YEAST

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7: 265-273 (1991)

CDCI.5, An Essential Cell Cycle Gene in Saccharomyces cerevisiae, Encodes a Protein Kinase Domain BERT SCHWEITZER* A N D PETER PHILIPPSEN liisiitut,fir Mikro- utid MolekularbioloRie der Cinivcwitut Giessen, Frankfurter Strosw 107. 0-6300 Giessen. F. R.G.

Received I 1 July 1990; revised 23 October 1990

The cell division cycle gene CDC1.5 is essential for the latc nuclear division in thc ycast Sacchuroinyces cererisiae. Thc amino acid sequence of the 974 amino aciddl 10 kDa C'DCl.5 gene product, as deduced from the nucleotidc scqucnce, includes an aminoterminal protein kinase domain which contains a primary sequence mosaic showing patterns spccific for protein serine/threoninc kinases besides those for protein tyrosine kinases. Many protein kinases non-essential for growth arc known. CDClS reprcscnts an csscntial protein kinasc like CDC7 and CDC28. A carboxyterminal deletion of 32 amino acids renders the protein inactive. KI:Y

w o m s - cell division cycle; Saccharomyws cerevisiae; c'DC15; protein kinase.

1NTRODUCTION Nuclear division in Saccharomyces cerevisiae depends on the function of several cell division cycle genes. of which CDCI4 and CDCI.5, and possibly C'DC'27 and CDC30, affect the late phase including chromosome separation and nuclear reorganization (Pringle and Hartwell. 1981). Heat-sensitive cdcl5 mutants arrest upon shift to restrictive temperature (36 C ) in the first cycle as singly budded, uninucleate. growing cells (Culotti and Hartwell, 1971) with complete, long spindles reaching to the extreme ends of the elongate nucleus (Byers and Goetsch, 1974). The CDCI.5 gene function is not needed for DNA synthesis (Hartwell. 1973. 1976; Wood and Hartwell. 1982). and cdc1.5 mutants sporulate at restrictive temperature, but a small percentage of eight-spore asci have been observed (Hirschberg and Simchen. 1977). A more detailed knowledge of the CDCIS gene function should be facilitated by the isolation and analysis of this gene. CDCIS is situated on the right arm of chromosome I . 6 centimorgans (cM) away from the centromere. and 2 cM centromere-distal from ADEl (Mortimer cf ul., 1989). Large cloned regions of chromosome I have been physically mapped and clones containing the CDCIS gene identified by complementation of the cdc1.5-1 allele (Steensma r f NI., 1987). We obtained two overlapping fragments *To whom corrcspondence should be addressed 0749 503X 91 03026509 505.00 1991 b) John Wiley & Sons Ltd

containing the CDCl.5 gene and report here the nucleotide sequence of CDCl.5 together with analyses of carboxyterminal deletions of the gene product. MATERIALS A N D METHODS Bucreriul struins Bacterial strains HBlOl (F-, L - ,ara-14, galK-2, hsdS20 ( r e - , mB-), IucYI, mil-1, proA2. recA13, rpsL20 (srrR),supE44, q 1 5 ) and XLI-Blue (endA1. gyrA96. h s d R 17, lac- . recA 1 , reIA 1 , supE44. [hi- 1. [F' lacIQ, lacZAMl5. proA+B+. TnlO ( r e f R ) ] ) were used as hosts for plasmid constructions. Bacterial media and general methods were as described by Sambrook et al. (1989).

Yeast strains S . cerevisiae media and general methods have been described by Sherman et al. (1986). Media components were from Difco or Gibco. S. crrevisiue transformation was performed by using the lithium acetate procedure (Ito ef ul., 1983) or the spheroplast method (Sherman c't ul., 1986). The S. cerevisiae strains 206-14B ( M A T a , cdcl.5-1. adel, adc2. tjv-1. lys2-2) and DK329-4D ( M A T a , cdcI.5-I, udrl-I. hi.s3-11,15, leu2-3,112, trpl-I, ura3-1) were gifts from G. Simchen and D. Kaback, respectively.

