Normal Chlamydomonas nuclear gene structure on linkage group XIX

JEFFERY A. SCHLOSS*

Molecular & Cell Biology Group, School of Biological Sciences, University of Kentucky, Lexington, KY 40506-0225 USA

and HENRIETTA BROWN CROOM

Department of Biology, The University of the South, Sewanee, TN 37375 USA

* Author for correspondence

Summary

The unusual Chlamydomonas linkage group XIX -called the uni linkage group for the uni mutants thatlack one of the paired flagellae of wild-type cells - hasbeen reported to be physically located exclusively atthe basal bodies. To learn whether the structure ofgenes on this linkage group differs from the structureof nuclear genes in this organism, we determined theprimary structure of a gene that maps to linkagegroup XIX. This analysis reveals the presence of nineintervening sequences; the nucleotides at exon/intron boundaries conform with nuclear gene intronjunction sequences. Also typical for C. reinhardtiinuclear genes are the position and sequence of theputative polyadenylation signal. These findings

suggest that transcripts from linkage group XIX arelikely to be processed in the nucleus. The openreading frame, which displays weak but easilydetected Chlamydomonas codon bias, potentiallyencodes a protein similar to a membrane anchor forcytoskeletal proteins. The observation that ex-pression of this gene is regulated during interphaseand in gametes is not consistent with the hypothesisthat linkage group XIX may be expressed onlyduring mitotic and meiotic processes.

Key words: uni linkage group, basal body DNA, flagellarregeneration, Chlamydomonas reinhardtii.

Introduction

Linkage group XIX of Chlamydomonas reinhardtii hasthree unusual characteristics: it is genetically circular,displays altered recombinational properties and everylocus identified by mutation to date affects processesinvolving microtubules (Ramanis and Luck, 1986;reviewed by Dutcher, 1989). Hall and coworkers (1989)presented evidence that linkage group XIX is physicallylocated at the basal bodies, renewing interest in the notionthat these structures might contain their own genome (cf.Wheatley, 1982). Furthermore, the DNA was detectedonly at the basal bodies and not in the nucleus (Hall et al.1989). If correct, these conclusions raise many questions,including several concerning the mechanisms by whichgenes of this linkage group might be transcribed and theRNA processed.

During an ongoing project to place molecular markerson the genetic map of C. reinhardtii (Ranum et al. 1988),the gene corresponding to cDNA clone pcf9-26 wasassigned to linkage group XIX (Ranum, 1989). This clonewas originally selected by differentially screening a cDNAlibrary for mRNAs that accumulate during flagellarregeneration (Schloss et al. 1984). The mRNA is enrichedin the polyadenylated fraction of total cellular RNA,prompting us to ask whether a typical C. reinhardtiinuclear polyadenylation signal was used for this gene and,furthermore, whether splicing would be required fortranscript processing. Knowledge of the structure of thisgene would also contribute to studies in our laboratory on

Journal of Cell Science 100, 877-881 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

the regulation of flagellar gene expression. We thereforeisolated genomic DNA clones and additional cDNA clonesfor this mRNA, and determined their nucleotide se-quences.

Materials and methods

Plasmid pcffl-26 DNA (Schloss et al. 1984) was nick translated andused to screen a genomic DNA library as described previously(Schloss, 1990). Three different clones were purified and restric-tion endonuclease cleavage site mapping demonstrated that eachcontained similar and mostly overlapping regions of the genome.One clone, A9-26A, containing a 13.8 kilobase (kb) segment ofalgal DNA, was analyzed further. A 3.8 kb Kpnl fragment fromthe center of the insert was shown to contain the gene of interestby hybridizing the cloned cDNA to Southern blots containingfragments (generated by restriction enzyme digestion) of thecloned genomic DNA, and by hybridizing DNA fragments isolatedfrom the genomic clone to Northern blots containing RNA fromcontrol or deflagellated Chlamydomonas cell cultures (data notshown). The available cDNA clone was clearly not full length,containing a 0.9 kb insert that hybridized to an RNA ofapproximately 2 kb (Schloss et al. 1984). Therefore, the 3.8 kbgenomic DNA fragment was used to screen C. reinhardtii gametecDNA libraries in Agtll (Adair and Apt, 1990), and severaladditional cDNA clones were identified. The DNA sequences ofthe entire 3.8 kb genomic DNA fragment (both strands) and of asubset of the cDNA clones, were determined by the chaintermination method (Sanger et al. 1977) using Sequenase™ kits(United States Biochemical) as described earlier (Schloss, 1990).The only discrepancies between cDNA clones were in the precise

