ICSU Short Reports UICSUU Volume 1

CIPRESS/"/

A d v a n c e s in Gene T e c h n o l o g y : H u m a n Genet ic D i s o r d e r s

Proceedings of the Sixteenth Miami Winter Symposium

Miami, Florida, U.S.A.

January 16-20, 1984

Edited by

Walter A. Scott , Fazal Ahmad, Sandra Black, Julius Schultz and William J . Whelan

CAMBRIDGE UNIVERSITY PRESS

Cambridge

London New York New Rochelle

Melbourne Sydney

Contents PREFACE xv

INTRODUCTION x v i i

ICSU SHORT REPORTS: A MESSAGE FROM THE ICSU PRESS x i x

SYMPOSIUM PRESENTATIONS

ONCOGENES

ACTIVATION OF CELLULAR TRANSFORMING GENES IN NEOPLASMS . . . . 3 Geoffrey M. Cooper

HUMAN ONC GENES, T-CELL GROWTH FACTORS (IL-2) AND THE FAMILY OF HUMAN RETROVIRUSES CALLED HTLV 7 R.C. Gallo, P.S. Sarin, W.C. Saxinger, M. Robert-Guroff and F. Wong-Staal

MECHANISMS OF c-njyc ACTIVATION IN AVIAN AND HUMAN B-CELL LYMPHOMAS 13 W. Hayward, C.-K. Shih, M. Goodenow, K. Wiman, A. Hayday, H. Saito and S. Tonegawa

TRANSLOCATIONS AND GENE ACTIVATION THE ROLE OF CHROMOSOMAL TRANSLOCATIONS IN ONCOGENE TRANSPOSITION INTO FUNCTIONALLY ACTIVE GENE REGIONS: A MECHANISM IN CARCINOGENESIS 19 George Klein

ACTIVATION OF C-MYC ONCOGENE BY CHROMOSOME TRANSLOCATION TO IMMUNOGLOBULIN GENES IN MURINE PLASMACYTOMAS 22 Kenneth B. Marcu, Lawrence W. Stanton, Jian-qing Yang, El?ine F. Remmers, Paul Fahrlander, Robert Greenberg, 1 ·»1 Eckhardt, Barbara K. Birshtein, Linda J. Harris

semary Watt

SOMAL BASIS OF HUMAN NEOPLASIA 27 jnis

i v

MECHANISMS OF GENE ACTIVATION DNA METHYLATION, X-INACTIVATION, AND CANCER 32 Arthur D. Riggs, Judith Singer-Sam, Douglas Keith and Brian I . Carr

STUDIES OF X-CHROMOSOME INACTIVATION IN HUMAN CELLS 36 Stanley F. Wolf and Barbara R. Migeon

DROSOPHILA HEAT SHOCK GENES AND THE CONTROL OF THEIR EXPRESSION 40 R. Voellmy, R. Lawson, R. M e s t r i l and P. S c h i l l e r

REGULATION OF TRANSCRIPTION BY GLUCOCORTICOIDS: IDENTIFICATION OF THE HORMONE REGULATORY ELEMENT AND ANALYSIS OF MECHANISM OF ACTION WITH EPISOMAL FUSIONS CONTAINING THE MMTV PROMOTER . . 45 Gordon L. Hager

THE SV40 "ENHANCER TRAP" 48 Frank Weber, Michael Boshart, Jean de V i l l i e r s , J u l i a n B a n e r j i , Christoph Α. Gehring and waiter Schaffner

THE INTRODUCTION OF METALLOTHIONEIN-GROWTH HORMONE FUSION GENES INTO MICE 52 R.E. Hammer, R.D. Palmiter and R.L. B r i n s t e r

THE MAJOR HISTOCOMPATIBILITY LOCI HUMAN HISTOCOMPATIBILITY ANTIGENS: GENES AND PROTEINS . . . . 58 Jack L. Strominger

STRUCTURE AND FUNCTION OF I-A MOLECULES 63 Hugh 0. McDevitt

STRUCTURE AND EXPRESSION OF HLA CLASS I GENES 67 Bertrand R. Jordan, Francois A. Lemonnier, Philippe Le B o u t e i l l e r , Marie Malissen, Zohair Mishal, Regis Sodoyer, Terry L. Delovitch, Tom Strachan, Michele Damotte, Catherine N'Guyen, Corine Layet, Jo e l l e Dubreuil, Andre J. van Agthoven, Jeannine Trucy and Danielle Cai1lol