266 Phage und Plasmids The M 13 derivative R408 (Russel et ul., 1986) was used as helper phage to obtain single stranded DNA from Bluescript plasmids. The vector pBluescript I1 S K + (Short et al., 1988) was from Stratagene (La Jolla. CA. U.S.A.), pRS314 and pRS316 (Sikorski and Hieter, 1989) from R. Sikorski, and pJS92 (pBluescript KS+, TRPl, 2) from J. Shero. The vectors pRS416(SK) and pJS92(SK) were constructed by replacing the PvuII fragment of pRS3 16 containing the multiple cloning site with the corresponding fragment from pBluescript I1 SK +. Analogously, pJS92(SK) was constructed from pJS92. Plasmids YCpSO(CDCl5)I IB and YCpSO(ADE1CDC15)7A were gifts from D. Kaback and Y. Steensma. Enzymes Restriction enzymes, DNA polymerases, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, U.S.A.). Standard recombinant DNA techniques were used (Sambrook et al., 1989). DNA seyuencinx

For sequencing, series of unidirectional deletions were obtained from fragments cloned in pBluescript I1 using exonuclease 111. From the resulting plasmids, single stranded DNA was made using helper phage R408. Sequencing was done according to the Sanger dideoxy method (Sanger et ul., 1977) using Sequenase (Tabor and Richardson, 1987; obtained from USB, Cleveland, OH, U.S.A.). Compuler methods DNA sequences were entered using DSI, a C program allowing keyboard input via redefined keys (B. Schweitzer, unpublished). Database searches were performed with FASTP and the evaluation of the results with R D F (Lipman and Pearson. 1985). Sequence analyses were carried out with the UWGCG package (Devereux ef al., 1984). RESULTS Localization oj'CDC 15 The partially overlapping plasmids YCp50 (CDCI5)I 1 B and YCp50(ADEl-CDC15)7A con-

taining fragments from chromosome I had been

B. SCHWEITZER AND P. PHILIPPSEN

shown to complement the cdcl5-1-allele and to contain the bona.fide CDCI5 wild type gene (Steensma et al., 1987). For orientation, a map is shown in Figure Ib. In a first step we isolated a 7.6 kb fragment from a partial HindIII digest of YCp50 (CDCIJ)1 1 B corresponding approximately to the region common to both plasmids. This partial HindIII fragment was subcloned into the Hind111 site of pRS316 and the resulting plasmid pBSl was shown to retain the complementing activity. Following a detailed restriction analysis of pBSI. a 4.05 kb PvuII fragment was cloned into the Smal site of pRS416(SK) and the resulting plasmid pBSY could complement the cdcl5 defect fully, too. The complementing activity of plasmids pBSl and pBS9 was shown to be plasmid-coupled by stability tests. D N A sequence For sequencing, the ends of a 6.2 kb Cia1 fragment from pBSl (one ClaI site genomic, one from polylinker) were rendered blunt with Klenow polymerase, cloned into the EcoRV site of pBluescript I1 SK + in both orientations, and after cutting with ClaI and KpnI a series of unidirectional deletions were obtained from these plasmids using exonuclease 111. From the resulting plasmids, singlestranded DNA was made using helper phage R408. Sequencing was done according to the Sanger dideoxy method using modified T7 DNA polymerase. Both strands of DNA contained within the PvuII fragment were sequenced as shown in Figure 1 b. The DNA sequence of the 4.05 kb PvuII fragment is shown in Figure 2. In this region, one large open reading frame (ORF) of 2922 bp was found which extends from position 401 to 3331 and which is capable of encoding a protein with 974 amino acids and a calculated molecular weight of 1 10 kDa. The first two methionine codons are situated at bases 411 and 420. According to the 'scanning model' (Kozak. 1978) we chose the first ATG in the O R F as the starting codon though presently we have no direct evidence to support this choice. Accordingly, the predicted CDCl5 gene product is a polypeptide of 974 amino acids. The next ATG codon at position 612 is very unlikely to be functional, as it is already inside the conserved protein kinase domain (see below). Both of the possible initiation codons are flanked by nucleotides which correspond fairly well to the consensus for preferred initiation sites. Three bases upstream of both there is a purine (in both cases an A) which is possibly important for initiation (Kozak, 1984). At position

267

CDCIS. AN ESSENTIAL CELL CYCLE GENE IN SACCHAROMYCES CEREVISIAE

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Figure 1. (a), Restriction map and positions of C E N I , A D E l and CDCl5 on the right arm of chromosome I. Bars represent subcloned fragments. H, HindII; P, PvuII; C, Clal. (b), Enlarged, inverted map ofthe CDC15 region. Arrows indicate sequences read from individual sequencing reactions.

