Gene. 91 (1990) 123426

Elsevier

123

GENE 03576

Cloning and nucleotide sequence of the CM’23 gene of Succhuromyces cereuisiw (Recombinant DNA; yeast spheroplasts; G2 phase; mitosis; cell cycle gene; sequence motif; repeated structure)

Akemi Doi and KenjiDoi Instituteof Scientificand Industriil Research, Osaka UniversQ. 8-l Mihogaoka,Ibaraki. Osaka 567 (Japan) Received by J. Manuur:2 January 1990 Accepted: 5 February1990

SUMMARY

As a preliminary step for studying the function and intracelhrlar behavior of the product of the CDC23 gene, a cell cycle gene of Succhuromycescerewidze,we cloned the CDC23 gene and determined its nucleotide (nt) sequence. The nt sequence contains an open reading frame (ORF) of 1878nt coding for a protein of 626 amino acids (aa). The CDC23gene is expressed as a 2.4- to 25kb major transcript. The putative protein product of the CDC23 gene has a potential Ca2+-binding site near its N terminus, and has four contiguous repeats of a consensus sequence consisting of 34 aa near its C terminus. Regions that can be regarded as consisting of repeats of the same consensus sequence were found in the published aa sequences of the products of three other yeast genes.

INTRODUCTION

In contrast to that of normal cells, progress through the G2 phase of the mitotic cycle of spheroplasts of wt strains of baker’s yeast, S. cereui&re, in liquid culture is significantly affected by reduction in nutrient concentrations of culture medium (Doi and Doi, 1982).We are interested in identification of G2 events that may be retarded in the spheroplast cell placed in medium of low nutrient strength, because they might represent G2 events of the normal cells’ mitotic cycle that are highly sensitive to potential changes in metabolite concentrations within the cell. One approach to identifying the proteins involved in these events might be

Correspondence to: Dr. K. Doi, Institute of Scientific and Industrial Research,OsakaUniversity,8-1 Mihogaoka,Ibaraki,Osaka 567 (Japan) Tel. (06) 877-S111; Fax (06)877-4977.

to search for candidates among the products of the CDC genes and study the functions and intracellularbehaviors of the candidates. The product of the CDC23gene (Hartwell and Smith, 1985) seems to be such a candidate,judging from the mitotic behaviorof spheroplastsof a ts mutant (A.D. and K.D., unpublished results). This gene product, however, has not been characterized biochemically, although it has been implicated in spindle elongation or chromosomesegregationin mitoticanaphase(Hartwelland Smith, 1985).The aimof the presentstudywas to clone the CDC23 gene, determineits nt sequence, and deduce the primarystructure of the CDC23 gene product, as a preliminarystep for characterizing the CDC23 gene product biochemically.

EXPERIMENTALAND DISCUSSION

Cloning of the CDC23 gene The CDC23gene was cloned on a 3.7-kbXhoI-BamHI fragment of chromosomal DNA from strain D22 (X2180-1A x X2 180-1B) ligated between the Sal1 and the BumHI sites of YRp7 to give pYX35 (Fig. 1). When cut

(a) Abbreviations:aa, amino acid(s); bp, base pair(s); COC, cell division cycle (gene); dCIPaS, deoxycytidine 5’-a-thiotriphosphate;ds, double strand(ed); kb, kiiobase(s) or 1000bp; nt, nucleotide(s); ORF, open reading Frame;S., Saccharomyces; SC., Schizosaccharomyces; ss, single strand(ed);fs, thermosensitive;wt, wild type. 0378-l119/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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121 GcAGG1.cTTGc~GAAGcT~TTGATG1.TGATcAAA~AcAcTCTTTAG~cGATGAATcGC~A~TAAGA~ATkAAcAAGGTGT~CcGAAAcAGATGTTTGAAAT~CAcAAA~GGGTTTGGC 414 G L A E A I D V D Q T H S L A DES P L R N K Q G V P KQf~FE I P Q N G F G 241 CTA•CAGAG•CTGAGTATGACCTGTACCTCCTTGGTTCT•CGTTGTTTGATGCT•A•GAGTTTGATCGATGCGTTTTTTTTCTAAA•GATGTCA¢TAATCCATACCTTAAGTTCTT•A•• 8 I L S E T E Y DL V L L G S T L FD AK E F D R C V F FL K D V T N PY L K FLK