GENES OF THE MAJOR HISTOCOMPATIBILITY COMPLEX Leroy E. Hood

70

ν

HUMAN GENETIC ORGANIZATION INTRODUCTION AND OVERVIEW 75 Will i a m L. Nyhan

GENETIC DIAGNOSIS BY DNA ANALYSIS 79 Yuet Wai Kan

THE MOLECULAR GENETICS OF HUMAN GLOBIN GENE EXPRESSION: IN-VITRO ANALYSIS OF SPLICING MUTATIONS 82 Adrian Krainer, Michael Green, Barbara Ruskin and Tom Maniat i s

HUMAN GENETIC LINKAGE STUDIES WITH DNA MARKERS 8 6

R.L. White, M. Leppert, D. Drayna, R. Leach and Ü. Barker

THE CHARACTERIZATION AND CORRECTION OF LESCH-NYHAN GENE DEFECTS BY RECOMBINANT DNA TECHNIQUES 90 C. Thomas Caskey, Thomas P. Yang, Pragna I . P a t e l , J. Timothy Stout and A. Craig Chinault

RANDOM GENE PROBES AND THE MAPPING OF MUSCULAR DYSTROPHY AND SIMILAR INHERITED DISEASES 94 Robert Williamson, Conrad G i l l i a m , Steve Hodgkinson, Caroline I n g l e , Mark Blaxter and Kay Davies

THE CLONING OF BLOOD COAGULATION FACTORS 100 Earl W. Davie, Dominic W. Chung, Sandra J. Friezner Degen, Kotoku Kurachi, Mark W. Rixon and S h i n j i Yoshitake

GENETIC DEFECTS IN LIPOPROTEIN RECEPTORS: A COMMON CAUSE OF ATHEROSCLEROSIS IN MAN 104 Michael S. Brown and Joseph L. Goldstein

vi

HYBRIDIZATION, IMMUNOLOGICAL AND

ENZYMIC TECHNIQUES QUANTITATION OF SPECIFIC GENE EXPRESSION IN HUMAN DISEASES BY QUICK-BLOT 108 David H. G i l l e s p i e and Joel Bresser

THE USE OF IMMUNOTOXINS FOR THE THERAPY OF CANCER AND THE MODULATION OF THE IMMUNE RESPONSE 112 Jonathan W. Uhr and Ellen S. Vitetta

THE T-CELL CIRCUIT - CLINICAL AND BIOLOGIC IMPLICATIONS . . . . 116 Stuart F. Schlossman

ENZYME REPLACEMENT THERAPY USING LIPOSOMES OF NOVEL COMPOSITION 120 Kunio Yagi

v i i

POSTER SESSION R E P O R T S

THE ISOLATION OF A CLONE CONTAINING THE COMPLETE HUMAN APRT GENE 126 Janet Arrand, E l l i o t Drobetsky and Anne Murray

LACK OF POLYMORPHISM FOR C-TYPE RETROVIRUS SEQUENCES IN THE SYRIAN HAMSTER 128 Sal l y S. Atherton, Robert D. S t r e i l e i n , and J. Wayne S t r e i l e i n

SYNTHESIS, CLONING AND EXPRESSION OF GENES CODING FOR HUMAN EPIDERMAL GROWTH FACTOR AND RAT TYPE I TRANSFORMING GROWTH FACTOR IN E^ COU 130 Allen Banks, Margery Nicolson and David Hare

ISOLATION AND CHARACTERIZATION OF SINGLE SEQUENCES FROM THE HUMAN CHROMOSOME 21 LIBRARY 1 3 2 D. J. Baro, S.M. McCutcheon and M.R. Cummings

STRUCTURE AND EXPRESSION OF MOUSE RENIN GENES 134 L.J. Beecroft, D.W. Burt, J.J. Mullins and 0. P i o l i

A NEW RESTRICTION FRAGMENT LENGTH POLYMORPHISM CLOSELY LINKED TO THE HUMAN HYPOXANTHINE-GUANINE PHOSPHORIBOSYLTRANSFERASE IHPRT) LOCUS 1 3 6

