The EMBO Journal vol. 1 0 no. 1 3 pp.4291 - 4299, 1991

The wis 1 protein kinase is a dosage-dependent regulator of mitosis in Schizosaccharomyces pombe

E.Warbrick and P.A.Fantes' Institute of Cell and Molecular Biology, University of Edinburgh, Darwin Building, Mayfield Road, Edinburgh, EH9 3JR, UK 'Corresponding author Communicated by A.P.Bird

The wisl + gene encodes a newly identified mitotic control element in Schizosaccharomyces pombe. It was isolated by virtue of its interaction with the mitotic control genes cdc25, weel and wini. The wisl+ gene potentially encodes a 66 kDa protein with homology to the serine/threonine family of protein kinases. wisl+ plays an important role in the regulation of entry into mitosis, as it shares with cdc25+ and niml+lcdrl+ the property of inducing mitosis in a dosage-dependent manner. Increased levels of wisl+ expression cause mitotic initiation to occur at a reduced cell size. Loss of wisl+ function does not prevent vegetative growth and division, though wisl- cells show an elongated morphology, indicating that their entry into mitosis and cell division is delayed relative to wild type cells. wislcells undergo a rapid reduction of viability upon entry into stationary phase, suggesting a role for wisl+ in the integration of nutritional sensing with the control over entry into mitosis. Key words: cell cycle/mitotic control/protein kinaseISchizosaccharomyces pombe

Introduction Recent studies have shown that entry into mitosis in a wide range of eukaryotes is controlled by similar mechanisms. Homologues of many of the genes involved in this control system, or of their products, have been isolated or identified in a range of species, suggesting that the same basic regulatory mechanism may operate in all eukaryotes (Nurse, 1990). Central to the control is a protein kinase complex containing a core kinase, p34cdc2, associated with a cyclin. In the fission yeast Schizosaccharomyces pombe, these components are encoded by the cdc2+ and cdc]3+ genes respectively (reviewed by Nurse, 1990; MacNeil and Nurse, 1989). Two major regulatory points exist in the S.pombe cell cycle: one in GI, which is analogous to start in S.cerevisiae, and a second in G2, controlling entry into mitosis. The GI control is evident at very slow growth rates, or in cell size mutants, though is cryptic under normal conditions of growth (Nurse, 1975). During exponential growth, the G2-M control is rate limiting and ensures that mitosis and cell division are permitted only when cells have attained a critical mass (Nurse and Fantes, 1981). The critical level is determined by the prevailing nutritional conditions and the © Oxford University Press

genotype of the cell. Changes in either of these factors may cause advancement or delay to the time of mitosis, reflected in reduction or increase of cell mass at division. Since

S.pombe cells grow throughout the cell cycle by length extension, this is reflected by an alteration of cell length at division. The regulation of entry into mitosis in S.pombe appears to be determined by the precise timing of the activation of the p34cdc2 protein kinase, which has been shown to be dependent on the dephosphorylation of a crucial tyrosine residue (Tyrl5) within the ATP binding domain (Gould and Nurse, 1989). Both positive and negative regulatory elements have been identified which control p34cd2 activity directly by affecting the phosphorylation state of Tyrl5. Several genes have been identified as playing a role in the negative regulation of p34cdc2 activity: among these are wee] + and mikl ± which encode homologous protein kinases. The wee]+ product belongs to a rare class of protein kinases capable of phosphorylating both tyrosine and serine residues, and there is strong evidence to suggest that it directly phosphorylates p34Cdc2 on Tyrl5 (Featherstone and Russell, 1990; Parker et al., 1991). Loss of either weel + or mikl + activity is not lethal, though loss of both activities leads to rapid dephosphorylation of Tyrl5 and to an apparently unregulated entry into mitosis, termed mitotic catastrophe, which is lethal to the cell (Lundgren et al., 1991). This evidence suggests that wee] + and mikl + share a redundant function which is required for tyrosine phosphorylation of p34Cdc2. The wee] + gene is a dosage-dependent inhibitor of mitosis: loss of wee]+ function advances mitosis, while over-expression delays mitosis (Russell and Nurse, 1987a). Loss of mik] + function, however, has no effect upon cell length at division (Lundgren et al., 1991). The niml + gene (allelic to cdrl+) also encodes a protein kinase, and is thought to regulate negatively the wee]+ product as part of a cascade control mechanism (Feilotter et al., 1991; Russell and Nurse, 1987b). Activation of p34cdc2 is brought about by the product of the cdc25+ gene (Moreno et al., 1989), which is a dosagedependent inducer of mitosis (Russell and Nurse, 1986). Recent results suggest that the cdc25+ gene product may be the tyrosine phosphatase responsible for p34cdc2 activation. Expression of a mammalian T-cell tyrosine phosphatase can compensate for loss of cdc25+ function in S.pombe (Gould et al., 1990), and cdc25+ homologue from both humans and Drosophila are capable of activating p34cdc2 by tyrosine phosphorylation in vitro (Kumagai and Dunphy, 1991; Strausfeld et al., 1991). A protein phosphatase has been identified from vaccinia virus which shows some sequence similarity with cdc25+ (Moreno and Nurse, 1991). Several sets of genes have been identified which show genetic interaction with the central components of the mitotic control discussed above. The six mcs genes were isolated 429 1