361 TT~TA~AGTAAATTTCTAT~GTGGGATA~GA~G~CA~G~AAGT~TGG~A~TATCTT~A~TAC~GGGAAGTTT~GACGA~ATGTACAGAGCT~Ck~GATGGGG~TGGT~GTGGG 121 L ¥ S K F L S V I) K K S Q ( S .'I ( N ! L T T G K F T 0 E 'I ¥ It A N K 8 G 0 G S . G

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Fig. 1. Restriction map ore CDC23 clone, nt sequence of'the CDC23 gone and adjacent regions and deduced aa sequence of the CDC23 gone product. A partial $ou3A library ofgenomic DNA of'strain D22 (X2180-1A x X2180-1B) on YRp7 was constructed as described (Nasmyth and Reed, 1980) and screened For plasmids with the activity of complementing the fs defect of a recipient strain !-12'/8 (a cdc23. I ,,rp] kb3) derived from strain fs9026 (Yeast Genetic Stock Center, Berkeley, CA,). A 3.'/-kb Xkol.BamHl fra~p~entderived from one such plasmid was ligated between the $~11 and the BamHl sites of YRp'/to give pYX3S, that retained the complementation activity. Yeast and bacterial transformations and manipulations of nucleic acids were conducted as described (Maniatis et el., 1982; Nasmyth and Reed, 1980; Sherman et al., 1983). Restriction sites are: AI, AJ/ll; Bin, BomHI; Ho, ~col; Np, NspV; Pt, Pstl; Se, Spel; Xa, XbaI; Xo, Xhol. Open bar, D22 sequence; hatched bar, YRp7 sequence. The 2.6-kb Pstl.Xbal fragment was used as the template for preparing a aSS-labeled probe using the Amersham rapid hybridization system-muitiprime (Amersham, UK) that hybridized to unique fragments or fragments ofthe expected sizes on Southern blots ofgenomic DHAs from congenic wt strains X2180-IA and X2180-I B digested with BamHI, Pstl, Xbal or Xkol, or their combinations, pYX35 linearized by Xkol cut was used to transform strain H2'/8 to give a stable integrant (On-Weaver etal., 1981), that was crossed with laboratory strains and subjected to tetrad analysis (Sherman et al., 1983) to confirm a tight link of its cdc2J + and trpl + phenotypes to the cdc23 locus. Subfragments ofpYX35 were cloned into pUC118 and pUC119 (Vieira and Messing, 198'/), serially deleted and sequenced using the dideoxy termination method (Biggin et al., 1983) with [0~.3SS]dCTP0~S (Amersham). The nt sequence of the 2.6-kb Spel-Xbal fragment (sequenced completely for both strands) is shown. The nt sequence data have been submitted to DDBJ, EMBL and GenBank and assigned the accession number D90081. The predicted aa sequence of the CDC2~ gene product is written below the nt sequence. A putative promoter sequence (nt -182 to -168, -112 to -109; Dobson etal., 1982) is overlined. A candidate for the Bennetzen and Hall (1982) sequence (nt 2282-2287) and two candidates for the Zaret and Sherman (1982) sequence (nt 1899-1901, 1919-1922, 1954-1956, and nt 1948-1950, 1997-2000, 2028-2030) are underlined. A putative Ca2 +-binding site (as 153-161) is underlined by a broken line. Four contiguous sequences each consisting of 34 aa (aa 399-432, 433-466, 467-500, 501-534) are indicated by horizontal arrows. The black-arrowed bar below the restriction map represents the protein-coding region and the direction of transcription. Lines and an arrow below the protein-coding region indicate, respectively, ds fragments and an ss fragment (with the arrow indicating its 5'-to-3' direction) cloned on pUC119( + ) strand, used as templates for preparing 3aS-labeled probes using the Amersham kit that hybridized to a 2.4- to 2.5-kb transcript on Northern blots of total RNA from logarithmically growing cells of X2180-1A.

125

with Xhol and integrated into the chromosomal DNA of a recipient strain by homologous recombination (Orr-Weaver et al., 1981), the plasmid DNA was found by tetrad analysis to lie close (,: 1.5 cM) to the cdc23 locus, conferming the authenticity of the cloned gene. Southern hybridizations showed that unique equivalents of this 3.7-kb fragment are present in the genomes of strains X2180- IA and X2180-1B, suggesting strongly that no gross rearrangement of the DNA structure occurred during cloning of the fragment (data not shown). Deletion analysis showed that the activity of complementing the cdc23-1 defect lies in a 2.l-kb SpelNco l fragment.