Barbara A. Boggs, Robert L. Nussbaum and Richard A. Lewis

A cDNA CLONE FROM AN ABUNDANT NONRIBOSOMAL 35S RNA IN SALIVARY GLANDS OF CHIRONOMUS TENTANS I 3 3

John R. Bower and Steven T. Case

MOLECULAR CLONING AND NUCLEOTIDE SEQUENCE ANALYSIS OF HUMAN c-sis/PLATELET-DERIVED GROWTH FACTOR GENE 141 Ing-Ming Chiu, Steven R. Tronick, Hisanaga Igarashi, E. Premkumar Reddy, Keith C. Robbins and Stuart A. Aaronson

ENHANCED EXPRESSION AND SUPPRESSION OF c - r a s H ONCOGENE DURING GROWTH AND REGRESSION OF HORMONE-DEPENDENT MAMMARY TUMORS. . . 142 Y.S. Cho-Chung and F.L. Huang

GENETIC RESISTANCE AND SUSCEPTIBILITY IN INSULIN-DEPENDENT DIABETES ANALYSED BY HLA CLASS I I ALLOGENOTOPES (DNA RESTRICTION FRAGMENT POLYMORPHISM) 144 D. Cohen and J. Dausset

v i i i

CHARACTERIZATION OF AN UNUSUAL ALU-RELATED FAMILY IN THE PROSIMIAN GALAGO CRASSICAUDATUS 146 Gary R. Daniels and Prescott L. Deininger

THE ISOLATION OF GENE SEQUENCES ON HUMAN CHROMOSOME 21. . . . 148 Je f f r e y N. Davidson, A l b e r t A. Davidson and Lee A. Niswander

HUMAN FACTOR IX CLONES ISOLATED WITH SYNTHETIC OLIGOMER PROBES 150 L.M. Davis, R.A. McGraw and D.W. S t a f f o r d

GENE TRANSFER AND AMPLIFICATION OF THE HUMAN GENE (S) ASSOCIATED WITH OROTICACIDURIA 152 R.E. Davis, J. Bleskan and D. Patterson

ISOLATION AND PRELIMINARY CHARACTERIZATION OF A cDNA CLONE FOR HUMAN THYMIDINE KINASE 154 Prescott Deininger, Harvey Bradshaw and Salvatore Carradonna

CHARACTERIZATION OF A MUTATION IN THE PR0o2(I)COLLAGEN C-PROPEPTIDE IN A CHILD WITH AN AUTOSOMAL RECESSIVE FORM OF OSTEOGENESIS IMPERFECTA 156 L.A. Dickson, T. Pi h l a j a n i e m i , S. Deak, F.M. Pope, A. N i c h o l l s , D.J. Prockop and J.C. Myers

IH VITRO PHOSPHORYLATION AND INACTIVATION OF DNA TOPOISOMERASES BY VIRAL AND CELLULAR TYROSINE-SPECIFIC PROTEIN KINASES . . . 158 Yuk-Ching Tse Dinh, Tai Wai Wong and A l l a n R. Goldberg

ISOLATION OF A HUMAN ADENINE PHOSPHOR IBOSYLTRANSFERASE (APRT) GENE, AND IDENTIFICATION OF AN ASSOCIATED RESTRICTION ENZYME POLYMORPHISM 160 M.K. Dush, P.J. Stambrook, J.C. Clark, J. T r i l l and J.A. T i s c h f i e l d

ISOLATION OF A MOUSE-LIVER ALCOHOL DEHYDROGENASE cDNA I 6 2

Howard J. Edenberg and Ke Zhang

POLYMORPHIC RESTRICTION ENDONUCLEASE SITES LINKED TO THE HLA-DR α CHAIN GENE: LOCALIZATION AND USE AS GENETIC MARKERS IN CONTROL AND INSULIN-DEPENDENT DIABETES POPULATIONS I 6 4

Henry A. Erl ich and Deborah S t e t l e r

A CELLULAR GENE HOMOLOGOUS TO v-mht IS EXPRESSED IN CHICKEN AND HUMAN CELLS 166 CS. Flordel l i s , N.C. Kan, M.C. Psal 1 idopoulos, K.P. Samuel, O.K. Watson and T.S. Papas

i x

LATENCY OF TUMOR INDUCTION BY METHYLCHOLANTHRENE IS HJ? LINKED 168 Herbert A. Freedman

ISOLATION AND CHARACTERIZATION OF HUMAN METALLOTHIONEIΝ-I AND I I PROCESSED GENES 171 Lashitew Gedamu and Umesh Varshney