E.Warbrick and P.A.Fantes

by their ability to suppress the mitotic catastrophe phenotype resulting from the combination of a wee] mutation with cdc2-3w (an activated allele of cdc2), and show a wide range of genetic interactions with other mitotic control genes (Molz et al., 1989). We have previously reported that the win]-] mutation reverses the suppression of cdc25 phenotype by loss of wee] + function (Ogden and Fantes, 1986). Interestingly, this property depends strongly on the nutritional conditions, and mcs4-13 is very similar in this respect (Molz et al., 1989). We report here the isolation of a previously unidentified gene, wis] +, by its ability, when present in multiple copies, to suppress the cdc phenotype of triple mutant cdc25 wee] win] strains. The wis] + gene is a new dosage-dependent regulator of mitosis which encodes a putative protein kinase.

Results Isolation and analysis of wis 1 The win]-1 mutation has previously been shown to be capable of reversing the suppression of cdc25t" by loss of wee] function (Ogden and Fantes, 1986). Various S.pombe gene libraries were screened for plasmids capable of suppressing the temperature-sensitive phenotype of a wee]-50 cdc25-22 winl-] strain. Several species of plasmid were isolated containing S.pombe insert sequences which fell into five classes. The five loci thus defined were termed wisl + to wisS+ (WIn Suppressing). This paper focuses on one of these, wisl+. Other wis genes will be described elsewhere. wis] + sequences were originally isolated in the plasmid pwisl-1, which consists of a 8.8 kb insert contained within the vector pDB262. Additional plasmids which all shared a 5.8 kb XbaI -HindIll fragment with pwis 1-1 were isolated from a number of gene libraries (Figure 1). Strains containing several integrated copies of pwisl-l were constructed by the transformation of S.pombe cells with pwisl-1 linearized at unique restriction sites within the insert sequence. Southern blot analysis of DNA obtained from both integrant and wild type strains probed with pwisl-1 sequences showed that the insert sequences contained within pwisl-1 were contiguous within the S.pombe genome, and that integration events had occurred by homologous recombination (data not shown). Genetic mapping experiments with these integrant strains were used to demonstrate that the sequences contained within pwisl-1 were not linked to either the win] or the cdc25 locus, showing that wis] is not allelic to either of these genes (data not shown). Comparison of the pwisl-l restriction map with that of previously identified S.pombe genes involved in the control over entry into mitosis revealed that wis] is not allelic to any of the following genes: cdc25 (Russell and Nurse, 1986), wee] (Russell and Nurse, 1987a), cdc2 (Durkacz et al., 1985), nimllcdr] (Russell and Nurse, 1987b; Feilotter et al., 1991) sucl (Hayles et al., 1986), cdc13 (Hagan et al., 1988; Booher and Beach, 1988), dis2 (Ohkura et al., 1988), ppal or ppa2 (Kinoshita et al., 1990). The function region within pwisl-I was delimited by the construction of various subclones, and the analysis of their functional capacity, defined by the ability to suppress the temperature-sensitive phenotype of a wee]-50 cdc25-22 win]-] strain. Transposon mutagenesis was used as a method to delimit further the pwisl-1 functional region. A library of pwisl-1 clones was prepared, each containing a single