877

position of the poly(A) tail, which began 13 (one clone), 14 (oneclone) or 15 (two clones) nucleotides past the putative polyadenyl-ation signal. Sequence data were analyzed using IBI Pustellsequence analysis software (International Biotechnologies, Inc.)and the Genbank FASTA Server.

Results and discussion

Gene structureThe structure of the gene corresponding to cDNA clonepcf9-26, which maps to linkage group XIX, appears to bethat of a typical C. reinhardtii gene. The gene contains atleast nine intervening sequences that are flanked byconsensus splice junction sequences (Fig. 1). If any featureof the gene structure is remarkable, it is the large numberof introns (the largest number reported to date for anygene in this organism). A putative polyadenylation signalTGTAA (Silflow et al. 1985) occurs 12-14 nucleotidesupstream of the polyadenylation site (Fig. 2). The lengthof the 3' untranslated region (426 nucleotides) is typicalfor nuclear genes of this organism. The 3.8 kb genesequence is 60% G+C (the introns, total cDNA andtranslated regions are 59%, 60% and 64% G+C, respect-ively), reflecting the base composition of C. reinhardtiigenomic DNA (63 %; Chiang and Sueoka, 1967). On thebasis of this gene structure analysis, this RNA is mostlikely to be spliced and polyadenylated in the nucleus.

The cDNA sequence is 1987 nucleotides long, excludingthe poly(A) tail. We estimate from Northern blots that themRNA is 2 kb long and thus believe that the longest cDNAclone is virtually full length. However, we have notconducted 5' end mapping experiments, and thereforecannot exclude the possibilities that the 5' untranslatedregion may be slightly longer or that another intron mightexist.

Predicted protein productThe major open reading frame is 432 codons long. Thispredicted translation product uses as the initiator codonthe first AUG (at position 466 of the gene sequence) that isin frame with the longest potential open reading frame.We selected this AUG for several reasons. Firstly, thecalculated molecular weight of the predicted polypeptide is46 860, in agreement with preliminary mRNA hybridselection and translation experiments that reveal a faint

TCGATG/gtaogg oag/GCTACC INTRON 1

GTAGCG/gtgagt aag/GTGCCG INTRON 2

TGTCAA/gtgggt cag/GGGCGC INTRON 3

CAGCCA/gtgagc cag/TCGACC INTRON 4

CCCGAG/gtacga cag/CTGCAC INTRON 5

GAGAAG/gtacgg cag/GTGCTG INTRON 6

AGGGCG/gtgagt oag/GGACCC INTRON 7

TCAAGG/gtgagt cag/GCAAGT INTRON 8

ACACAG/gtgcgg cag/CGATGC INTRON 9

Fig. 1. Exon/intron boundaries of a gene on linkage groupXIX. Boundaries are shown for nine intervening sequences.Exon nucleotides are uppercase, intron nucleotide arelowercase, and each junction position is shown with a slash.Nucleotides that match the C. reinhardtii consensus sequences(Zimmer et al. 1988) are in bold type.