GENETIC LINKAGE STUDIES OF HUMAN CHROMOSOME 11 172 D.S. Gerhard, K.K. Kidd, J.K. Kidd, J. Gusella and D. Housman

INSERTION OF A SELECTABLE MARKER INTO VARIOUS SITES ON HUMAN CHROMOSOME NO.11 174 Tom Glaser and David Housman

DIFFERENTIAL EXPRESSION OF TWO CLUSTERS OF MOUSE HISTONE GENES 176 Reed A. Graves, Susan E. Wellman and W.F. Ma r z l u f f

HEMOGLOBINOPATHIES: DETECTION OF DEFECTIVE HUMAN &-GLOBIN GENES BY DIRECT AND INDIRECT DNA RESTRICTION ANALYSIS 178 Jürgen Horst, Renate Oehme, Enno Kleihauer and Elisabeth Kohne

ISOLATION AND PRELIMINARY CHARACTERIZATION OF FERRITIN HEAVY CHAIN cDNA CLONE 180 Swatantra K. Ja i n , Dana Boyd, K. Barret, J. Crampton and Jim Drysdale

LOCALIZATION OF c-abj, c - s i s AND c-fes ONCOGENES TO HUMAN GERMLINE CHROMOSOMES 182 Suresh C. Jhanwar, Benjamin G. Neel, William S. Hayward and R.S.K. Chaganti

REGULATION OF METALLOTHIONEIΝ GENES IN MENKES' DISEASE . . . . 184 L. Jolicoeur-Paquet, A. Zelinka, L.-C. Tsui and J.R. Riordan

A RANDOM CHROMOSOME 6 DNA PROBE AND RFLP ANALYSIS OF HLA-TYPED FAMILIES 186 K.W. Kl i n g e r , A.W. Muir and C. Gi1 lam

STUDIES ON DRUG-INDUCIBLE GENE SEQUENCES 188 J.A. Koch, C.W. Fisher, A.G. D i l e l l a , M. Haska, A.W. Steggles and J.F. Wong

CLONING OF cDNA ENCODING ORNITHINE DECARBOXYLASE (ODC): STUDIES ON ODC-mRNA INDUCTION BY ANDROGENS IN NORMAL AND ANDROGEN-INSENSITIVE (Tfm/Y) MICE 190 K. Kontula, C.W. Bardin and O.A. Janne

χ

MOLECULAR AND CYTOGENETIC CHARACTERIZATION OF FLOW-SORTED MAMMALIAN CHROMOSOMES 192 G. Langer, K.J. Hutter, J. Barths and N. B l i n

MOLECULAR CLONING OF THE FACTOR IX GENE 194 Don E. Lavelle and Pudur Jagadeeswaran

ANALYSIS OF SINGLE BASE SUBSTITUTIONS IN HUMAN DNA 196 L.S. Lerman, N. Lumelsky and S.G. Fischer

POSSIBILITY OF PRENATAL DIAGNOSIS OF CLASSICAL PKU BY RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF THE PHENYLALANINE HYDROXYLASE GENE 198 Alan Lidsky, Fred Ledley, Anthony D i L e l l a , Kathryn Robson and Savio L.C. Woo

ISOLATION, PARTIAL CHARACTERIZATION AND CELL CYCLE REGULATION OF HUMAN Hl HISTONE GENE IN A CLUSTER WITH CORE HISTONE CODING SEQUENCES 200 F. Marashi, M. Plumb, L. Green, J. Stein and G. S t e i n

RESTRICTION ENZYME FRAGMENT LENGTH POLYMORPHISM (RFLP) OF HUMAN PRE- PRO-PARATHYROID GENE IN ISOLATED CLONES AND IN A POPULATION SURVEY 202 Hubert Mayer, Georg Widera and Erich Breyel

RESTRICTION ENZYME SITE POLYMORPHISMS OF CLASS I I HISTOCOMPATIBILITY GENE SEQUENCES IN WILD MICE 204 Thomas J. McConnell and Edward K. Wakeland

THE EXPRESSION OF A PRO-OPIOMELANOCORTIN-LIKE GENE IN MURINE LEYDIG TUMOR CELLS 206 Michael H. Meiner and David Puett

CONSTRUCTION OF HUMAN cDNA LIBRARIES FOR ISOLATION OF THE TERMINAL DEOXYNUCLEOTIDYLTRANSFERASE GENE 208 S.K. Moore, L.K. Ri l e y , M.S. Coleman and R.C. Dickson

V-MOS EXPRESSION IN CELLS INFECTED WITH A MUTANT OF MUSY IS ACTIVATED BY A TEMPERATURE-SENSITIVE RNA SPLICING EVENT . . . . 210 Michael A. Nash, B i l l L. Brizzard and Edwin C. Murphy, J r.

METAPHASE CHROMOSOME TRANSFER OF INSERTED SELECTABLE MARKERS: A TOOL FOR GENE MAPPING AND ISOLATION 212 David L. Nelson and David E. Housman

LOCATION OF MURINE LEUKEMIA VIRUS SEQUENCES WITHIN THE MURINE MAJOR HISTOCOMPATIBILITY COMPLEX, H-2 214 Chr i s t i n e Pampeno, Ruth Kornreich, Anthony Rossomando, Andrew L. Mellor, Elizabeth Weiss, Richard F l a v e l l , Angel P e l l i c e r and Daniel Meruelo

xi

CARRIER DETECTION BY GENE ANALYSIS IN A FAMILY WITH HAEMOPHILIA Β(FACTOR IX DEFICIENCY) 216 I.R. Peake, B.L. Davies, A.L. Bloom and G.G. Brownlee

NONRADIOACTIVE DETECTION OF EUKARYOTIC GENES ON SOUTHERN BLOTS 219 R. P e r g o l i z z i , C. Bra k e l , R. Lang, M. Solanki and S. Mowshowitz

ORGANIZATION AND EXPRESSION OF ENDOGENOUS MMTV PROVIRUSES IN C3Hf/He AND C3Hf/Ki MICE 220 Brian J. Popko and Robert J. Pauley

ISOLATION AND CHARACTERIZATION OF A SINGLE-COPY PROBE FOR A HUMAN DNA REPAIR GENE 222 Jaime S. Rubin, V a l e r i e Prideaux, Gordon F. Whitmore and Alan Bernstein

A POSSIBLE Β CELL GROWTH FACTOR PRECURSOR 224 CG. Sahasrabuddhe, J.W. Morgan, D.A. Wright, R. Adlakha and A. Maizel

SEQUENCE ANALYSIS OF ALPHA-1-ANTITRYPSIN DEFICIENCY ALLELE, PiS 226 Daniel Schindler, Barbara WalIner-Phi1ipp, Richard T i z a r d , Russell Chan, William K e l l e y , and Richard F l a v e l l

NUCLEOTIDE SEQUENCE OF CLONED cDNA ENCODING RAT PREPRO-PARATHYROID HORMONE 228 Hans-Jurgen Schmelzer, Gerhard Gross and Hubert Mayer

CHARACTERIZATION OF A STEROID-REGULATED GENE OF DROSOPHILA . . 230 R.A. Schulz, L. Cherbas, M.M.D. Koehler and P. Cherbas

EXCLUSION LIMITS OF DNA RESTRICTION FRAGMENTS ON GEL FILTRATION MEDIA 232 Cheri Seitz

HIGH LEVEL EXPRESSION OF v-mos PROTEIN OF MOLONEY MURINE SARCOMA VIRUS IN E^ COLI 234 Arun K. Seth and George F. Vande Woude

TRANSFORMANTS ISOLATED FOLLOWING T24 DNA GENE TRANSFER EXPRESS A RECEPTOR BINDING Fc£ 236 K.D. Somers, A.E. Campbell, M.L. Beckett, M. Patten-Aardrup and G.L. Wright, J r .

LINKAGE CONSERVATION AND ONCOGENE MAPPING IN THE CHINESE HAMSTER 238 R.L. S t a l l i n g s , A.C. Münk, J.L. Longmire, J.H. J e t t and B.D. Crawford

x i i

A HUMAN-LIKE PREPROINSULIN LEADER SEQUENCE DIRECTS PROTEIN SECRETION IN YEAST 240 P.O. Stepien, R. Brousseau, R. Wu, S. Narang and D.Y. Thomas

MECHANISMS OF DEXAMETHASONE AND BLEOMYCIN REGULATION OF PROCOLLAGEN SYNTHESIS AND TYPE I PROCOLLAGEN mRNAs 243 Kenneth M. S t e r l i n g and Kenneth R. Cutroneo