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integrated copy of the transposon Tn5, and the functional capacity of each clone determined as described above. The results of these experiments are shown in Figure 1. These results indicate that the functional region within pwisl-l is located between the PstI and the right hand XbaI sites (Figure 1). The sequence of this region was determined (Figure 2). Sequence analysis of wis1+ The sequence of a 3.0 kb region was determined (see Materials and methods), and a single open reading frame of 1815 nt identified (Figure 2). The ATG codon believed to initiate translation is preceded by a region of 847 nt containing no ATG in any frame. No sequences matching that of the 5' consensus for S.pombe introns (G/TGTANGT/A) were found within the proposed wisl ± open reading frame, suggesting that no introns are present. The position of this open reading frame corresponds to the wisl + functional region as defined by subcloning and transposon mutagenesis. Properties of the predicted wis 1 + gene product The wisl+ ORF described above predicts a gene product of 605 amino acids with a relative molecular mass of 66 kDa. The homology search algorithm FASTA (Lipman and Pearson, 1985) was used to search both the NBRF and EMBL protein sequence data bases for proteins showing similarity to the predicted wis] + gene product. The results of these data base searches show that the predicted wis] + product is related to the protein kinase family of polypeptides, as the 50 highest scoring matches were all kinase, or kinase related, proteins. The sequence elements defined by Hanks et al. (1988) as highly conserved protein kinase domains are all present in the predicted wis] + product (Figure 3). Those proteins sharing the highest degree of homology with wis] + are the S. cerevisiae genes PBS2 and STE7 and the S.pombe gene byrl , though none of these -

A H

P4 X W

I lA

B

pwisl-lA

-

pwisl-lB pvi1l-1C

-

HZ

III

X

HAl

A

1

-

pwisl-BD pIRT-X1

+

Fig. 1. Subcloning and mapping of wisi +. Panel A: The restriction map of the S.pombe genomic insert of pwisl-l: (B = BglII, E = EcoRI, H = HindIII, P = PstI, X = XbaI). The shaded region indicates sequences common to all wisl isolates and the black region the wis] open reading frame. The arrow indicates direction of transcription. The circles beneath the restriction map indicate the position of integrated TnS transposons, and their effect upon wisi function. Shaded circles indicate the positions of transposons causing loss of wisi function. Panel B: The ability of subclones derived from pwisl-l to complement the temperature-sensitive phenotype of a weel-50 cdc25-22 winl-l strain is indicated. pwis1-lA, pwis1-lB and pwisl-IC consist of HindIII fragments from pwisl-l subcloned into pDB262. pwisl-l was digested with BglIl and subsequently religated to give pwisl-BD. The 4.1 kb XbaI fragment from pwisl-l was subcloned into pTZ18 to give pXl. This fragment was subsequently subcloned into pIRT2 (Booher and Beach, 1986) using flanking polylinker sites to give pIRT-XI.

S.pombe wisl protein kinase

show significant homology with regions of wisi + outside the protein kinase domain (Figure 3). The PBS2 and STE7 genes have previously been assigned to a subfamily of protein kinases (Hanks et al., 1988). Sequence comparisons indicate that the predicted wisl + product belongs to the serine/threonine family of protein kinases, judging from a motif found in the conserved protein kinase domain VI, which is the most striking indicator of amino acid specificity. In the case of the predicted wisi + product this motif is Lys-