band at 45 000 molecular weight (J. A. Schloss, unpub-lished observations). Secondly, this AUG has the essentialpurine at - 3 (Kozak, 1989); two upstream AUGs and thefirst two AUGs downstream of position 466 have pyrim-idines at —3. The AUG at 1091 matches more closely theinitiator codon context we have compiled for Chlamy-domonas mRNA sequences, with an A at —3 and G at +4.However, the subsequent open reading frame is relativelyshort (135 codons) and exhibits none of the bias generallyobserved in Chlamydomonas open reading frames. Thelong open reading frame we have identified exhibits weakChlamydomonas codon bias. For example, the percentageof codons with A in the third position is 0.1 % for the fourtubulin genes, that display strong bias (Youngblom et al.1984; Silflow et al. 1985), 8% for the arylsulfatase gene,that displays the weakest bias reported to date (de Hostoset al. 1989), and 9% for this 9-26 gene.

The predicted protein product of this open reading frame(hereafter referred to as the linkage group XIX openreading frame [Igl9orf]) is not readily identified bycomparison (Pearson and Lipman, 1988) with entries inthe GenPept or Swiss-Prot (releases 64.3 and 18) data-bases. The best match is to the Drosophila segmentpolarity protein armadillo (Riggleman et al. 1989). Thearm protein contains 13 repeated segments, each 42 aminoacids long. The region of Igl9orf from amino acid residue182 to 345 can be aligned either with arm protein repeatsegments # l - # 4 or #3 -#6 (Fig. 3), and each of thesealignments yields a similar score: about 20 % identity and50 % similarity (sum of identities and conservative aminoacid changes). The regions of these polypeptides that lieN-terminal to the repeat domains are nearly 50 % similar.Some general characteristics of the polypeptide structuresare also conserved: the N-terminal domain of eachpolypeptide is acidic and hydrophilic, and the centralregion (which is much smaller in the algal polypeptide) isbasic and hydrophobic (cf. Riggleman et al. 1989). TheC-terminal domains are both acidic, although this regionis hydrophobic in the algal and hydrophilic in the flyprotein. We also note that introns 4, 6 and 7 are eachlocated a few amino acids past the positions in the Igl9orfproduct with which the beginning of the repeat segmentsalign, consistent with the concept that repeated proteinsegments may have arisen by duplication of an ancestralDNA segment, with subsequent maintenance of intronpositions in the Chlamydomonas gene.

Armadillo is the fly homolog of mammalian plakoglobin(Peifer and Wieschaus, 1990; Franke et al. 1989), acomponent of cell membrane-associated plaques in whichintermediate filaments and microfilaments are anchored(Cowin et al. 1986). The armadillo protein also localizes toplasma membranes and sometimes colocalizes with actinfilaments (Riggleman et al. 1990). This prompts theintriguing speculation that the Igl9orf product may alsobe a component of a membrane-associated cytoskeletalanchor. In this case the cytoskeletal component might beaxonemal outer doublet or central pair microtubules thatterminate in specific structures at the distal tip of theflagellar membrane (Dentler, 1980). Consistent with thisproposed identity is the observation that the abundance ofthis mRNA is 30- to 40-fold lower than that of tubulinmRNA (see below), as expected for an mRNA whoseproduct is present in a small region of the axoneme. Analternative interpretation, that the Igl9orf product isrelated to arm only as a consequence of the presence ofsimilar repeat segments, is not favored because thesimilarity between Igl9orf and arm is more apparent than

878 J. A. Schloss and H. B. Croom

11012013014011

5011260146701798019190195

1001114110112312011561301140115815011711601189170119218011901208200123021012201236230125624012682501275260130827013412801

290135530013101388320139633014303401350136013701

I I I I I I I I 1 Iggtacctaagcgtgcccaggtatgcacactacacgcggtatactactgagagtgcacggttttgaggcgtgcgtttgccgttgacttgccgtgctggcggcagcacacagctttgcattggacggcccacgcgccccgcccgctagcgtcgctttcatcgcgaagggaccggtagcaagccgacatagcccctggcgttc

MetPheIleGlyLeuCysArgGlyArgLysA3nLe5CGAAGTCAGCTTCCCTTTCCGAAACAGCGCCGCTT

uAspAlaValAlaProThrProSerThrSerSerLysGlySerProSerGlnAlaSerThrProAlaLysSerAlaSerLeuSerGluThrAlaProLeu

ThrValGlyProSerSerThrSerProMetAlaAlaAlaAlaGluAlaGluHisLeuPheAspLeuArgAlaHisGlySerValProGlyGlyArgCysMTGTATATCCAGTCTGGTGACACCAGCGACTTCGATGgtacgggggcgaagtcagggggggaatctaggttgcccatagggtagatactagcacacaagggetTyrIleGlnSerGlyAspThrSerAspPheA3pGctgtttgggaggactgtccggagataacagtcctggcacccatcgccactgacaccctacacgacgaaaccgtggccgcaaccgcagGCTACCTTATCGC

lyTyrLeuIleAljtgagtacttgtccagcggtcaggcttttggq

aAlaAlaGlyHi3ValIleGlnArgGlnThrProAspPheGluTyrValValAlagcccaaacatgggtgacaggctgtgacacgcatcaaaggtcactggtgtgcggcgtcccggtgtcctatgaagGTGCCGGAACGGCGAGCATGGCGGGAC

ValProGluArgArgAlaTrpArgAsp

LysAspGluProAspThrAsnLysHisAspProThrTyrSerGluGluValLeuAlaThrSerGlyAlaLeuLeuArgHisLeuCysProAspAlaMetLTTGTCAAgtgggtggacatgatgcgatgataggcgcgtgtatgggattgcgggctgaaggagatgccggagtgttggagctggtgaagggcacgcggggceuValLyccttgtcatggatgtggcagctgttccggcgtcgcacttaagctggaggaacgcaagccctcaacaggcagacggcactacagccccgcgcccgtgtgtgtaactgtaagcatgcacatggtggtgcaagttctctgagttgactatcgaaccacctgcagGGGCGCACTCAACCAGCGCAACATCATCCCCTGGAAGAT

sGlyAlaLeuAsnGlnArgAsnllelleProTrpLysMe

tMetPheAsnGluProGluAsnTyrGlyProLeuIleArgAspLeuProAlalacgatgcatggaagtgttgctgtgccaagcccctgcttgtggtaattgtatgtgtgatgaatgaagcggctctcttggattgtcgctgcagTCGACCCTC

leAspProAGGTGGAGTTCCCTGGAGCAGCTGGCGCAGCGCATCCTCAGCCCCGAGgtacgacatgcagtgcctgcgtagcgcctgctgtgttgttttggtctgtggttrgTrpSerSerLeuGluGlnLeuAlaGlnArglleLeuSerProGluacgacttatgtgacagccaggaccatgagctagccgaaatggaacgtgttgaggcacggctcaccatcctacagctgagctgtggcaaaggacctgacgacgccacccacattgacaccggctgccccgcagCTGCACTCGCTGGTGTTGGACATGAACGGCTCCATGGGCTACCTGGACGCACTGGTCAACCTGATCGG

LeuHisSerLeuValLeuAspMetA3nGlySerMetGlyTyrLeuA3pAlaLeuValAsnLeuIleGlGCCGGAGGGCGAGAAGgtacggcaacagagatagggagggcaatggggcctggcgagtggcgagaggttgttgcgggtttggcggggagagctgtgcaggyProGluGlyGluLysagggctgggcacattatgacatgacaatggagcagagtatgcgcatggggattgagttatcagttcttacaaatattagctgatgttcatgctaacgctccagcttgtgccatgctgtgctctactccgccgccgctcagGTGCTGGGCGCCAAGATGAAGGCGTCGGGACTGCCGCTGGTCATTATGGCGGGCGTGCAG

ValLeuGlyAlaLysMetLysAlaSerGlyLeuProLeuVallleMetAlaGlyValGlnGCTGAGGTGGTGGCACAGACGTTGCCGCTGCCAGGGCGgtgagttgccgacggcgctgacttatcgttcgtgtcactacggcacacacagtcccccatccAlaGluValValAlaGlnThrLeuProLeuProGlyAracgcccgtagctacctcgcccgccctctcacgctcttgccacgccctcctcccttgcttcttccccgctgcaccccgcagGGACCCCCGCGCCACCAAAA

gAspProArgAlaThrLysA:AACAACGTGTGCAACAAGCT

snAlalleTyrTyrProAlaAlaValArgValLeuLeuArgLeuAlaArgSerHisGlylleProLeuLeuPheValThrAsnAsnValCysAsnLysLe

uLeuLy3PheGlnA3pAlaProGluValAlaValLysLeuGlyLeuGlnGlyLeuLeuArgLysValGlyAspThrTrpPheSerLeuProTyrLeuLysGgtgagtgcggaggcgtgggtgctgcgggcttggctttggggatgcctgttactgttagtcagcacacacgtgcggcaggtggtccagggcaggggcgctGtgagcttgaagggctggtgggctgctgcgctcgctgtcttacgccgcccgctgcaccgcagGCAAGTGCGTGCCGTTCGACTGGGTGGCGTTCGCGGCCA

lyLysCysValProPheAspTrpValAlaPheAlaAlaMiCAAGGACGATCCCTCAGTGCTGGTGCTGCGGGACACAGc

etLeuLeuTyrGlyArgLysProAlaSerMetAlaLeuAlaHisGlnGluLeuTrpValGlyLysAspAspProSerValLeuValLeuArgAspThrAtgcggccggcggagtcgcgcgcacatgtgcatgcggtgtacgtctgggcgagctgaataggagagggcaaggcgagggtgaggtttcttcgctggggtgtggtgtccacataatgaaggcatgtgccaaaactccagcccacacacccgattgctaccctcgtgccattccgcagCGATGCCGGCGGATGACGTGGTAGC

laMetProAlaAspAspValValAlGCTCGCCAAGCACGCCTGCTCGCAC

aAsnAsnValAlaAsnThrGluArgTrpGlyValValGluSerValValGluIleAsnlleGluMetMetLeuGlnLeuAlaLysHisAlaCysSerHisACAGCCGTCTAGCThrAlaVal

TGGCCTTACCCAGCCAGCATGTAAGCGTCACCTTTCCGggtgctctccagcctctcctttaggtccgaggtacc

I I I I

Fig. 2. DNA and deduced protein sequences encoded by a gene on linkage group XIX. The sequence of a 3774 nucleotide-long Kpnlfragment is shown. The cDNA sequence is shown in upper case letters; flanking and intervening sequences are in lower caseletters. The putative polyadenylation signal is underlined. The polyadenylation sites (indicated with asterisks) were determinedfrom four independently derived cDNA clones; two end at nucleotide 3738, the third at 3737, and the fourth at 3736. The sequenceis available from the EMBL/Genbank/DDBJ databases under accession number X62135.

uni linkage group gene structure 879

Igl9orf

Dro arm

QASTPAKSASLSETAPLTVGPSSTSPMAAAAEAEHLFDLRAHGSVPGGRCMYIQSGDTSDFDGY 92. I I . . .:I. I . . I I . I I : I : : : I I . I : I I . : 7

MSYMPAQNRTMSHNNQY—NPPDLPPMVSAKE-QTL—MWQQNSYLGDSG—IHSGAVTQVPS- 56

I . .. : : : I : . I : . . .I. : I I . : . : : .. : I . :"::.. . . : . I I .Dro arm LSGKEDEEMEGDPLMFDLDTGFPQNFT-QDQVD-DMNQQLSQTRSQRV-RAAMFPETLEEGIEIPSTQFDPQQPTAVQ-RLSEPSQ—M 139

repeat #3 \/ repeat #4 \/Dro arm LLAI-FKSGGIPALV-KLLSSPVESVLFYAITTLHNL—LL-H-QDGSKMAV—RLAGGLQ-KMVTLLQRNNVKFLAIVTDCL-Q 310

III : . .: I : : I I . :.I::: .: I I: : I. I . . : : I : I. :I. : IIgl9orf LIRDLPAIDPRWSSLEQLAQRILSPELHSLVLDMNGSMGYLDALV-N-LIGPEGEK—VLGAKMKASGLPLVIMAGVQ-AEWAQTL-P 2 64

I : : T . : I I I : I I I I : I : : I : : : I I . T T : . . . I I . : I . :Dro arm LKHAWNL-INYQDDAELATRAI-PELIKLLNDEDQVWSQAAMMVHQLSKKEASRHAIMNSPQMVAALVRAISNSND-LESTKAAVGT 225

\ r e p e a t HI A r e p e a t #2_ I

repeat #5 \ / repeat #6Dro arm ILA-YGNQESKLIILASGGPNELVRIMRSYDEYKLLWTTSRV-LKVLSVCSSNKPAIVDA—GGMQALAMHLGN—MS-PRLVQNCL 390

: . . . . I I . . . . I : I : I I . I I I . . I I : I : I : I : I . I : I : I I I . I :I g l 9 o r f LPG-RDPRATKNAIYYPAAVRVLLRLARSHGI-PLLFVTNNVCNKLLKF—QDAPEVAVKL—GLQGLLRKVGDTWFSLPYLKGKCV 34 5

I ~ . I I I : . . . : I : : I I . : I I . : . I I I I . . : I I : I I I I : : : : : ~ I :Dro arm LHNLSHHRQGLLAIFKSGGIPALVKLLSSPVE-SVLFYAITTLHNLLLH—QDGSKMAVRLAGGLQKMVTLLQRNNVKFLAIVTDCL 309

\ r e p e a t #3 A r e p e a t #4 /

Fig. 3. A region (amino acids 29 to 345) of the deduced Igl9orf product is aligned with amino acids 140-309 of the Drosophila armpolypeptide (Peifer and Wieschaus, 1990). The arm sequence is shown below the Igl9orf sequence, and arm repeat segments # l - # 4are indicated. A second alignment (shown with the arm sequence above the Igl9orf sequence) demonstrates that arm repeatsegments #3-#6 and # l - # 4 align with the same region of the Igl9orf product. Vertical lines indicate amino acid identities, twodots indicate a PAM250 score +2 or greater, and one dot indicates a score of +1 (Dayhoff et al. 1978). Intron positions in theIgl9orf are shown by underlining: the intron interrupts the codon of single underlined amino acid residues, and separates codonswhen adjacent amino acid residues are underlined.

the similarity between repeated domains within theIgl9orf product.

Gene regulation in growing and differentiated cellsPreliminary characterization of pcf9-26 included thedemonstration that the corresponding mRNA accumu-lates in deflagellated gametic cells (Schloss et al. 1984).Fig. 4 confirms this observation and extends the analysisto vegetative cells, in which the mRNA is also expressedand induced approximately 5- to 10-fold upon deflagella-tion. We estimate that this mRNA is 30-fold lower inabundance than a-tubulin mRNA in deflagellatedgametes (based on quantitative RNA dot hybridizationsused to generate Figs 5 and 6 of Schloss et al. 1984), and40-fold lower than a--tubulin mRNA in deflagellatedvegetative cells (unpublished observations).

ConclusionThis is the first reported nucleotide sequence analysis of agene on linkage group XIX, and it raises several importantissues pertinent to the reported basal body localization ofthe DNA. The transcript from this gene is spliced andpolyadenylated. If the gene resides in basal bodies and notin the nucleus, then RNA transcription and processingenzymes would likely need to exist in basal bodies.Alternatively, Hall and coworkers (1989) proposed thatDNA of linkage group XIX might be quiescent duringinterphase, and replicated and expressed only duringmitotic replication and meiotic reorganization, when thebasal bodies themselves are reorganized (the cytologicallocation of basal body DNA at these times was notspecified). However, their proposal is inconsistent with theobservation that 9-26 mRNA accumulates during flagellarregeneration in experiments that are conducted in the

middle of interphase, and in gametes, which are differen-tiated cells not progressing through the cell cycle. Suchaccumulation requires either an increase in transcriptionrate, or continuous transcription accompanied by changesin mRNA turnover rate.

Two cytological studies have recently presented strongevidence against the presence of DNA in the C. reinhardtiibasal body (Johnson and Rosenbaum, 1990; Kuroiwa et al.1990). While this manuscript was in preparation, Johnsonand Dutcher (1991) reported molecular evidence againstthe presence of linkage group XIX DNA in the basal body.They showed that the copy number per cell of linkagegroup XIX and nuclear linkage groups is the same (thenuclear genome is haploid, but each cell has two basalbodies), and that this remains true in mutant strainslacking basal bodies. The present study does not directlyaddress the cytological location of linkage group XIXDNA, but places constraints on the cellular machinerythat must be able to act on that DNA and its product, andon the timing when that machinery must act. These dataneed to be incorporated into any future evaluation ofpotential relationships between linkage group XIX andthe presence or function of basal body DNA.

We thank L. Ranum, P. Lefebvre and C. Silflow for communicat-ing the results of RFLP mapping studies, M. Gillette for therestriction map of genomic clone A9-26B, S. Adair and S.Waffenschmidt for supplying cDNA libraries and P. Lefebvre andC. Silflow for helpful comments on the manuscript. A. Bhatprovided excellent technical assistance. H.B.C. was a Fellow ofthe Faculty Scholars Program, University of Kentucky. Thisresearch was also supported by NIH grant GM-34837; BRSG S07RR07114-21 awarded by the Biomedical Research Support GrantProgram, Division of Research Resources, NIH; and MajorEquipment grants from the University of Kentucky GraduateSchool to J.A.S.

880 J. A. Schloss and H. B. Croom

_ A

B

DC

NDF 20 40 60 80 100 120Time after deflagellation (min)

Fig. 4. Gene expression during flagellar regeneration. Totalcell RNA was isolated from gametes or vegetative cells beforeand at various times after deflagellation and analyzed by RNAblot and RNA dot hybridization as described previously(Schloss et al. 1984). Vegetative cells were used 6 h after thedark/light transition and gametes were used after 18 h incontinuous light. The probe was nick translated cDNA clonepcf9-26. (A) Northern blots were prepared using 5 fig of RNAisolated from gametes before deflagellation (a), and at 15 (b),35 (c), 55 (d), 85 (e), 120 (f) and 160 min (g) after deflagellation,and from vegetative cells before (h) and 45 min (i) afterdeflagellation. Assay conditions were different for the gameteand vegetative cell RNAs, so band intensities are notcomparable. The relative migrations of denatured size markerDNA fragments are indicated to the left (lane s; from top tobottom, fragments are 2936, 2293, 2069, 1631 and 1426nucleotides long). (B) Dot blots were loaded with duplicate 8 ngsamples of total vegetative cell RNA isolated from cells beforedeflagellation (j) and 10 (k), 25 (1), 40 (m), 60 (n), 90 (o) and120 min (p) after deflagellation. (C) Relative abundance wasdetermined by scintillation counting to quantify hybridizationintensity from the dot blot shown in panel (B). This set ofexperiments demonstrates that the mRNA from a gene onlinkage group XIX is expressed in both vegetative cells andgametes, and accumulates transiently during flagellarregeneration.

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(Received 25 July 1991 - Accepted 30 August 1991)

uni linkage group gene structure 881