+ 4 of the second possible initiation codon is a guanine which is supposed to prevent bypassing the initiation site (Kozak, 1981). Another characteristic feature is a pyrimidine at position + 6 of both AUG codons. At position 371 to 376 the sequence CACACA is found which appears near the initiation codon in many S. cerevisiae genes (Dobson et al., 1982). In the 5’ region of the ORF TATA boxes were found 166, 85 and 6 bases in front of the predicted translational start. Of these, the first sequence TATAAT (at base 254) matches with the eukaryotic consensus sequence TAT(A/T)A(A/T). The other

TATA boxes are less well conserved. The TATA box of eukaryotic genes is normally located 20 to 30 bp in front of the transcription initiation; but in S. cerevisiae there are also TATA boxes up to 200 bases upstream of the transcription initiation codon (Kozak, 1984). None of the other conserved promoter sequences known for S . cerevisiae (Dobson et al., 1982; Guarente, 1984) was found. Since the functional subclone pBS9 carries only 400 bp of the 5’ flanking region, these seem to be sufficient as promoter. Located 39 bp after the termination codon (at base 3371) is the sequence AATAAT which is similar to the conserved polyadenylation

268

B. SCHWEITZER AND P. PHILIPPSEN PVUII

I C1G~Gl1G1GCTCTllTlCGGCITTICTGIGGTCGTlllGlCllGGCIGIIClIllCGCTIGIIGCClTTlTGTlGCTTTGClClTCTClIlICGGTllGGlTGICGTITGTClTc~l 120 GCGGCG11CTlCIG111IGIllIllTGICCTCTGGCGICGIGTlTGlllllGUGGClIllTCllllII6GGCCTlTCllc1ClTTlGClCTCIlTGCTlTlCTGTlGTGCTCTGTGCG~ 2 4 0 CTTTGCTGTGCCl~~IC~TTCClC~IGCGlTGGTCTTTCCGCT~I~IGCIGITlIIITTGTlTlCGGGlGlI~GCCTClCllllGGI~CIICIlCITGGlG6CIffilG H B S H l D I D P V B L T P I ~ P l S B K S V 360 GllGG1CllllClClClIllGClCTlCCClTITIC~GGIllGlCT~~lIGllClGIlTGGCCGlIlCCGlTlGlGICllCTTGlCTCCClICClGlGGGClTCIGlGlllTCCGTG

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480 C1lIlCClCTTG11GClGGIClTTGGGlG6GG~CTTlCGGGGIlG~IlClllGCClIIllTlllClTlCIGlCCllGTCGTGGCllTlllGGlGGTCGIGTlCGllllTGlTGlGGll ~ ~ L U D I H A B I S L L K E L B H B B I V K Y H G F I P ~ S Y B L Y I L L B Y C A 600 CIT11TG1C1TT1TGGClGlllTTlGCITGITllllllTTTlllCClIllCllTlTIGTTlllTlCClCGGCIIClIlCGlllllGCTlIGllTTGIlTlTCCIC~CGllTl~GCGCI I O ~ ~ G S L P B L I S P S S ~ G L S B Y K ~ K T Y V T ~ T L L G L K Y L H G ~ 720 llTGG1TCTT~l6G1GGCIClIIICllGGlGCTCTlCCGGlTIllGTGllllTGllTCGllllCCTlTGTGlClClGlClCTlTTGGGGCIGlllIlIITlClCGGIGllGGlGTClTC I ~ ~ ~ P D I K ~ A ~ I L L S ~ D ~ I ~ K L A D F G ~ ~ I I ~ 840 ClC1GGGlClTCllGGCGGCTllClTC~GCTGlGTGCIGlIllClCTGIClUC~GCIGlIITTGGCGIIICClCTlTTGIGllCICClGCGCCTTllCGc1l6CGGGClClCICllI 1 8 4 U ~ 1 P B I L G E 1 G 1 S 1 L S D I U S L G 1 1 V V B ~ L 1 K E P P Y H E L 1 D

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960 IGG1IGGC1CClGlGlTCCTGGGCllClGGGGlGC~CIlCGCIClGCGlClTITGGTCTCTlGGTGCClCTGTlGIIGlllTGCTClClllGllTCClCCCIlCClCllI~GlClGlC

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1080 GCC11IlTCTlCIlCGC1GTIGllllIGlTlCCTlCTlCCClCc1lGCICTIICTCTGlGCClCTlllGGlITICIIlTCTlllTGCIITGTGlllllClIGTlCUGlGG~GlClGCC

2 6 4 D ~ L L K H V W I 1 S 1 B B V K V D K L U K P K B D F I D l D ~ H W D l D F Q B 1200 GlCC1GTTlCTC11GC1TGIGIGGlTCllCTCIlCffillllIGTGllGGICGlCllG~CllCllGITCll~lGGl~ITlCffilCGCTGlIIlTClTTGGGlIGCCGlT~TCllGll 3 0 4 B K L B I S P S K F S L P 1 1 P 1 P W 1 B B B ~ B L D L H P P 1 B S ~ L L S ~ L 1320 GlGl11CTlllTlTlTClCCCTCTlllTTClGTCTICGlGCGGc1C~GCTCCCTGGGClGllllCllI~lGllCTlGl~TllIGCCCCCClCIGlllGTCllTIG~GlGCCllTIG 344K S S S K P L I D L H V L P S V C S L E B I A D I I I B C L S L T T V D K P L I 1440 11G1GTTClTCTllGCCI~GlCGGlCTIGClIGTGCTTTTClGTGTTIGCICCCTCGlGllClTCGCTGlTlCllTIlTCGlGIGTCTGTCGC6ClCllCTGTTGlTlllCGlTTUTl 3 8 4 2 1 F G S I P V Y D 1 ~ B B H S P L P L K F 1 1 M G G 1 P L 1 1 K F B E L l K B 1560 lCIGClITTGGCTCClIITITGTTIlCGlTlCCClGClTllCClCTCTlGGIIGCGlc1Glll~ClICGCIlI~GlGGllIICClCTGlIClIIlllTTCGllClI~lGCClllGlG 4 2 4 F V I D Y P q 1 L I B C G I M Y P P E F 1 S L K T P K Y 1 L B L V Y ~ F Y D L 1 1680

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TICGIClTCGlC1lCCCIClGlCT~llITGllTGTGGllTllIGTlTCc1CCGllI~T6ClTCG~GllllC~ClllGTlTl~ITlGUCTCGICTlTlGG~CTlCGl~TUCl 4

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1800 TCClClGCCTICT6GTGTCGCIGGTGTTTClllCl~TCGlIlTlTClCICCTTCTGllIllClICClIGlllGllGlGCCCllICClIlCTlCTlllGCIlTCGIClTlT6ClCClTGG

5 0 4 S F B K I L P S L I D S K L K K K I L I S P ~ 1 1 Y V V F K S 1 U Y H 1 1 1 E D 1920 TCTTTTG1Gll11~T1GCCCTCITTllIIGlCICTllGCTll~llGllll~IIllIClGTCCTClllTTlC~lffiIlGTc1TCl~TCllIlllCIlTlIGlTllCClCGllIGlC 5 4 4 D K I H K S l I P S S S S L P L S S S P T P B S P V B S V q S P S P S P V H S L 2040 G111111I1ClCllGTCC6CTlTICCTIC~CIICCTCTCTlCCGTTlTCCTCCTClCCClCGlGGUCIClCClGTGll~CGGIlClGT~CClTCllGGTCCCCTGIIClIICTIIG 5 8 4 H l T P P S S P H P H K S I S E F P H L I I S S K S P L L I B L P B 6 F F I U L

2160 lI6GCllCGCGTCCCTCIICICCllIG~lClCllGlGClIITClllCI~CCCClT~GlCClIlICIICllllICllGlCIlCIllITGllITlCCGGlGGGTITCIIIlCCIGGCTl ~ ~ ~ I S F F ~ D R ~ ~ I K D L S ~ L K Y F T K L ~ Y L Y ~ H I ~ 2280 1ClTc1T1ITTTG11G1ClIGGCCClllTClllGlTTIlICIGITIIlllGTlCTTClCCllGCTTTGTTlCCIIlClGTlClTlIlllClGClCTIIITTlllIGlICIGCIIGlCllC 6 6 4 D 1 F F 1 F I L B I D 1 I I P P I D D 1 K I l l F 1 U K Q 1 T 1 1 C V B H S L D 2400 G1IGc1TTII~GCTTTTlICCGGllIlIIGlIlCClTClTICCCT~lICGlCGlCGClllGlClGClGCTTTClTTTGGlllClllTClCTGCIlIlIGCGITGlllIGlGITI6GlI ~ ~ ~ R D ~ ~ S ~ S L F S I A ~ ~ F I B K K B ~ I ~ I ~ G 2520 1TGG1CC1l1TGlG1GCITCTITlITITCIlClGCTlIGll~IClIClGllllllGllIllClCCTCClIllGIGGlCT6GlGlIClIlCTGllCI6CITGClITTIlCGTTlCGCllI ~ ~ ~ V B D D V ~ P T ~ G S S B S H S V F L I K ~ E ~ D A ~ I E L P I 2640 GT111TG11G1TGTGGCTCCIlClGTGGGCIClIClGlGTCTClTlGTG~TICCIClIlllGGICllCllIGlCGCTGCIlTTGllTTlCCGlITGlTCllIIlGTTGlCCIGTIIIlT