Pro-Thr, which is closely related to the Lys-Pro-Glu consensus for serine threonine protein kinases, and distinct from the Arg-Ala-Ala or Ala-Ala-Arg consensus for tyrosine protein kinases (Hanks et al., 1988). One notable exception to these rules is the wee]+ product, which shares homology with serine/threonine kinases, but has been shown to belong to a rare class of protein kinases capable of phosphorylating serine and tyrosine residues (Featherstone and Russell, 1990). The N-terminal half of the proposed wisl + product

-359

CCTGTTTCTTCCAGTTTCTGGCTTTTGCTGTTAAATTTAAACCCTTCCAAACCTCCTTTTTTTCCGTGGCATTTACCTACACAAAGCTAC

-270

-269

TCGTAGGTGATTGCCTTTAAACTTCCTTTTTTTTTCTTGGAATATCCTTCCTGCGGACTTTTTAGACCACCGCTTTTTTTTTCCTTCTTC

-180

-179

GTCGGAGACGACTCGTAATTGATTGTCTAATTTTAATTGTTCTTTTCACCCAAGATACCTTTTTTGTATTGCCATCTCACTTTCGTTCAT

-90

-89

CTTCACTTTTGCTTCATTTATATTACCGGAATTTAGTTTACCTAATTTTTTTTTCTTTTTTTTTAAGTTTGTGAAGCACATTTATTTTAT

0

1

ATGTCTTCTCCAAATAATCAACCCTTGTCTTGCTCATTGAGACAGCTGTCTATTTCTCCTACCGCACCTCCCGGTGATGTTGGTACTCCC

90

M 91

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Y

1800

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GGTGAAAAAAGAAGCTAAAGGTTCGCCTGCTTTCTAATTGCCTGCTCTGTTTTAAAGTACCCATGCGCATTGGTGTTTGTCTTTAATTTC G

180

S

CATGAGTTGGCTAACCATCCATGGTTGTTAAAATATCAAAATGCAGATGTGGACATGGCTTCATGGGCAAAAGGCGCTCTTAAAGAGAAA H

1801

P

CCACCTTCTCTCCCCGATTCATTTTCTCCCGAAGCTCGTGATTTTGTAAACAAGTGTTTGAATAAAAACCCGTCTTTGCGTCCCGATTAT P

1711

S

ATTTTAGAAATGGCTTTAGGAGCTTATCCGTATCCACCTGAATCATATACTTCAATATTTGCACAACTATCGGCGATTTGCGATGGCGAT I

1621

I

ATGGCTCCTGAAAGAATTCGTGTTGGTGGACCTACCAATGGCGTCTTGACTTACACCGTACAGGCTGATGTGTGGTCTCTAGGCCTTACC M

1531

S

TCTAATGGCCAGGTTAAGTTATGTGACTTTGGCGTGAGTGGGAATCTTGTGGCTTCTATATCCAAAACGAACATTGGATGTCAATCTTAC S

1441

L

GCTTATGCTGTAGTGCAAGGCCTCAAAACTTTGAAAGAGGAGCATAATATCATTCATCGTGACGTTAAACCTACTAATGTTTTGGTAAAT A

1351

Q

GTTTTTATTTGTATGGAATATATGGATGCTGGTAGCATGGACAAACTGTATGCTGGTGGTATCAAAGACGAAGGAGTTTTAGCTAGAACT V

1261

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AATCAAATTATAATGGAATTGGATATTTTACATAAAGCAGTTAGTCCTTATATCGTTGACTTTTATGGTGCCTTTTTTGTGGAAGGTTCT N

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TATGGTGTTGTGTATAAAGCATTGCATCAACCGACTGGTGTCACTATGGCCTTGAAGGAAATTAGGTTGTCCTTAGAAGAAGCAACATTT Y

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AATTCAGAAGGTGTTAACTTTTCATCTGGCTCTTCGTTTCGTATTAATATGTCAGAGATTATTAAGCTTGAAGAACTTGGAAAAGGTAAC N

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CAGGCTGTTTCGGAAACCCCTTTTTCCACATTTTCGGATATTTTGGATGCAAAATCAGGCACCTTAAATTTTAAAAACAAAGCCGTGTTA Q