(b) Nucleotide sequence of the CDC23 gene The nt sequence of a 2.6-kb SpeI-Xbal fragment encompassing the CDC23 gene was determined (Fig. 1), and was found to contain an ORF of 1878 nt that is of significant length and overlaps the SpeI-Ncol fragment exhibiting the activity of complementing the cdc23-1 defect. The ORF starts at an ATG codon that lies in a favorable environment (Kozak, 1984), and the 5' and 3' sequences flanking the ORF contain the ones believed to be characteristic of yeast promoters (Dobson et al., 1982) and terminators (Bennetzen and Hall, 1982; Zaret and Sherman, 1982) (Fig. 1). The ORF does not contain the consensus sequence for splicing mRNA of S. cerevbiae (Langford and Gallwitz, 1983). We conclude that this ORF represents the coding sequence for the CDC23 gene product. Total RNA from the logarithmically growing cells of strain X2180- IA contained a molecular species 2.4 to 2.5 kb in size that hybridized to probes (including a strand-specific one) covering the coding region of the CDC23 gene, suggesting strongly that the molecular species is a major transcript of the CDC23 gene (data not shown). (c) Primary structure of the CDC23 gem product The putative CDC23 gene product (Fig. 1), that consists of 626 aa, is an acidic protein, but has a slightly basic region near the C terminus. Near its N terminus there is a sequence (NKDGDGSGN) homologous to the Ca 2 +-binding sites of known Ca2+-binding proteins (Kretsinger, 1980; Vyas et al., 1987; Takada and Helmer, 1989), suggesting that the CD¢23 gene product might be involved in a Ca 2+modulated process (Kretsinger, 1980). Visual inspection of the slightly basic region revealed that it contains four contiguous sequences each consisting of 34 aa that are homologous to one another in the positions of several conserved aa rather than in the overall aa sequence. Thus, this region can be said to contain four repeats of a consensus sequence defined by these conserved aa (Fig. 2). The CDC23 gene product did not show close sequence homology to any known protein when examined on a protein sequence database of the Protein Research Foundation, PRF/SEQDB,

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XWXXLGXXYXXXXXXXXAXXXYXRAXXLXPXXXX COIl Fig. 2. Repetitive aa sequences of four yeast gene products. The aa sequences of the products of the CDC23 gene (aa 399-$34)° the CDCI6 gene (aa 533-'/43) and the SSN6 8ene (aa 48-399) of S. cerevbiae, and of the nut2 + 8ene (aa 433-$68) of Sc. pombe are written in blocks of 34 to 41 aa so as to align the periodically occurring L(1)-G(A)-X2-Y(F) sequences and homologous ones and to place the highly conserved Gly (Ala) residue at the 6th position in each block, The consensus sequence (Con) represents the positions of aa conserved in the CDC23 sequences and in the nuc2 + sequencesas well. Positions of the conserved aa in the consensus sequence are indicated by vertical lines, The letters in the consensus sequence should be read as follows: A, Ala or Set; G, Gly or Ala; L, Leu, lie, Val or Met; P, frequently Pro; R, At8 or Lys; W, frequently Trp; X, unconserved residue; Y, Tyr or Phe.Asterisks indicate that the sequences of the first 34 aa in the blocks deviated from the consensus sequence by insertions or extensive replacements.

but the same examination revealed that the published aa sequences of the nuc2 ÷ gene product of Sc. pombe (Hirano et al., 1988), the CDCl6 gene product (Icho and Wickner, 1987) and the 55N6 gene product (Schultz and Carlson, 1987) of S. cererbiae contain regions that can be regarded as consisting of contiguous or nearly contiguous repeats of this consensus sequence (Fig. 2). Thus, the 21 sequences each consisting of 34 aa in these four yeast gene products (excluding the sequences marked by asterisks in Fig. 2) are of the form X4-L(1)-G(A)-X2-Y(F)-XsA(S).X3.Y(F)-X-R(K).A(S)-X2-L(1)-Xz, or of homologous or slightly divergent forms. The highly conserved hydrophobic aa in these sequences might play a role in folding them individually into structures similar in conformation. In analogy with the functions of some proteins with repetitive structures (Lewis et al., 1988; Miller et al.,

126 1985), we speculate that the regions in question ofthese four yeast-gene products might constitute binding sites for a specific class ofmacromolecules. DNA may be a candidate for such macromolecular class, because, apart from that of the CDC23 gene product, the functions of the other three yeast gene products have been discussed in the context of their possible binding to DNA (Hirano et al., 1988; Icho and Wickner, 1987; Schultz and Carlson, 1987).