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7 8 4 1 L U D D D V U L S K L I S I P I K I C S L P G F B B L 1 1 B 1 1 F H P U F Y B 2760 GClTIG11TGlCG1TG1CGIIllCCTClGIlllCIllIIlGCl~IIClCGllGlTlIGCIClITGCCCGGITTTGllllCCITlCllIIllTlTClTlIITClCCCGllC~IIlIGll 8 2 4 K I V S F F D T Y F U S L L I ~ I D L L K F 1 K L 1 F S K S L L K L Y D Y T G ~ 2880

‘l1G1~GTTI~TTC~IG1IlCCTlTTICllClG~TlCIIlITCUlTCGlICTlIIGlllITClIlllGCIllIlITCTCllllICGITlTIGllG~lIlTGlCIlClClGGlClG

~ ~ ~ P D P I K ~ I B P I P P ~ K ~ I ~ P K L P ~ I L ~ ~ I T 3000 CCGGl1CCTl1lllGClllCCGllCClllICGTCGTllTllGGCClCTGITTTClllCIICGI6CClTI~lGTlClllIllCGGlGTTITTlllCllCllCIGGllCllCGGlTGICCl 9 0 4 K B E S B ~ V G G D S V L I C ~ L C H D 1 P S L S K K 6 S L ~ K V S S V 1 1 1 1 3120 1A11GG11IIClllTCAlGTTGGGGGGGlCTClGTTIIGlTCTGTClGCIlTGTGlGGlTAICCGIIClIIlIClllllAlGGllGCCIGClllllGTTTCllGCGIClCIGClGCllTT ~ 4 G S S P T K D E P S B L P S S R D K S D G F S V P I I T F Q T * 3 2 4 0 G611G1TCTCCllCll11GlIGlGCGTlGIllIIIGCGlICCICClllGlTllllGTGlCGGCTTIICCGICCCClTIlCllClTITClllClIllIGIlGGllIIGClIGIGClTlClI 3360 1ClTlI1T1G1G~lClGCl~lClITllClCTlTlIlIlGllIIGIGllCCTTCCllTlCTGllllCGClTCTGI~GGllllGlGlTClGllllGI~I~llTIIlllGlGGC 3480 ClGTllIGlIllllllGClTlTCTlIlIlTGCTTTTTTTCTTTTTCTTTTTTTTT~CllIIG~GlIIlllIGIlTGllCCGlCICTlGllGGIGGClITIIGITlllIGTTTCTlGCI 3600 lT1G1TCllTlllTCTG11lTlCGlTlICIGlClIGlIIGClGIGGClClGTCIICCGlTlGIIlClGCCllGIITTTTI~IllGGTTICIGllIT~IIClIIlCG~ICCTllTIlG 3720 GT1111CGT111I1111GCGlCTI~CllllGllTlTClllTClTlTllClTGllllGlGCllGlTTClCllTlCIllTTICllTGlGlGIGCTlTTTGATllIIGllICllTICTCIGT 3840 G1G1G1l1GClIlGlCG1C1G~IITCCllGllIl~GTlGICCIClGClGClGClGGGlClCCClCCllGGTClGllIlCCllGTTCICGllGlllIIGGGlGl6GITClTITGGGICI PYUII 3960 GT1CGl11lG1ClICClIlIlCCIlCCll6lllCTTTIGGTTlGlllGGlTl~ClllIlT6GCClIlIGllIlGClllGlGAGlCAlClGCIG

Figure 2. Nucleotide and deduced amino acid sequences of CDCIS. The predicted amino acid sequence is shown in single-letter code, TATA boxes and termination sequence are underlined.

sequence AATAAA of eukaryotes. 25 bp further downstream a sequence similar to the postulated threepartite consensus sequence for transcription termination in S. cerevisiae (TAG/TATGT/TTT) (Zaret, 1982) is present.

Deduced amino acidsequence When the deduced amino acid sequence of the CDCIS gene product (Figure 2 ) was compared with

protein sequences in data banks using the program

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