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS IN ACHONDROPLASTIC DWARFISM AND THANATOPHORIC DWARFISM 244 Charles M. Strom and Charis E.L. Eng

ALLELE-SPECIFIC HYBRIDIZATION USING OLIGONUCLEOTIDE PROBES OF VERY HIGH SPECIFIC ACTIVITY: DISCRIMINATION OF THE HUMAN tA AND &S-GLOBIN GENES 246 Anna B. Studencki and R. Bruce Wallace

AN ENZYME-IMMUNOBINDING ASSAY FOR FAST SCREENING OF EXPRESSION OF TISSUE PLASMINOGEN ACTIVATOR cDNA IN E^ COLI 248 John C.-T. Tang and S h i r l e y H. L i

MOLECULAR BASIS OF HEREDITARY TYROSINEMIAS: PROOF OF THE PRIMARY DEFECT BY WESTERN BLOT 250 R.M. Tanguay, C. Laberge, A. Lescault, J.P. V a l e t , J.L. Duband and Y. Quenneville

USE OF CHEMICALLY MODIFIED NUCLEIC ACIDS AS IMMUNO-DETECTABLE PROBES IN HYBRIDIZATIONS 252 P. Tchen, R. Fuchs, Ε. Sage and Μ. Leng

EFFECT OF CHEMICAL CARCINOGENS AND VIRAL ONCOGENES ON THE NEOPLASTIC PROGRESSION OF SYRIAN HAMSTER EMBRYO CELLS: EVIDENCE FOR A THREE-STEP PROCESS 254 David G. Thomassen, Mitsuo Oshimura, Tona Gilmer, Lois Annab and J. Carl B a r r e t t

THE HUMAN ALDOLASE Β GENE: STUDIES TOWARD UNDERSTANDING HEREDITARY FRUCTOSE INTOLERANCE 256 Dean R. Tolan, William H. Rottmann and Edward E. Penhoet

CHARGE HETEROGENEITY OF PROTEINS SYNTHESIZED IN CELL-FREE PROTEIN SYNTHESIS AND E^ COLI MINICELLS 258 K. Trin k s , K. Beyreuther and P. Habermann

Alu and Kpnl SEQUENCES ARE FOUND ADJACENT TO ALPHOID DNA IN THE HUMAN GENOME 260 P.W. Turk, G.J. Graham, T.J. Hall and M.R. Cummings

PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES THAT DISCRIMINATE AMONG INDIVIDUAL SlOO POLYPEPTIDES 262 Linda J. Van Eldik

x i i i

BIOCHEMICAL AND GENETIC STUDIES ON CELLS PRODUCING ABNORMAL IgM 2 6 4

L.M. Vazquez, M.P. Beckmann, M.J. Shulman and W.J. Grimes

STUDIES OF THE HUMAN TRANSFERRIN GENE 2 6 6

Funmei Yang, J.B. Lum, John R. McGill, Charleen M. Moore, Peter H. van Bragt, W. David Baldwin and Barbara H. Bowman

NONRADIOACTIVE COLONY HYBRIDIZATION 2 6 9

Huey-Lang Yang and Norman Kelker

MOLECULAR CLONING OF MAMMALIAN ADENOSINE DEAMINASE GENE SEQUENCES THROUGH GENE AMPLIFICATION 2 7 0

C. Y. Yeung, D.E. I n g o l i a , M.R. Al-Ubaidi, A.G. Hook, D. A. Wright, E.G. Frayne and R.E. Kellems

AUTHOR INDEX 273

48

THE SV40 "ENHANCER TRAP"

Frank Weber, Micha e l Boshart, Jean de V i l l i e r s , J u l i a n B a n e r j i , C h r i s t o p h Α. Gehring and Walter S c h a f f n e r

I n s t i t u t für M o l e k u l a r b i o l o g i e I I der Universität Zürich, Hönggerberg, CH-8093 Zürich, S w i t z e r l a n d

INTRODUCTION T r a n s c r i p t i o n a l enhancers are s h o r t DNA segments t h a t

a c t i v a t e t he t r a n s c r i p t i o n o f l i n k e d genes, i n e i t h e r o r i e n t a t i o n and over d i s t a n c e s of many k i l o b a s e p a i r s ( k b ) , which were o r i g i n a l l y d i s c o v e r e d i n v i r a l genomes ( B a n e r j i e t a l . , 1981; de V i l l i e r s and S c h a f f n e r , 1981; Moreau e t a l . , 1981; f o r r e c e n t reviews see Khoury and Gruss, 1983, and a l s o Gluzman and Shenk, 1983). T i s s u e -s p e c i f i c c e l l u l a r enhancers have r e c e n t l y been found i n immunoglobulin genes ( B a n e r j i e t a l . , 1983; G i l l i e s e t a l . , 1983; Neuberger, 1983; Queen and B a l t i m o r e , 1983; P i c a r d and S c h a f f n e r , 1983).

We reasoned t h a t i t i s p o s s i b l e t o g e n e r a l l y s e l e c t f o r f u n c t i o n a l enhancers by assaying f o r t h e r e s u r r e c t i o n of enhancerless SV40, a f t e r c o t r a n s f e c t i n g i t w i t h s h o r t random DNA fragments i n t o monkey c e l l s . I n t h i s way we c o u l d s e l e c t f o r the growth o f recombinant v i r u s e s which had i n c o r p o r a t e d DNA w i t h enhancer f u n c t i o n . T h i s technique o f f e r s c l e a r advantages over t h e d i r e c t and i n d i r e c t approaches used p r e v i o u s l y t o i d e n t i f y enhancers ( B a n e r j i e t a l . , 1981; Lusky e t a l . , 1983; F r i e d e t a l . , 1983; Folger e t a l . , 1983).

RESULTS We have t r a n s f e c t e d monkey CV-1 c e l l s w i t h non­

i n f e c t i o u s , l i n e a r SV40 DNA, l a c k i n g t h e 72 bp repeated enhancer r e g i o n . I n f e c t i o u s v i r u s was re c o v e r e d from t h i s

49

"enhancer t r a p " upon c o - t r a n s f e c t i o n w i t h enhancer DNA segments from v a r i o u s v i r u s e s such as SV40, polyoma, c y t o m e g a l o v i r u s and Rous sarcoma v i r u s . The enhancer DNA segments a p p a r e n t l y became i n t e g r a t e d i n t o t h e enhancerless SV40 DNA by i n t r a c e l l u l a r r e s e c t i o n / l i g a t i o n / r e p a i r processes. A t r u n c a t e d polyoma "semi-enhancer" ( B e l l t o P v u I I fragment; de V i l l i e r s and S c h a f f n e r , 1981) was i n c o r p o r a t e d as a dimer and r e t a i n s t he host c e l l p r e f e r e n c e (de V i l l i e r s e t a l . , 1982) of the complete polyoma enhancer. C o - t r a n s f e c t i o n o f the "enhancer t r a p " w i t h fragmented DNA of mouse, monkey or human o r i g i n , y i e l d e d no recombinant v i r u s w i t h i n t e g r a t e d c e l l u l a r sequences, w i t h one p o s s i b l e e x c e p t i o n . This i n d i c a t e s t h a t t h e r e are no more than a few hundred segments per mammalian genome w i t h the s t r o n g enhancer a c t i v i t y demanded by our assay. I n some t r a n s f e c t i o n experiments w i t h o u t added v i r a l enhancer DNA, SV40 v a r i a n t s were generated which have a segment of t h e i r f l a n k i n g " l a t e " DNA d u p l i c a t e d t o s u b s t i t u t e f o r the d e l e t e d 72 bp r e p e a t . I n one o f these v a r i a n t s (SV7.2, see Fi g u r e 1) an 88 bp d u p l i c a t i o n c r e a t e s a s t r o n g enhancer from t h i s p r e v i o u s l y i n a c t i v e DNA r e g i o n . Both the polyoma enhancer fragment and t h e spontaneously c r e a t e d enhancers l a c k the p h y s i c a l sequence f e a t u r e s which have been a s s o c i a t e d w i t h enhancer elements, namely the "GTGG(A/T)-box" (Khoury and Gruss, 1983), the "CACA-box" (Lusky e t a l . , 1983) or s t r e t c h e s of a l t e r n a t i n g p u r i n e s - p y r i m i d i n e s (Nordheim and Rich, 1983). I t t h e r e f o r e appears t h a t these sequence m o t i f s are not u b i q u i t o u s l y a s s o c i a t e d w i t h , and may even be redundant f o r , t h e f u n c t i o n of t r a n s c r i p t i o n a l enhancers. REFERENCES B a n e r j i , J., Rusconi, S. and Sc h a f f n e r , W. (1981) C e l l 27, 299-308 B a n e r j i , J., Olson, L. and Sc h a f f n e r , W. ( 1 9 8 3 ) . C e l l , 729-740 · de V i l l i e r s , J. and S c h a f f n e r , W. (1981) Nucl.Acids Res.9, 6251-6264

50

de V i l l i e r s , J., Olson, L., T y n d a l l , C. and S c h a f f n e r , W. (1982) Nucl.Acids Res. 10, 7965-7976 F o l g e r , K.R., Wong, Ε.Α., Wahl, G. and Capecci, M.R. (1982) M o l . C e l l . B i o l . 2, 1372-1378 F r i e d , M., G r i f f i t h s , M., Davies, B., B j u r s e l l , G., LaMantia, G. and Lan i a , L. (1983) Proc.Natl.Acad.Sei.80, 2117-2121 G i l l i e s , S.D., M o r r i s o n , S.L., O i , V.T. and Tonegawa, S. (1983) C e l l 33, 717-728 Gluzman, Y. and Shenk, T. E d i t o r s (1983) Enhancers and e u c a r y o t i c gene e x p r e s s i o n . Cold S p r i n g Harbor L a b o r a t o r y , Cold Spring Harbor, New York Khoury, G. and Gruss, P. (1983) C e l l 33, 313-314 Lusky, M., Berg, L., Weiher, Η. and Botchan, Μ. (1983) M o l . C e l l . B i o l . i n press Moreau, P., Hen, R., Wasylyk, B., E v e r e t t , R., Gaub, M.P. and Chambon, P. (1981) Nucl.Acids Res. 9, 6047-6068 Neuberger, M.S. (1983) EMBO J. 2, 1373-1378 Nordheim, A. and Rich, A. (1983) Nature 303, 674-679 P i c a r d , D. and S c h a f f n e r , W. (1983) Nature, i n press Queen, C. and B a l t i m o r e , D. (1983) C e l l 33, 741-748 Tooze, J. Ed. (1981) DNA tumor v i r u s e s (Cold S p r i n g Harbor, New York: Cold Spr i n g Harbor, New York

LEGEND TO FIGURE 1 SVA50: This v a r i a n t v i r u s has a 54 bp d e l e t i o n around t h e Kpnl s i t e . I t was o b t a i n e d from a c o t r a n s f e c t i o n of enhancer t r a p DNA w i t h the enhancer b e a r i n g r e s t r i c t i o n fragment and an excess of s o n i c a t e d c a r r i e r DNA.

SVP2M2: This v a r i a n t was o b t a i n e d from an experiment u s i n g the mixed-in s o n i c a t e d p l a s m i d pPySVklOO- where the SV40 enhancer was p r o v i d e d f l a n k e d by polyoma v i r u s sequences (de V i l l i e r s e t a l . , 1982).

S V 1 5 - : This i s a v i r u s c o n t a i n i n g t h e SV40 enhancer i n t h e o p p o s i t e o r i e n t a t i o n , o b t a i n e d from an experiment w i t h t h e same t r a n s f e c t i o n p r o t o c o l as i n t h e case of SVA50.

SVFE2 and S V 7 . 2 grew out of c e l l s t r a n s f e c t e d w i t h SV40 enhancer t r a p and c a r r i e r DNA w i t h o u t enhancer added. The t r a n s f e c t e d SV40 DNA was c i r c u l a r i z e d in. v i v o . 77 bp and 88 bp o f the v i r a l " l a t e " r e g i o n around t h e H p a l l s i t e were d u p l i c a t e d i n SVFE2 and SV7.2, r e s p e c t i v e l y , t h e r e b y g i v i n g r i s e t o v a r i a n t s which grow s l o w l y (SVFE2) or i n t e r ­m e d i a t e l y f a s t (SV7.2).

51

FIGURE 1 : Maps o f SV40-derived enhancers

Δ 5 0 recombination?

sj / i B * " D

• I

exonuclease I h

268 322 V

Bgl I Hpall

P 2 M 2 90 103

Bg/I Hpall

1 5 -

EcoRI linker

inker DNA

Hpall

F E 2 duplicated fragment > ι

99 294 376 300

linker DNA

Hpall HpaH

7.2

Bg/I

duplicated fragment 1 I

99 ! 295 379! 295

linker DNA

Hpall linker DNA

Hpall