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CTCGATTTATCCAATTCCAATCCCACCAGCCCTGTCAGTCCGTCTAGCATGGCTTCTCGCCGTGGCCTAAACATTCCTCCCACCCTTAAA L

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AAATCACTTGCTGCTGCTAGGAATCCTTTACTCAACCGTCCAACGTCCTTCAATCGACAAACGAGAATCCGTCGTGCACCACCTGGAAAA K

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GGAAGTTATCCTTTGCCTAACTCTCAACTTGCTGGTCGATTAAGTAATTCGCCCGTAAAGTCTCCGAATATGCCAGAGTCCGGGCTTGCA G

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GCCTTCCATGCATCTAGATCAAAATCAATGCCGGAAGTAGTCAACAAGATCAGTAGTCCAACTACCCCTATTGTCGGTATGGGTCAACGA A

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GCAGCTGGTCAGAACAATATTGGATCTCCTCCTACTCCACCGGGCCCATTTCCTGGAGGACTTTCAACTGATATACAGGAGAAATTGAAG A

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AGTGGCAGTGACAATGACTCAAAGGTTTCTTCTCCTAGTCGTGAAATACCTTCCGATCCCCCTCTTCCCCGTGCCGTGCCTACGGTCAGA S

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GGCTCGCTCCTTTCTCTTTCGTCTTCAAGTTCTTCAAACACCGATTCTTCTGGTTCTTCCTTGGGTTCCTTGTCTTTAAATTCTAACAGT G

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GAATGCATGACTATTACGTGATCCATAATTATGTTTCAGCAGAACCGACGCTATTTTGCATTTGTGCTTTTTCATAAATTTAATAATTTG

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GGTATGATTCCGTATAACGGTAGTTGATGTTTGCATTTTTGCTTTAAATTAAAACGGGTATTTAATGTGTTATTACATTTGTTTAAGGCA

2070

Fig. 2. Nucleotide and predicted amino acid sequence of the i*isl+ gene. The first nucleotide of the predicted open reading frame is numbered 1. An asterisk marks the position of the termination codon.

4293

E.Warbrick and P.A.Fantes

PBS2 Wisl

PBS2 Wisl

49 0 *ME DKFANLSLHE KTGKSSIQLN EQTGSDNGSA VKRTSSTSSH MSSPNNQPLS CSLRQLSISP TAPPGDV... ....GTPGSL LSLSSSSSSN 99 50 YNNINADLHA RVKAFQEQRA LKRSASVGSN QSEQDKGSSQ SPKHIQQIVN TDSSGSSLG.

..........

...

Table I. The effect of increased wisl + expression upon cell length Cell length at division of strains containing various levels of wisl + expression

SLSLNSN SSGSDNDSKV SSPSREIPSD

STE7 PBS2 Wisl

149 MFQRKTLQR. RNLKGLNLN. ......... LHPDVGNN.. KPLPP ..... LPVAGSSKV SQRMSSQ WQ ASSKSTLKNV LDNQETQNIT PPLPRAVPTV RLGRSTSSRS RNSLNLDMKD PSEKP..RRS LPTAAGQN..

STE7 PBS2 Wisl

199 150 ..GQLQEKTE THQGQSRIEG HVMSNINAIQ NNSNLFLRRG IKK..... DVNINIDTTK ITATTIGVNT GLPATDITPS VSNTASATHK AQLLNPNRRA . .NIGSPPTP PGPFPGGLST DIQEKLKAFH ASRSKSMPEV VNKISSP...

Strain

No. of copies

Cell length/tm (SD) Mean

972 INT3 OP2 (-thiamine) OP2 (+thiamine)

1 2 3 5-10 see text see text

13.0 11.3 11.1 10.8 10.7 13.8

(0.8) (0.3) (0.6) (0.6) (0.4) (0.6)

leul.32 (pDB262) leul.32 (pwisl-l)

1 see text

13.6 10.1

(0.7) (2.0)

100

249 KLTLD AFGDDQAISK PNTVVIQQPQ NEPV.... LV LSSLSQSPC. PGGMSLKMPT PRRPLSTQHP TRPNVAPHKA PAIINTPKQS LSARRAVKLP Wisl .....TTPIV GMGQRGSYPL PNSQLAGRLS NSPVKSPNMP ESGLAKSLA. 200

STE7 PBS2

.

299

250

Byrl STE7 PBS2 Wisl

.....KSSD. MFK RRRNPKGLVL ... NPNASV. TPSTI AYSNNFGL SPSSTNS ... .VSSSSSLST PCIID ..... KTAQQPQQFA PSPSNKKHIE TLSNSKVVEG KRSNPGSLIN GVQSTSTSSS .AARNPLLNR PTSFNRQTRI RRApPGKLDL SNSNPTSPV. ....SPSSM .....

..

349

300

Byrl STE7 PBS2 Wisl

Byrl STE7 PBS2 Wisl Byrl STE7 PBS2 Wisi Byrl STE7 PBS2

Wisl

KSFESNVEA LPPLEESLS QGLSNIATPV ENEHSIS ... LFANFSKYVD SNSGSSGGGG TEGPHDTVGT TPRTGNSNNS ASRRGLNIPP TLKQAVSETP .FSTFSDILD .N

DHKEELINNQ ..........

.

..........

350 LD RPAWISD ... DTLS GTSNGNY.IQ KLSLSSKGID FSNGSSSRIT KAVLNSEGVN FSSGSSFRIN ........

.

.

I E G LQDLVQLGKI A S;G, 1'VK LDELEFLDEL G H,Y;V1SK MSEIIKLEE L GK,Y.VYK NSSLEV RH

III 400 II RTVGSD SKLQ.KQILR ELGVLHHCRS K K TIIPVEQN NSTIINQLVR ELSIV.KNVK TEVRLELD EAKF.RQILM E LEVLHKCNS LK EI RLSLE EATF.NQIIM ELDILHKAVS

IV

FMEQCAHMNR PAAADLK... IKSGSLNFAG AKSGTLNFKN 399 SLVKHRNIFM ALHVPDSKIV VLHKPTNVIM

ALHQPTGVTM 449

PY.. tVGFYG PHENIITFYG PY..IVDFYG PY.. L VDFYG

AFQ...YKNN AYYNQHINNE AFF...IEGA

450 V ISLCMEYMDC GSLDA.I..L ...R.EGG.P ..I ....PLD IIILMEYSDC GSLDKILSVY KRFVQRGT.V SSKKTWFNEL VYMCMEYMDG GSLDK.I..Y D..E..SSEI GGI....DEP V?ICMEYMDA GSMDK.L..Y ...A..GG.I ..K....DEG

499 ILGKIINSMV TISKIAYGVL

500

VI

XGLIWYIYNVL LHI YRQY STE7 PBS2 LIGKEIKEQH Wisl S CULTI KEEH Byrl

HRDL

PS HII KIIIHRDI NIIIHRD PT PT NII

KPS

QLAFIANAVI VLARTAYAVV

549 VII ELVNSVAQTF I I ICDFGJSK KLINSIADTF

CDFGSSG QIILCSANQGT VIICDFGJSG VLVNSN.GQ VI4qpfGSG VVNSR.GE VLINSK.GQ

AFF...VEGS

NLVASLAKTN NLVASISKTN

599 IX RIRGG ..... .. KYTVK!I ji LIS IIEL ATQELPWSFS VGTSTYMSPE RIQ.G .. N. V ..YSIKqDV WW GLCI IIEL VTGEFPLG.. IGCQSYMAPE RIKSLNPDRA TYTVQ!DII W ILGISILEM ALGRYPYP.. prILEM ALGAYPYP.. IGCQSYMAPE RIRVGGPTNG

550 VIII

Byrl VGTSTYMSPE STE7 PBS2

Wisl

.

.

.