ACKNOWLEDGEMENTS

We thank Dr. Yasuhiko Seto (Peptide Institute, Minoh, Osaka) for assistance with aa sequence comparisons and homology searches and for helpful discussions.

REFERENCES Bennetzen, J.L. and Hall, B.D.: The primary structure of the $acc.~aromyces ¢erevbiae gene for alcohol dehydrogenase I. J. Biol. Chem. 257 (1982) 3018-3025. Bi88in,M.D., Gibson, TJ. and I-long, G.F.: Buffergradient gels and 3sS label as an aid to rapid sequence determination. Proc. Natl. Acad. Sci. USA. 80 (1983) 3963-3965. Dobson, M.J., Tuite, M.F., Roberts, N.A., Kingsman, A.J. and Kingsman, S.M.: Conservation of high efficiency promoter s(:quences in Sacckaromyces cerevblae. Nucleic Acids Res. 10 (1982) 2625-2637. Doi, A. and Doi, K.: Progress in the mitotic cycle of yeast spheroplasts in liquid culture. Exp. Cell Reg. 137 (1982) 95-110. Hartwell, L.H. and Smith, D.: Altered fidelity of mitotic chromosome transmission in cell cycle mutants of Sacckammyces cerevbtae. Genetics 110 (1985) 381-395. Hirano, T., Hiraoka, Y. and Yanagida, M.: A temperature-sensitive mutation ofthe $chi:osaccharomycespombegene nuc2 + that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase. J, Cell Biol. 106 (1988) 1171-1183. Icho, I". mtd Wickner, R.B,: Metai-binding, nucleic acid-binding finger

sequences in the CDCI6 gene of Sacckaromyces ceret~siae. Nucleic Acids Res. 15 (1987) 8439-8450. Kozak, M.: Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 12 (1984) 857-872. Kretsinger, R.H.: Structure and evolution of calcium-modulated proteins. CRC Crit. Rev. Biochem. 8 (1980) 119-174.

Langford, C.J. and Gallwitz, D.: Evidence for an intron-contained sequence required for the splicing ofyeast RNA polymerase I! transcripts. Cell 33 (1983) 519-527. Lewis, S.A., Wang, D. and Cowan, NJ.: Microtubule-associated protein MAP2 shares a microtubule binding motif with tan protein. Science 242 (1988) 936-939. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Miller, J., McLachlan, A.D. and Klug, A.: Repetitive zinc-binding domains in the protein transcription factor IliA from Xenopus oocytes. EMBO J. 4 (1985) 1609-1614. Nasmyth, K.A. and Reed, S.I.: Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc. Natl. Acad. Sci. USA. 77 (1980) 2119-2123. On-Weaver, T.L., Szostak, J.W. and Rothstein, RJ.: Yeast transformation: a model system for the study of recombination. Proc. Natl. Acad. Sci. USA. 78 (1981) 6354-6358. Schultz, J. and Carlson, M.: Molecular analysis of 8$N6, a gene functionally related to the SNFI protein kinase of Saccharomyces cure. vb[ae. Mol. Cell. Biol. 7 (1987) 3637-3645. Sherman, F., Fink, G.R. and Hicks, J.B.: Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1983. Takada, Y. and Helmet, M.E.: The primary structure of the VLA-2/ collagen receptor ~ subunit (platelet 8pla): homology to other integrins and the presence of a possible collagen-bindingdomain. J. Cell Biol. 109 (1989) 397-407. Vieira, J. and Messing, J.: Production of single-stranded plasmid DNA. Methods Enzymol, !$3 (1987) 3-11. Vyas, N.K., Vyas, M.N. and Quioeho, F.A.: A novel calcium binding site in the galactose-bindin8 protein of bacterial transport and chemo. taxis. Nature 327 (1987) 635-638. Zaret, K.S. and Sherman, F.: DNA sequence required for efficient transcription termination in yeast. Cell 28 (1982) 563-573.

NOTE ADDED IN PROOF

After this paper was accepted for publication, we noticed the following two reports (Hirano et al., 1990; Sikorski et al., 1990) that describe the repeats of 34 aa in several yeast proteins including the CDC23 gene product. Our nt sequence data for the 2.6-kb Spel-XbaI fragment encompassing the CDC23 8ene are identical to those described in one of the reports (Sikorski et al., 1990).

Hirano, T., Kinoshita, N., Morikawa, K. and Yanagida, M.: Snap helix with knob and hole: essential repeats in $.pombe nuclear protein nuc2 ÷. Cell 60 (1990) 319-328.

Sikorski, R.S., Boguski, M.S., Goebl, M. and Hieter, P.: A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis.