H-tetrodotoxin. Mol. Gen. Genet., in press.
Kato, M., Tanouye, M. A., Ferrus, A., Thomas, J. B. and Wyman, R. J. (1981) The morphology of the cervical giant fiber neuron on Drosophila. Brain Res. 221, 213-217.
Mogami, K., Fujita, S. and Hotta, Y. (1982) Identification of Drosophila indirect flight muscle myofibrillar proteins by means of two-dimensional electrophoresis. J. Biochem. 91, 643-650.
Shotwell, S. L. (1981) Cyclic adenosine 3',5'-monophosphate phosphodiesterase (cAMP-PDE) and its role in learning in Drosophila. Soc. Neurosci. Abstracts 1, 352.
Tanouye, M. A., Ferrus, A. and Fujita, S. C. (1982) Abnormal action potentials associated with the Shaker complex locus of Drosophila. Proc. Nat. Acad. Sci. USA 78, 6548-6552.
Tanouye, M. A. and Wyman, R. J. (1981) Inhibition between flight motoneurons in Drosophila. J. Comp. Physiol. 144, 345-355.
Wyman, R. J. and Tanouye, M. A. (1981) Drosophila flight motor pattern: The evidence from interspike intervals. J. Exp. Biol. 96, 413-416.
Support: The work described in the following research reports has been supported by:
E. S. Gosney Fund National Institutes of Health, USP HS Pew Memorial Trust Evelyn Sharp Fellowship Whitehall Foundation
Summary: Our research combines genetic, physiological
and behavioral techniques to study the control of rhythmic
behavior in Drosophila. We have identified several
genetic loci that affect the periodicity, expression and
temperature compensation of circadian rhythms and
courtship song rhythms. The aim of our current research
is to understand how different cell types utilize these
genes and gene products to regulate their rhythmic
functions on different time scales.
237. THE CHRONOTRANSPOSON HYPOTHESIS
Investigator: Ronald J. Konopka
Two mutations that we have previously isolated alter
the period of the Drosophila activity rhythm by 1.5 hours.
One mutation (andante) lengthens the period by this
amount, while the other (clockKOG) shortens the period by
this amount. The andante mutation maps to the
miniature-dusky region and has a dusky phenotype. We
have formulated a hypothesis which states that the 1.5
hour difference in period is due to a transposable element
of DNA; depending on the site of insertion, the period is
either lengthened or shortened by this amount. To test
this hypothesis, a cross was made in an attempt to cause
the transposable element to move from its site of
integration in the dusky region; this should result in
recovery of flies with normal wings and a wild-type
rhythm. Accordingly, males containing P-factor elements
were crossed to andante females; while the majority of F1 males were andante, a few males were recovered with
normal wings and a wild-type rhythm, consistent with the
chronotransposon hypothesis. Additional tests of this
hypothesis are in progress.
238. CLONING THE PER LOCUS
Investigator: Janine Perlman
During the past year, a set of clones flanking and
containing the white locus at bands 3Cl-2 was made
available by R. Levis and coworkers. By using these
clones to screen a wild-type Drosophila library, it should
be possible to "walk" to the DNA located at the
breakpoints of deficiencies that place the per locus close
to the white region. Additional "walking," making use of
available deficiencies whose rhythm phenotypes have been
defined (Smith and Konopka, 1981), will define the limits
of the per locus. Subsequently, comparison of the DNA
from per mutant strains with that of wild-type strains will
165
reveal the molecular basis of the altered periodicity
produced by the per mutants.
Reference: Smith, R. F. and Konopka, R. J. (1981) Molec. Gen. Genet.
183, 243-251.
239. ISOLATION OF NEW CLOCK MUTANTS
Investigator: Ronald J. Konopka
As a result of screening several hundred stocks that
had been mutagenized with ethyl methanesulfonate, two
mutations were recovered that affect the adult locomotor
activity rhythm. Both are X-linked. One, designated E20,
produces arrhythmic activity in the majority of individuals
tested. The other, designated E1283, produces weak
rhythmicity with variable periodicity. The mapping of
these mutants and the characterization of their
interaction with other clock mutants are in progress.
240. TEMPERATURE DEPENDENCE OF PERIOD IN FIVE CLOCK MUTANTS
Investigators: Dominic Orr, Ronald J. Konopka
One of the general properties of circadian clock
systems is the relative temperature independence of the
clock period. Three lines of evidence have suggested that
the periodicity of circadian clocks is temperature com
pensated rather than temperature independent: (1) in some
organisms, the period slows down the increasing
temperature, (2) circadian clocks can be phase-shifted by
temperature steps and pulses, and (3) in many cases, the
temperature insensitivity of period is restricted to a
limited temperature range. In this study, our aim is to ask
whether this homeostatic property of circadian systems is
subject to perturbation by five clock mutants at three
genetic loci. The results of measurements of activity
rhythm periods at 17°, 22" and 25° indicate that two of the
mutants at different loci, andante and clockKOG, do not
significantly affect the temperature compensation mecha
nism. The per locus mutants, however, do have significant
effects on temperature compensation of period; the Q10 of per" is greater than that of wild type, while the Q10 of
per1 and per12 are less than that of wild type. One
hypothesis to account for these results is that the genetic
elements utilized in the temperature compensation
mechanism are a subset of those that affect periodicity.
Alternatively, effects of temperature compensation may
be significant only when the genetic alteration of period is
greater than a certain threshold value of about 3 hours.
166
241. MOSAIC STUDillS OP CIRCADIAN AND COURTSHIP SONG OSCILLATIONS
Investigators: Ronald J. Konopka, Jeffrey Ball*, C. P. Kyriaeou**
Mutations at the per locus affect the periodicity of the
circadian locomotor activity rhythm as well as a short
term oscillation in the interpulse interval of the male's
courtship song. In order to determine the behavioral foci
of these effects, mosaic flies were constructed whose
nervous tissue was partly wild type and partly hetero
zygous for the per8 mutation. A histochemical marker
(acid phosphatase) was used to distinguish between these
two tissue genotypes. The flies were homozygous for a
transformer gene so that all mosaics would be
behaviorally male. The results of measurements of both
the activity rhythm and the courtship song oscillation
indicate that the foci for the effe~ts of per8 on the two
rhythms are some distance apart. It appears likely that
the activity rhythm focus is in the brain, while the song
rhythm focus is in the thoracic ganglia. Analysis of the s distribution of wild type and heterozygous per nervous
tissue in each mosaic is in progress. These histochemical
results will further localize the cells responsible for the
effects on the two rhythms.
*Department of Biology, Brandeis University, Waltham, Massachusetts. **Department of Psychology, University of Edinburgh, Scotland.
242. GENETIC STUDillS OP SENSORY NEURON PROJECTION PATl'HRNS IN DROSOPHILA MELANOGASTER
Investigator: Steven IL Green
The projections of genetically produced ectopic
thoracic sensilla into the brain of D. melanogaster were
studied by cobalt and HRP filling. These were compared
with the normal projections of these sensilla into the
ventral ganglion as well as the projections into the brain
of the local sensilla that normally occupy the new
locations of the ectopic neurons. The ectopic sensilla
studied were the tarsal and tibial bristles of antenna! legs
produced by the genotype Antp Df(3)sbd/Pc ss and dorsal
thoracic and wing margin bristles produced by the
mutation, ey. The mutations cause these sensilla to
appear on the head by replacing or being added to sensilla
that are normally present.
The normal antenna's projection has also been
described by Stocker and Lawrence (1981). It consists of a
bilateral olfactory lobe (OL) component organized into
glomeruli, and a mainly ipsilateral component in the
posterior deutocerebrum and SEG which can be further
subdivided into three major branches. The head macro
chaetes all arborize in an identical manner in the
subesophageal ganglion (SEG). The routes they take to
the SEG, however, vary according to their positions on the
head. The arborization is L-shaped with an ipsilateral,
longitudinally oriented branch and a medially directed
branch that crosses the midline. One branch of the non
OL antenna! component precisely matches the head bristle
projection.
The antenna! leg projection is identical to the antennal
for the non-OL component but in the OL the axons
arborize randomly, are not organized into glomeruli, have
few contralateral branches and have adventitious projec
tions into the protocerebrum and SEG. Some of these
latter are consistent from fly to fly. Mechanosensory
fibers alone can make up the entire projection.
Thoracic and wing bristles on the head and eye project
in the brain and optic lobes. In the brain they often reach
the SEG but ramify irregularly. Similarly, their branching
in the optic lobes is irregular and unlike that of retinular
or intrinsic axons.
1 conclude that the ability of ectopic axons to be
guided by local pathways or branch in local neuropil is not
like that of local neurons and depends on the modality or
type of ectopic sensilla that is being studied. In general
only the initial projection of the ectopic sensilla is like
the local sensilla. The projections of ectopic sensilla into
"foreign" neuropil bears no resemblance to their normal
projections.
Reference: Stocker, R. and Lawrence, P. (1981) Devel. Biol. 82,
224-237.
PUBlJCATIONS
Smith, R. F. and Konopka, R. J. (1981) Circadian clock phenotypes of chromosome aberrations with a breakpoint at the per locus. Molec. Gen. Genet. 183, 243-251.
Smith, R. F. and Konopka, R. J. (1982) Effects of dosage alterations at the per locus on the period of the circadian clock of Drosophila. Malec. Gen. Genet. 185, 30-36.
Smith, R. F. and Konopka, R. J. (1982) Andante, a new circadian clock mutant of Drosophila melanogaster. Malec. Gen. Genet., submitted for publication.
DEVELOPMENTAL GENETICS
Edward B. Lewis
Professor: Edward B. Lewis Visiting Associate: Jeffrey R. Powell Gosney Research Fellow: Ian W. Duncan Researcl\ Fellows: Shigeru Sakonju, Margit Schardin Research Staff: Rollin H. Baker, Su-Ming Chiang, Loring
G. Craymer III, Josephine Macenka, Ker-kwa Susan Ou Laboratory Staff: Helen Alvarez, Ana L. Gharzeddine,
Nancy Harris, Gertrude Jordan, Eva Westmoreland
Support: The work described in the following research reports has been supported by:
Deutsche Forschungsgemeinschaft E. s. Gosney Fund National Institutes of Health, USPHS National Science Foundation Edwin H. Schneider Fund Helen Hay Whitney Foundation Yale University
Summary: In many higher organisms, abdominal and
thoracic body segments are highly differentiated from one
another internally and externally. In Drosophila, primary
control of such differentiation resides largely within the
bithorax gene complex (BX-C). BX-C regulates many
other genes in the sense that organisms lacking entirely
one or more BX-C genes show embryonic patterns of
development in which differences between thoracic and
abdominal segments become blurred; the more BX-C
genes that are deleted, proceeding distally in the
chromosome, the more the segmentation pattern of the
abdominal segments approaches the primitive thoracic
state. The complex itself is under both cis- and trans
regulation. Thus, a series of cis-dominant gain-of
function mutants is scattered throughout the complex.
Such mutants presumably alter regions within BX-C that
are under negative control of one or more trans
regulatory genes. Of particular interest is the trans
regulatory Polycomb (Pc) gene, since mutants and
deletions of this gene not only exert derepression of wild
type BX-C gene functions, but they intensify the gain of
function associated with several cis-regulatory BX-C
mutants.
The genes of the complex obey the following rules:
the more posterior the segment of the organism, the more
genes of the complex are derepressed (or activated}.
Secondly, the order of the genes in the chromosome
parallels the order in which they are derepressed (or
activated) during embryonic development. In order to test
the generality of these rules, we hope to saturate the
complex with mutants, especially of the cis-regulatory
type. The strategy is to look for derepression of a gene in
regions of the body where it is normally kept in the
repressed state; that is, to look for mutants of the
169
bithorax complex that result in active function of such a
gene in one or more segments anterior to the segment in
which it normally becomes active. An example is the
strategy we are using to search for cis-regulatory mutants
that extend the easily scored black pigmentation of the
fifth and sixth abdominal segments of the male to more
anteriorly located segments. Extension of pigment to the
fourth segment has already been achieved in the case of a
spontaneous mutant, Miscadastral pigmentation (Mcp).
Madeline Crosby, who found Mcp, has shown that it is cis
regulatory; namely, it derepresses only in cis an
abdominalizing gene, infra-abdominal-5 (iab-5), that is
normally only active in the fifth and sixth segments. It
follows that there should exist an iab-4 gene that when
active in the third segment of an Mcp animal might cause
the pigment to extend forward to the third segment. The
strategy therefore has been to mutagenize Mcp flies with
ENU and to analyze mutants which produce black pigment
on the third through the sixth segments. One such
mutant, tentatively designated Ultra-abdominal-4
(Uab-4), has now been isolated by Dr. Sakonju, who has
found that it maps within the bithorax complex.
A bonus of such mutation analyses is that mutants of
trans-regulatory genes of the bithorax complex, such as
the Polycomb (Pc) gene, are also detected. Thus
inactivation of Pc leads presumably to a reduction in a
repressor-like substance with concomitant activation of
bithorax genes in segments more anterior to those in
which they are normally expressed. Ultimately, the cis
regulatory and trans-regulatory mutants should tell us how
the bithorax genes come to be turned on in an orderly
manner during embryogenesis and should enable us to test
the validity of our current specific hypothesis that there
is an antero-posterior gradient along the embryo in the Pc
substance and a gradient along the chromosome in the
affinity of cis-regulatory regions for that substance, such
that genes most proximally located in the complex have
the lowest affinity and those most distal the highest
affinity.
243. LOCALIZATION OF BITHORAX COMPLEX (BX-C) AND ANTENNAPEDIA COMPLEX (ANT-C) GENE ACTIVITIES ALONG THE BODY AXIS OF DROSOFIIlLA
Investigator: Ian W. Dwtcan
At least two major gene complexes (BX-C and ANT-C)
are involved in controlling body segment differentiation in
Drosophila. Analysis of cuticular patterns in embryos
170
having various combinations of mutants within these
complexes reveals that in some segments, genes from both
complexes are required for normal development. In
embryos deficient for only the bithorax complex (BX-C),
the third thoracic (T3) to seventh abdominal (A7) seg
ments are transformed both dorsally and ventrally to T2;
the head, Tl, and T2 appear unaffected. AS is more
complicated: in the anterior portion a Tl-like setal belt
forms ventrally and a T2-like belt forms dorsally while in
the posterior portion, sclerotized chitinous plates (CP),
which appear to be mouth-hook material, form both
dorsally and ventrally. This complex behavior of AS
occurs in all segments from T2 through AS in BX-C
animals that are hemizygous for certain Antp mutants
(Antp WlO, Antp07, AntpB, AntpScx). Antp +, therefore,
appears to function in T2 through A 7 to suppress the
formation of Tl-like setal belts and CP. Hence, one or
more genes of the Antennapedia complex (ANT-C) is
active more posteriorly in the body than had previously
been thought. Tl-like setal belts and CP provide useful
markers for assaying the activities not only of Antp +, but
also of the BX-C genes. The CP are of particular value in
this connection because they mark the posterior portions
of segments. When added to BX-C- AntpScx hemi
zygotes, DP-100 causes suppression of CP in T2 to AS
(and partial suppression of Tl setal belts from T3 to AS).
Similarly, AntpScx Df-Ubx109 hemizygotes show suppres
sion of CP in A4 to AS and the corresponding Df-PlO and
Df-100 hemizygotes show suppression of CP in Al to AS.
Therefore, with respect to setal belts and chitinized
plates, the BX-C genes present in Dp-100 are expressed in
middle but not in terminal body segments and the BX-C
genes remaining in Df-Ubx109, Df-PlO, and Df-100 are
shown here to be active more anteriorly in the embryo
than had previously been known. For descriptions of
BX-C mutants see Lewis (197S) and of ANT-C mutants
see Kaufman et al. (19SO).
References: Kaufman, T. c., Lewis, R. and Wakimoto, B. (19SO)
Genetics 94, llS-133. Lewis, E. B. (197S) Nature 276, SSS-S70.
244. A SEARCH FOR A NEW CIS-REGULATORY REGION WITHIN THB BITHORAX COMPLEX
Investigator: Shigeru Sakonju
The current model for interpreting regulation of genes
within the bithorax complex postulates the existence of
cis-regulatory regions for each of the major genes of the
complex. A new mutant, possibly of the cis-regulatory
type, has now been identified that causes the third
abdominal (A3) segment to be transformed into one
resembling the fourth (A4). In the wild-type fly these two
segments are for all practical purposes indistinguishable.
A way of selecting for mutants that might transform the
third segment into the fourth presented itself when
Madeline Crosby discovered some years ago a dominant
mutant, Miscadastral pigmentation (Mcp), also known as
Male-chauvinist pigmentation, which causes an extension
of the uniform black pigmentation normally found in
segments AS and A6 of the male to A4 (cited in Lewis,
197S). Furthermore, she showed that Mcp is cis
regulatory, derepressing (or activating) in cis, the wild
type allele of a gene designated infra-abdominal-S (iab-S).
In normal development iab-5 + is assumed to be inactive in
A4 and active in AS (and presumably A6) where it leads to
uniform black pigmentation. In Mcp flies iab-5 + is
assumed to be active in A4, as well as AS and A6, but not
to be active in A3. If, by analogy with iab-S, there exists
another gene, iab-4, which is involved in the normal
differentiation of A4, then one might expect that a cis
regulatory mutation affecting iab-4 would tend in an Mcp
background to cause male pigmentation to form on
segment A3, as well as on A4, AS and A6. Accordingly,
Mcp males were mutagenized with the chemical ethyl
nitroso urea (ENU). These males were then mated to Mcp
females carrying suitable flanking markers. It was
anticipated that extension of Mcp pigmentation into A3
could also occur if dominant mutants occurred at the loci
of certain genes that are known to act as negative trans
regulators of the bithorax complex, such as Polycomb
{Lewis, 1978) and certain other genes {Duncan and Lewis,
19S2). Indeed, from a total of 42 cases in the first
generation of males with extended pigmentation on A3, all
but one proved to be dominant mutants of the trans
regulatory type. The remaining case has turned out to
map to the bithorax complex and therefore is a strong
candidate for the sought after cis-regulatory mutant,
which is tentatively being called Ultra-abdominal-4
(Uab-4). The mutant is fortunately not associated with
any detectable chromosomal rearrangement in the
salivary gland chromosomes. It remains to determine by
recombination analysis the precise location within the
bithorax complex of this mutant and to induce revertants
of it in order to determine whether it alters the regulation
of either an iab-4+ gene or the iab-5+ gene.
References: Duncan, J. and Lewis, E. B. (1982) In: Developmental
Order: Its Origin and Regulation. S. Subtelny (Ed.), Proceedings of the 40th Symposium of the Society for Developmental Biology, Boulder, Colorado, June 8-10, 1981. Alan R. Liss, Inc., New York, in press.
Lewis, E. B. (1978) Nature 276, 565-570.
245. MITOCHONDRIAL DNA POLYMORPHISM IN DROSOPIIlLA
Investigator: Jeffrey R. Powell
Stemming from pioneering studies by Professors A. H.
Sturtevant and Th. Dobzhansky 50 years ago at Caltech,
Drosophila pseudoobscura and its close relative, D.
persimilis, have contributed more to our understanding of
evolutionary genetics than practically any other species.
Among other findings, studies on these species revealed
two fundamental and unexpected phenomena: (1) that
evolutionary changes occur on a time scale short enough
to be experimentally observed and manipulated; and
(2) different species may be morphologically identical; the
crucial aspect of speciation is reproductive isolation
which leads to genetic isolation and thus, evolutionary
independence.
During this past year, I have been studying variation in
mitochondrial DNA (mtDNA) in D. pseudoobscura and D.
persimilis. mtDN A is maternally inherited in egg cyto
plasm; thus, variation in this DNA allows one to follow
maternal lineages which are of great potential usefulness
to population biologists. Variation is detected by changes
in recognition sites for restriction endonucleases. One of
the main goals and accomplishments of my research
program was to simplify techniques so that variation in
mtDNA could be routinely studied on a population level
with a minimum of cost and time. i have surveyed 60
strains for variation in restriction site patterns for nine
restriction enzymes.
171
The most important and unexpected result of this
• study is that the two species studied appear to be sharing
mtDNA genomes in areas where they live together, i.e.,
are sympatric. Sympatric strains of the different species
have essentially identical mt genomes. D. pseudoobscura
has a wider geographic range than D. persimilis, and
where it exists in the absence of D. persimilis the mtDNA
genome has diverged considerably. Thus, the similarity in
restriction site patterns in flies collected from locations
where the two species are sympatric is not due to slow
evolution of this DNA. The similarity of sympatric
populations is probably due to mtDNA exchange between
species via occasional hybridization. However, much
previous data indicates that nuclear genes are not
exchanged between species. This is the first observation
of cytoplasmic gene flow between species in the absence
of nuclear gene flow.
PUBLICATIONS
Craymer, L. (1981) Techniques for the manipulation of chromosomal rearrangements and their application to Drosophila melanogaster. I. Pericentric inversions. Genetics 99, 75-97.
Duncan, I. and Lewis, E. B. (1982) Genetic control of body segment differentiation in Drosophila. In: Developmental Order: Its Origin and Regulation. S. Subtelny (Ed.), Proceedings of the 40th Symposium of the Society for Developmental Biology, Boulder, Colorado, June 8-10, 1981. Alan R. Liss, Inc., New York, in press.
Lewis, E. B. (1981) Developmental genetics of the bithorax complex in Drosophila. In: Developmental Biology Using Purified Genes. ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. XXJI. D. D. Brown and C. Fred Fox (Eds.), pp. 189-208. Academic Press, New York.
Lewis, E. B. (1982) Control of body segment differentiation in Drosophila by the bithorax gene complex. Progress in Clinical and Biological Research, BSA, in press.
172
THE FOLLOWING REPORTS ARE BY GRADUATE STUDENTS IN THE DIVISION OF BIOLOGY WHO ELECTED TO DO THEIR THESIS WORK IN THE DIVISION OF CHEMISTRY AND CHEMICAL ENGINEERING.
246. A CLUSTER OP DROSOPIULA CUTICLE GENES
Investigators: Michael P. Snyder, Michael W. Hunkapiller, David Yuen•, Leroy E. Hood,
Support:
Donald J. Silvert••, James Pristrom, Norman Davidson***
National Institutes of Health, USPHS
We are studying the sequence organization and
expression of the larval cuticle genes of Drosophila. Five
major cuticle proteins are synthesized and secreted by the
epidermal cells of late third instar larvae in order to
provide a protective pupal coat. This gene system is of
interest for the study of evolutionarily-related genes,
their hormonally-induced developmental expression, and
the coordinate control of this expression.
A 50 kb DNA segment of the Drosophila genome which
resides at chromosomal locus 44D. and contains genes for
several larval cuticle proteins has been cloned (Snyder
et al., 198la). Gene mapping and DNA sequencing
techniques have shown that five cuticle-like genes are
clustered within 7,9 kb of DNA (Snyder et al., 198la,b,
1982). Amino acid sequences of four of the five major
third instar cuticle proteins have been determined. These
four sequences are identical with those predicted from the
sequences of four of the five genes of the 440 cluster.
Two of these genes are transcribed in one direction while
two are transcribed in the opposite direction. The fifth
cuticle-like gene is believed to be a pseudogene because
several features of its structure and the absence of
detectable transcripts suggest that it is non-functional.
Sequence comparisons indicate that it arose by an unequal
crossing-over event involving two closely related and
adjacent cuticle genes. The structures of the four cuticle
genes have several interesting features. Each contains a
signal peptide coding sequence which is interrupted by a
short intervening sequence (about 60 bp) at a conserved
site. Conserved sequences occur in the 5' mRNA
untranslated region, in the adjacent 35 bp of upstream
flanking sequence, and at -200 bp from the mRNA start
position in each of the cuticle genes. These sequences
may control the expression of these genes; moreover, gene
clustering may be important in the mechanism by which
these genes are expressed coordinately.
10 kb away from the third instar cuticle genes lie
three genes which are related in sequence and expressed
in second instar larvae. Further analysis will determine
whether this multigene family encodes second instar
larval cuticle proteins and test the hypothesis that these
sets of structural genes are arranged on the chromosome
in order of their developmental expression.
References: Snyder, M., Hirsh, J. and Davidson, N. (1981a) Cell 25,
165-177. Snyder, M., Hunkapiller, M., Yuen, D., Silvert, D.,
Fristrom, J. and Davidson, N. (198lb) In: Developmental Biology Using Purified Genes. D. Brown (Ed.), pp. 125-133. Academic Press, New York.
Snyder, M., Hunkapiller, M., Yuen, D., Silvert, D., Fristrom, J. and Davidson, N. (1982) Cell, submitted for publication.
*Undergraduate, California Institute of Technology. **Department of Genetics, University of California, Berkeley. ***Division of Chemistry and Chemical Engineering, California Institute of Technology.
247. CHARACTERJZATION OP GENES ENCODING MUSCLE PROTEINS IN DROSOPHILA MELANOGASTER
Investigators: Scott Falkenthal*, William W. Mattox, Vann P. Parker, Norman Davidsml*
Support: Muscular Dystrophy Association of America National Institutes of Health, USPHS
A major event during muscle development is the rapid
accumulation of both myofibrillar proteins and glycolytic
enzymes. Since the onset of this accumulation appears to
be simultaneous for many of these proteins, a common
mechanism for their temporal regulation has been
hypothesized. The expression of some myofibrillar pro
teins appears to be restricted to muscle tissue and even to
particular muscle types.
In order to understand the mechanisms of tissue
specific coordinate regulation of muscle protein genes, we
have isolated 17 independent genomic clones containing
gene sequences which are abundantly expressed in the
adult muscle of Drosophila melanogaster. In vitro
translation of hybridization-selected polyadenylated RNA
reveals that we have isolated genes encoding proteins with
the molecular weights and isoelectric points expected for
myosin light chain and tropomyosin (both myofibrillar
structural proteins) as well as triose phosphate isomerase
(TPI) and aldolase (both glycolytic enzymes). Screening
the cloned DNAs with a 32P-labeled chicken glycer
aldehyde-3-phosphate dehydrogenase (G3PDH) cDNA
clone identified one of our clones as cross-homologous.
This Drosophila clone hybridization selects an RNA which
directs the in vitro translation of a polypeptide with the
characteristics expected for G3PDH. Thus we have
probably isolated the genes coding for at least two
myofibrillar structural proteins and three glycolytic
enzymes.
These clones and the other 12 muscle gene clones were
used as probes for in situ hybridization to Drosophila
polytene chromosomes. This analysis revealed that our
presumptive aldolase and TPI clones have cytogenetic
locations of 97 A and 99E, respectively. This is consistent
with the localizatioris of these enzyme genes previously
derived from cytogenetic analysis of deficiency
chromosomes (Voelker et al., 1979). In situ hybridization
also showed that the cytogenetic location of G3PDH,
myosin light chain, and tropomyosin are 53F, 98B and 88F,
respectively. All 17 clones hybridized to distinct
chromosomal sites, indicating that the muscle genes are
dispersed. This is in contrast to some other coordinately
regulated genes in Drosophila which are tightly clustered.
Preliminary results indicate that in wild-type flies
both the myosin light chain and glycolytic enzyme genes
encode multiple polyadenylated transcripts and that the
concentrations of these RNA species are developmentally
regulated in the pattern expected for genes which encode
muscle proteins. We are now fully characterizing these
genes and studying their expression in two flightless
mutants, raised and wings-up B.
Reference: Voelker, R. A., Ohnishi, S. and Langley, C. H. (1979)
Biochem. Genet. 17, 769-783.
*Division of Chemistry and Chemical Engineering, California Institute of Technology.
248. MOLECULAR BIOLOGY OF THE ACETYLCHOLINE RECl!PTOR FROM TORPEDO
Investigators: Katharine S. Mixter, N. Davis Hershey*, Daniel Noonan•, Toni Claudio•,
Support:
Norman Davidson•
Muscular Dystrophy Association of America National Institutes of Health, USPHS
The electric ray, Torpedo, has been widely used to
study the protein components that make up the
cholinergic synapse because of the higJ:ily specialized
electroplaque cells that make up the electric organ.
Studies in the laboratory of Professor Michael A. Raftery
at Caltech as well as those by other laboratories have
conclusively shown that the acetylcholine receptor is an
173
integral membrane protein complex composed of four
different polypeptide subunits (Raftery et al., 1980).
These peptides-40 kd, 50 kd, 60 kd, and 65 kd-are found
in a 2:1:1:1 stoichiometric ratio in native receptors. In
order to investigate further the structure and action of
this important class of neuroreceptors, we have set out to
clone the genes for these four subunits.
We have constructed a double-stranded cDNA library
from electric organ poly(A)+ RNA by standard methods,
using the plasmid vector, pBR322. Five thousand colonies
of this library were screened with 32P-labeled cDNA from
electric organ and counter-screened with liver and brain 32 P-cDNA to identify clones corresponding to abundant
transcripts specific to the electric organ.
These clones (about 150) were then screened by hybrid
selection as follows: DNA from each clone was bound to
nitrocellulose filters and used to select complementary
mRNA by hybridization. This mRNA was then translated
in a reticulocyte lysate system. The resulting proteins
were immunoprecipitated with antibodies raised against
SOS-denatured receptor subunits. SDS-polyacrylamide
gels were used to identify clones that enhanced the
translation of specific proteins known to be receptor
subunits. Two such clones were identified. One, pT37, is
a 1.0 kbp cDNA clone that selects mRNA specific for the
65 kd subunit. The other, pT24G, is a 625 bp cDNA clone
specific for the 50 kd subunit.
The current directions of this project are aimed at
(1) sequencing these two genes, (2) generating full-length
cDNA copies of these two and the other two receptor
subunits, and (3) identifying and characterizing genomic
clones of the genes that encode all four subunits.
Reference: Raftery, M. A., Hunkapiller, M. W ., Strader, C. D. and
Hood, L. E. (1980) Science 208, 1454-1457.
*Division of Chemistry and Chemical Engineering, California Institute of Technology.
249. DEVELOPMENTALLY REGULATED EXPRESSION OF DROSOPIHLA ACTIN GENES
Investigators: Beverley J. Bond, Eric A. Fyrberg*, Norman Davidson**
Support: National Institutes of Health, USPHS
Our work in the pa.st year has been directed toward
identifying the developmental stages in which each of the
six Drosophila actin genes is expressed.
This analysis required the construction of probes that
174
could be used to identify the specific messenger RNAs
from each gene. Since DNA fragments from the protein
coding portions of the genes will cross-hybridize, we chose
to use DNA fragments from the 5' and 3' untranslated
regions of the genes as probes. The untranslated regions
have been shown by heteroduplex analysis to be non
homologous between different actin genes. These
nonhomologous, gene-specific DNA fragments were sub
cloned in pBR322.
Polyadenylated RNA was isolated from synchronized
animals at nine time points during Drosophila
development. RNA blots with these samples were probed
with nick-translated gene-specific probes. The six actin
genes exhibited several different patterns of expression
over the time points studied.
Two of the Drosophila actin genes, DmA2 and DmA3,
are expressed at low to intermediate levels in all stages of
d~velopment studied as well as in the Kc line of cultured
Drosophila cells (in collaboration with David Price and
Carl Parker). Therefore, they are the cytoplasmic actins.
The DmAl and DmA6 actin genes are expressed at high
levels in late pupae when the adult flight muscles are
being synthesized. These are candidates for indirect
flight muscle and tubular muscle specific actins. DmA4 is
expressed abundantly in larvae and is a reasonable
candidate for the larval intersegmental muscle actin.
DmA5 is expressed at several different stages but it is not
clear as yet what its particular function is.
Now that we have shown that the Drosophila actin
genes represent a differentially regulated gene family, we
wish to examine whether DNA sequences 5' to the gene
may function in controlling these differences in
expression. We plan to modify the sequences 5' to the
genes in vitro and examine their function in vivo by
injecting the modified genes into Drosophila embryos.
*Division of Biology, Johns Hopkins University. **Division of Chemistry and Chemical Engineering, California Institute of Technology.
250. DO DROSOPHILA ACTIN GENES REQUIRE INTRONS FOR EXPRESSION?
Investigators: Lei Yu, Beverley J. Bond,
Support:
El'lc A. Fyrberg*, Norman Davidson**
California Foundation for Biochemical Research
National Institutes of Health, USPHS
We wish to address the question of whether there are
classes of structural genes in a multigene family for which
all of the primary transcripts require the removal of an
intron by processing, in order for export to the cytoplasm
and subsequent translation to protein. We and others have
shown that four of the actin genes of Drosophila have
introns, either in their protein coding region or in the 51
untranslated region. For the other two genes, DmA3 and
DmA5, there is no information at present as to whether
they have introns in either the 5' or 3' untranslated region.
Sequence data and electron microscope heteroduplex
experiments indicate that these two genes do not have
introns within their respective protein coding regions, but
the possibility of a very short intron therein has not been
rigorously excluded. Therefore, various appropriate kinds
of Berk-Sharp experiments for identifying introns in these
two genes are now being carried out.
*Division of Biology, Johns Hopkins University. **Division of Chemistry and Chemical Engineering, California Institute of Technology.
GRADUATES
FINANCIAL SUPPORT
177
GRADUATHS
Twenty-six students in Biology were awarded the B.S., M.S. or Ph.D. degree in June 1982. Names, degrees conferred, and titles of doctoral theses are as follows:
Bachelor of Science
Cynthia Juyne Beegle, B.S. Catherine Ann Kirschvink, B.S.
Roberta Jane Brandenburg, B.S. with honor. Linda Beth McAllister, B.S. with honor.
Duke P. Briscoe, B.S. Phillip Andrew Patten, B.S. with honor.
Joseph Anthony Garcia Jr., B.S. Geoffrey David Rubin, B.S. with honor.
Nak-Hui Hwang, B.S. with honor. Juanito Serrano Villanueva, B.S. with honor.
David Stone Kamins, B.S. Thiennu Huy Vu, B.S. with honor.
Master of Science
David Alan Myers, M.S.
Doctor of Philosophy
David Lynn Gard, Ph.D. Intermediate Filaments and Myogenesis in vitro.
Bruce Leslie Granger, Ph.D. Composition and Function of Intermediate Filaments in Avian Muscle Cells and Erythrocytes.
Steven Haym Green, Ph.D. Genetic Studies of Neuronal Development in Drosophila melanogaster.
Kent Richard Jennings, Ph.D. Studies of Excitability in a Model Peptidergic System: The Roles of Cyclic AMP, Protein Phosphorylation and Serotonin During Afterdischarge in the Bag Cell Neurons of Aplysia californica.
John Henry Richard Maunsell, Ph.D. Functional Organization and Connections of the Middle Temporal Visual Area in the Macaque Monkey.
Dominic Ping-Yim Orr, Ph.D. Studies of a Circadian melanogaster.
Behavioral Neurogenetic Clock in Drosophila
Jing-hsiung James Ou, Ph.D. Structure and Replication of Alphavirus RNAs.
Steven Elery Petersen, Ph.D. Visual Response Properties of Neurons in Extrastriate Cortex of the Owl Monkey.
James William Posakony, Ph.D. Studies of the Organization and Expression of Individual Repetitive Sequence Families of the Sea Urchin Genome.
Antonio Arevalo Reyes, Ph.D. Application of Synthetic Oligonucleotides in the Isolation of Murine Transplantation Antigen cDNA Clones.
Loveriza A. Sarmiento, Ph.D. Developmental Regulation in Drosophila melanogaster.
Sandra Lee Shotwell, Ph.D. A Biochemical and Genetic Analysis of the Cyclic AMP Phosphodiesterase Defect in dunce, a Memory Mutant of Drosophila.
Randall Forrest Smith, Ph.D. Genetic Analysis of the Circadian Clock System of Drosophila rnelanogaster.
178
FINANCIAL SUPPORT
The financial support available for the work of the Division of Biology comes from many sources: from general Institute endowment and from special endowment funds for broad areas of work; from grants or contracts with individuals, companies, foundations, and U.S. governmental agencies for specific projects; from unrestricted annual gifts; from fellowships for the support of individual scholars; and from contributions to general funds provided by Industrial Associates and Institute Associates, as follows:
Fund--Reseerch SUpport
American Cancer Society
American Heart Association
Anonymous Gift Fund
John E. Barber Fund
Louis D. Beaumont Foundation
Beckman Instruments, Inc.
Bing Endowment Fund, Inc.
Biology Research in Neuroscience
Biomedical Research Support (NIH)
The James G. Boswell Foundation
The James G. Boswell Foundation
Ethel Wilson Bowles and Robert Bowles
Mrs. Leah Hills Bruce
Norman Chandler
Louisa Jane Church Fund
Norman W. Church Foundation
The Commonwealth Fund
Charles B. Corser Fund
Roberta Crutcher
The Deafness Research Foundation
Dreyfus Foundation, Teacher-Scholar
Mrs. Mary Bruce Delbriick
Josephine V. Dumke Fund
E. I. DuPont de Nemours & Co.
Fairchild Foundation
Lester and Irene Finkelstein Endowment Fund
Gloria Gartz Fund
E. s. Gosney Fund
The Hearst Foundation
Frank P. Hixon Fund
Cancer research
Cardiac research
Research in educational programs
Biological research, particularly as related to the brain
Neuroscience research
Funds for equipment; Research in developmental biology
Professorship in behavioral biology
Neuroscience research
Biomedical research
Professorship in biology
Virus research
Professorship in chemical biology
Research in biology
Professorship in cell biology and chemistry
Research in biology
Research in chemical biology
General support
Research in chemical biology
Research in biology
Deafness research
Biochemistry research
Research in biology
Cancer research
Interferon research; General support
Fairchild scholars program
Interferon research
Research and education in biology
Research in genetics
Research in auditory anatomy and physiology
Neurobiology, physiological psychology, and related research
Irma Hoef!y Fund
The Henry J. Kaiser Family Foundation
The Kroc Foundation
Lasker Award Fund
MacArthur Foundation
Louis B. Mayer Foundation
The McKnight Foundation
Merck and Company, Inc.
Monsanto Company
Francis Mosley Fund
Muscular Dystrophy As.sociations of America, Inc.
The National Foundation - March of Dimes
National Institutes of Health, USPHS
National Science Foundation
The Ann Peppers Foundation
Pew Memorial Trust
Gustavus and Louise Pfeiffer Research Foundation
The President's Fund
President's Venture Fund
Prince Charitable Trusts
The Rockefeller Foundation
Albert Billings Ruddock Fund
Damon Runyon-Walter Winchell Cancer Fund
Edwin H. Schneider Fund
Norton Simon
Alfred P. Sloan Foundation
Alfred P. Sloan Fund for Basic Research
Spencer Memorial Fund
Richard Steele
The Stone Foundation
A. H. Sturtevant Memorial Fund
179
Cancer research
Research on the antiviral and antitumor properties of human interferon
Research in biology
Chemical genetics
Human interferon research
Medical research program
Neuroscience research
Research in biology
Instrumentation development
Cancer research
Research in biology
Research in birth defects
Studies in basic experimental biology, animal physiology, biochemistry, biological systems analysis, biophysics, developmental biology, genetics, neurobiology, plant and cell biology, psychobiology, and virology; graduate research training, biomedical sciences support grant
Studies in animal physiology, biochemistry, biophysics, developmental biology, genetics, neurobiology, plant and cell biology; undergraduate research participation program
Research in auditory physiology and anatomy
Neuroscience research program
Neuroscience research program
Research and development at JPL
Medical science
Interferon research
Chemical biology research
Professorship in biology
Cancer research
Study and research in genetics
Cancer Center
Neuroscience research
General support
Research in neurobiology
Grace C. Steele professorship of immunology
Research in psychobiology
General support
180
Sundry Donors
Walter and Sylvia Treadway Fund
Albert Tyler Memorial Fund
Unallocated Gifts
Joseph G. Venable Fund
The Del E. Webb Foundation
Martin Webster Fund
Jean Weigle Memorial Fund
The Weingart Fund
Whitehall Foundation
Robert E. and May R. Wright Foundation
The Zanetti Grant
Cancer research
General support
Annual lectureship in biological research
General support
Arthritis research
Neuroscience research program
Immunology and virology basic to the problems of multiple sclerosis
General support
Gene isolation; General support
Neurobiology
Medical science
Biochemistry, cancer research
181
l'und--Fellowship Support
American Cancer Society The Anna Fuller Fund
American Heart Association E. S. Gosney Fund
The American-Scandinavian Foundation Lawrence A. Hanson Foundation
Earle C. Anthony Fellowship International Union Against Cancer
Arthritis Foundation Mackenzie Foundation
The James G. Boswell Foundation The Helen G. and Arthur Mccallum Fund
British Heart Foundation Muscular Dystrophy Associations of America, Inc.
California Foundation for Biochemical Research National Institutes of Health, USPHS
Cancer Research Institute, Inc. National Multiple Sclerosis Society
Carnegie Institution of Washington National Research Council
Centre National de la Recherche Scientifique, France National Science Foundation
Lucy Mason Clark Fund Prince Charitable Trusts
The Jane Coffin Childs Memorial Fund for Medical Research Gordon Ross Medical Foundation
Council for International Exchange of Scholars Damon Runyon-Walter Winchell Cancer Fund
Albert and Kate Page Crutcher Science Research Council, England
Delegation Generale a la Recherche Scientifique et Technique Evelyn Sharp Fellowship
Deutsche Forschungsgemeinschaft Swedish Natural Science Research Council
Deutsche Krebsforschungszentrum Walter and Sylvia Treadway Fund
The Camille and Henry Dreyfus Foundation, Inc. Vern Underwood Undergraduate Scholarship
European Molecular Biology Organization Veterans Administration
Fairchild Scholars Program The Del E. Webb Foundation
Federal College Work-Study Program Helen Hay Whitney Foundation
John E. Fogarty International Research Fellowship Program for Advanced Study in the Health Sciences (NIH)
182
Adler, H. L., 54 Agmon, N., 83 Allman, J. M., 123, 126, 130 Anderson, c., 33 Asai, D. J., 82 Arias-Ortiz, c. F ., 67 Attardi, G., 17-20
Baker, J. F., 130 Bell, J. R., 66, 68, 69 Bennett, M. K., 106 Benzer, S., 157-159 Beratan, D. N., 84 Berg, H. C., 75, 78, 79 Berson, B. J., 39, 41 Bertani, L. E., 55 Birt, D. L., 139 Block, S. M., 75, 76 Bond, B. J., 64, 173, 174 Bond, M. W ., 41, 58 Braun, M., 34 Breckler, J., 87 Britten, R. J., 25, 26, 29 Brockes, J. P., 99-101 Brokaw, c. J., 80-82 Brown, J. P ., 34 Burkhalter, A., 150
Cabrera, C. V ., 26, 160 Calame, K. L., 39, 41 Capetanaki, Y ., 85 Carpenter, J. D., 138, 139 Cartier, P. K., 49 Chabala, L. D., 108-110 Chapman, B., 41 Chariang, G., 53-55 Chomyn, A., 20, 21, 23 Claudio, T ., 17 3 Clegg, K. B., 30 Colaco, c. A. L. S., 87 Conley, M. P., 79 Connolly, M., 151 Corsaro, C. M., 93 Crews, S. T., 38, 39 Cronin-Golomb, A. M., 140, 141 Crosby, M. A., 60 Crowley, T. E., 57, 58
Dalgarno, L., 65, 67 Davidson, E. H., 24-30 Davidson, N., 46, 64, 172-174 Davie, J. M., 41 Dervan, P ., 84 Doersen, C.-J. W ., 19 Doolittle, R. F., 34 Dreyer, w. J., 31-35 Dubin, D. T., 19 Duncan, L w., 169
Eakle, K. A., 45 Eatock, R. A., 103 Edens, J., 95 Elliott, J., 117 Ellison, J. W., 41, 42 Engelman, D., 95
AUTHOR INDEX (by page number)
Erlanger, B. F., 110 Erondu, N. E., 107
Falkenthal, S., 172 Ferrus, A., 160 Fisher, D. A., 46 Flitz, L. L., 50 Flytzanis, c. N., 26, 28 Frelinger, J. A., 45 Fristrom, J., 172 Fritz, L. c., 99 Fryxell, K. F., 100 Fujita, S. c., 157-159 Fyrberg, E. A., 173, 174
Gaines, G. L., 19 Gao, B., 29 Gard, D. L., 86 Garfinkel, M. D., 58, 59 Geller, G., 84 Gibbs, G. G., 19 Giffin, C. E., 35 Gilbert, J., 113 Gogol, E., 95 Gomer, R. H., 90 Goodenow, R. S., 45, 46 Gordon, H., 152 Gordon, H. W ., 144 Granger, B. L., 87, 89 Green, s. H., 166 Greengard, P., 106 Griffin, J. A., 39, 45 Grim, L. B., 93, 95 Gros, D., 92, 93 Grula, J. W ., 25, 26
Hall, J., 166 Hamilton, c. R., 145-147 Heggelund, P., 125, 133 Hellstrom, I., 34 Hellstrom, K. E., 34 Herman, K. G., 116, 117 Hershey, N. D., 173 Hewick, R. M., 34, 35 Holton, T., 103 Hood, L. E., 35, 37, 45, 51, 172 Hopfleld, J. J., 83, 84 Horowitz, N. H., 53, 54 Hough-Evans, B. R., 29, 30 Howard, J., 30 Huang, H. V., 50, 51, 66 Hudspeth, A. J., 102, 103 Hudspeth, R. L., 29 Hunkapiller, M. w., 35, 49, 50,
94, 172
!fune, C. K., 145 Ishihara, A., 78 ltakura, T., 137
Jacobs, H. T., 26, 27 Johnson, S. A., 25 Jonsson, G., 123, 126 Joran, A. D., 84
Kasamatsu, T., 123-126, 128, 129 132, 133 '
Katz, L. c., 135 Kauvar, L. M., 162 Kennedy, M. B., 105-107 Khan, S. M. M., 7 8, 95 Kim, S. K., 42 King, D. G., 160 King, M. P., 20 Kintner, c. R., 100, 101 Konishi, M., 134, 136 Konopka, R. J., 159, 164-166 Kraig, E. B., 43, 93 Kronenberg, M., 43 Krouse, M. E., 110 Kuppermann, B., 128, 152 Kurumiya, S., 137 Kwong, S.-K., 113 Kyriacou, c. P., 166
Lane, J., 138 Lapidus, !. R., 7 9 Lazarides, E., 84, 87 Leahy, P. S., 28 Lee, J. L., 28 Lemke, G. E., 100 Lenches, E. M., 67 Lester, H. A., 107, 108, 112 Leutwiler, L. s., 61 Lewis, E. B., 169 Lewis, R. s., 104 Livant, D. L., 40 Lo, D. C.-T., 111 Lopez-Charreton, s., 69 Luck, D. J. L., 81
Macchi, M. J., 45' Mailheau, S. L., 117 Manson, M. D., 7 6 Margoliash, D., 136 Mariottini, P ., 20 Masters, J. N., 21, 22 Mattox, W. M., 172 Maunsell, J. H. R., 148-150 Maurer, B. J., 22 Mayne, J. T., 68 Mccasland, J. s., 134 McGuinness, E., 123, 126 McGuinness, T., 106 Mcintyre, J., 117 McKnight, s., 71 McMahon, A. P ., 28 McMillan, M., 46, 49 McNicholas, J. M., 43 Meyer, D. J., 94, 95 Meyer, P. W., 80 Meyerowitz, E. M., 56, 59-61 Miezin, F. M., 126 Milgram, A. E., 72 M~namide, L., 115, 116 Minard, K., 43 Mitchell, H. K., 62-64 Mixter, K. S., 173 Moiseff, A., 136 Montoya, J., 18, 19 Moore, B., 107
Moore, H.-P. H., 99 Moore, K. W ., 45 Mottes, M., 23 Myers, D. A., 82 Myers, J. J., 141
Nahm, M., 41 Nakai, K., 126, 128 Nakamura, G., 26 Nargeot, J., 112 Nerbonne, J. M., 108, 110-113 Ng, B., 53-55 Ngai, J. J., 86 Nicholson, B. J., 92-95 Nicolson, M. A., 46 Niman, H., 29 Noonan, D., 173
Olds, M. E., 137-139 Omoto, C. K., 82 Qpresko, L. K., 81 Orn, A., 47, 48 Orr, D., 165 Ou, J.-H. J., 65, 66
Parker, V. P., 172 Pecht, M. P ., 46 Perlman, J., 165 Perlmutter, R. M., 39, 41 Petersen, N. S., 62-64 Pik6, L., 30 Pine, J., 113 Posakony, J. W., 26, 27 Powell, J. R., 171 Price, M. G., 88 Pruitt, R. E., 61
Raftery, M. A., 99 Ramachandran, V. S., 128 Readhead, c., 40 Reel, C., 71
Reinitz, J. B., 161 Reiter, H., 129 Repasky, E. A., 89 Revel, J.-P., 91-95 Rice, C. M., 65-68 Roach, A., 51 Roach, J., 62 Roberts, J. W ., 25, 26, 29 Roman, J. M., 32, 33 Rose, S. J., 28 Royden, c., 130
Sakonju, S., 170 Sandoval, 1. V., 87 Schl_echte, F., 117 Scholler, E., 133 Schopf, T. J. M., 30 Segall, J. E., 76 Shaffer, E. E., 112 Shepherd, K., 123 Sher, B. T., 45 Sheridan, R. E., 108-110 Shott, R. J., 28 Shotwell, S. L., 157, 163, 164 Siddiqi, 0., 161, 162 Silver!, D. J., 172 Siu, G. J. M., 39 Sivertsen, D. w., 131 Smith, J., 138 Smyth, R. D., 79, 80 Snyder, M. P., 172 Sperry, R. W ., 140 Springer, E., 38, 39 Stadler, D. R., 55 Steinmetz, M., 43, 44 Stephens, D. A., 115, 116 Strauss, E. G., 65, 66, 68 Strauss, J. H., 65, 66, 69 Stroynowski, I., 46, 47 Strumwasser, F., 51, 111, 114-118 Stryker, M., 129 Stygall, K. A., 101
Sun, Y. H., 45 Suzuki, N., 159
Tanouye, M. A., 160 Teng, E. L., 142 Teplow, D. B., 34 Thomas, T. L., 25, 28, 30 Timko, K. D., 19 Trent, D. W ., 65
Umemoto, M., 137, 138
Van Essen, D. C., 148, 150-152 Venkatesh, T. R., 158 Vermeire, B. A., 145-147 Villeneuve, A. M., 60, 61
Wall, J. B., 40 Wang, C., 90, 91 Wassermann, N. H., 110 Watabe, K., 132, 133 Wathey, J. c., 131 Weinstock, M. M., 109 Wek, R. C., 27, 28 Winoto, A., 43, 44 Wold, B. J., 42, 70-72 Woolum, J. C., 115 Wright, P. C., 131 Wu, C.-F., 159, 160
Yancey, s. B., 93 Yang, J. K., 21 Yeakley, J. M., 118 Yu, L., 174 Yuen, D., 172
Zaidel, E., 142-144 Zipursky, S. L., 158, 159 Zuniga, M. C., 42, 47, 48
183
184
FINANCIAL SUPPORT INDEX (by page number)
American Cancer Society, 24, 37 American Heart Association, 84, 107 The American-Scandinavian Foundation, 37 Earle c. Anthony Fellowship, 37 Applied Molecular Genetics, Inc. (AMGen), 37 Arizona State University, 56 Arthritis Foundation, 37 Australian National University, 65
Baylor College of Medicine, 37 Bing Chair of Behavioral Biology, 134 Biomedical Research Support Grant (NIH), 17, 24, 31, 62,
65, 75, 84, 114, 123, 140 Cenci Bolognetti Foundation, Rome, 17 The James G. Boswell Foundation for Virus Research, 157 Ethel Wilson Bowles and Robert Bowles Professorship, 37 British Heart Foundation, 84
California Foundation for Biochemical Research, 24, 37, 62, 174
Cancer Research Institute, Inc., 37 Centre National de la Recherche Scientifique, 84 Norman Chandler Professorship in Cell Biology, 24 Charles B. Corser Fund for Biological Research, 65, 70 The Norman w. Church Fund, 105
Deutsche Forschungsgemeinschaft, 37, 169 Deutsche Krebsforschungszentrum, 24 The Camille and Henry Dreyfus Foundation, Inc., 84 Josephine V. Dumke Fund, 62
European Molecular Biology Organization, 24, 84, 91
Fairchild Foundation, 37, 53, 157 Finkelstein Support for Interferon Research, 37 Fogarty International Research Fellowship, 24, 148 Fulbright Fellowship, 37, 107
E. s. Gosney Fund, 24, 65, 84, 164, 169
Lawrence A. Hanson Foundation, 157 Frank P. Hixon Fund, 140 Hoag Foundation, 37
The Henry Kaiser Family Foundation, 37 The Kroc Foundation, 99
L. S. B. Leakey Foundation, 123
MacArthur Foundation, 37 Louis B. Mayer Foundation, 37 The McKnight Foundation, 99 Monsanto Co., 37 Francis Mosley Fund for Cancer Research, 37 Muscular Dystrophy Association of America, 84, 99, 107,
172, 173
National Institutes of Health, USPHS, 17, 24, 31, 37, 53 56, 62, 65, 70, 75, 80, 84, 91, 99, 102, 105, 107' 114, 123, 134, 137, 140, 148, 157, 169, 172, 173, 174
National Multiple Sclerosis Society, 99 National Research Council, Washington, D.C., 37 National Science Foundation, 24, 31, 37, 56, 65, 75, 83,
84, 123, 134, 148, 157, 164, 169 National University of Mexico, 65
Ann Peppers Foundation, 102 Pew Memorial Trust, 99, 102, 105, 107, 114, 123, 134,
140, 148, 157, 164 Gustavus and Louise Pfeiffer Research Foundation, 75,
99, 102 Prince Charitable Trusts, 37
The Rockefeller Foundation, 62 Gordon Ross Medical Foundation, 37, 91, 102, 105, 114 Albert Billings Ruddock Fund, 91
Edwin H. Schneider Fund, 169 Science Research Council Fellowship, England, 24 Evelyn Sharp Fellowship, 164 Alfred P. Sloan Foundation, 56 Alfred P. Sloan Fund for Basic Research, 70, 105 Stanford University, 37 Sundry Donors for Cancer Research, 37 Sweden's National Science Foundation, 84
University of Alberta, Canada, 114 University of California, Los Angeles, 140 University of Chicago, 24 University of GOteborg, Sweden, 5 3 University of Southern California, 37, 140
Veterans Administration, 24
The Del E. Webb Foundation, 99, 102, 107, 134 Weingart Foundation, 37, 99 Weizmann Fellowship, 83 Whitehall Foundation, 134, 164 Helen Hay Whitney Foundation, 134, 157, 169
Yale University, 169
Antibody Decoration of Chicken Erythrocyte Intermediate Filaments
Sonication of erythrocytes attached to a glass substrate leaves elliptical patches of adherent plasma membrane; intermediate filaments associated with these patches can be visualized by transmission electron microscopy after low angle rotary shadowing with platinum (upper left). Incubation of patches with synemin preimmune serum prior to shadowing does not alter this image (upper right). Incubation with anti-vimentin (lower left) or anti-synemin (lower right) specifically decorates the corresponding antigen and reveals its spatial distribution within the filament. Width of each panel represents 9.5 micrometers.
(B. L. Granger and E. Lazarides)