VLTYTVQ1V~

XI 649 FPEDLRLFVP ACLHKDPTI R YSKEMTDFVJ RCCIKNERE1R FSSDAQDFVS LCLQKIPEP R FSP EARDFVq KCLNKNPSI R

Wisl

600 NIDDSIG..I .GHNDTPDGI .PETYDN..I .PESYTS..I

Byrl

ASPQQLCAMP YFQQALMINV D.LAS.WA.S NFRSS

STE7 PBS2

Wisl

SSIHELLHHD LIMKYVSPSK DDKFRHWCRK IKSKIKEDKR IKREALDRAK PTYAALTEHP WLVKYRNQDV H.MSE.YI.T ERLERRNKIL RERGENGLSK PDYHELANHP WLLKYQNADV D.MAS.WA.K GALKEKGEKR S

STE7 PBS2

LEKKQSERST H NVPALHMGGY SVNIQIKANR HVNITTKKKQ TKSYCGNDAA NTKKPYIYIC

PBS2

750 LL

Byrl STE7 PBS2

X

LDLLHCIVQE LDLLQRIVNE FSQLSAIVDG FAQLSAICDG

EPPRLP.SS. PSPRLPKDRI PPPRLPSDK. DPPSLP.DS.

650

700

Fig. 3. Amino acid homology between the predicted wisi + product, the PBS2 and STE7 genes of S.cerevisiae, and the byrl + gene of S.pombe. The protein sequences were aligned using the BESTFIT program from the UWGCG package. Roman numerals indicate highly conserved protein kinase domains as defined by Hanks et al. (1988) and boxes indicate the conserved residues within these domains. PBS2 shows 49% identity, byrJ+ 39% identity, and STE7 32% identity with wisi, in this alignment.

is particularly rich in serine, and also in threonine, proline and glutamine residues. Clusters of these amino acids, often flanked by positively charged residues, appear to be charac4294

D6X1-2 D6X1-3

For details of strains, see Materials and methods.

teristic of many unstable proteins (Rogers et al., 1986). The PBS2 gene was identified in * cerevisiae by its ability to confer resistance to the antibiotic polymyxin B (Boguslawski and Polazzi, 1987). Since wisl + and PBS2 share a high degree of homology, the ability of wisl + to confer resistance to polymyxin B in S.pombe was tested. Transformants containing either pDB248 or pwisl-l showed a similar sensitivity to polymyxin B: cells were resistant to a concentration of 0.2 mg/ml, but sensitive to 0.3 mg/mil polymyxin B indicating that wisl + is not capable of conferring resistance to polymyxin B on S.pombe. wis 1 + acts as an inducer of mitosis The effect of multiple copies of wisl + upon the cell length of an otherwise wild type strain was examined. Multiple copies of wisi +, either plasmid borne, or integrated into the S.pombe genome, resulted in a reduction of cell length at division of -20% (Table I), a phenotype which has been termed 'semi-wee' (Russell and Nurse, 1986). This effect was not shown by the other four wis genes. In order to investigate this phenomenon more precisely, strains were constructed containing either one, two, or multiple extra copies of wisl + integrated into the S.pombe genome. Relative levels of the 2400 nt wisl + mRNA, as determined by Northern blotting, were found to increase concomitantly with wisl + copy number (Figure 4). Examination of the cell length at division of these integrant strains showed that a single extra copy of wisl + resulted in a reduction in cell length of 15% compared to wild type. Further increase in wisl + copy number resulted in further small decreases in cell length (Table I). To determine the effect of a very high level of expression of wisi +, the wis] + promoter was replaced with the regulatable nmtl + promoter in a plasmid construct, pwis 1-RIP (Maundrell, 1990; see Materials and methods). Stable integrant strains were constructed by the transformation of a ura4-294 leul-32 h- strain with the plasmid pwis 1-RIP linearized at a unique restriction site within the wisi + sequence. The phenotypes of two independent integrant strains (OP2 and OP4) were examined upon a shift from thiamine-containing to minimal medium. pwisl-RIP integrant strains continue to grow for at least 48 h following a shift to minimal medium, showing that a very high level of wisl + expression is not lethal. Following 48 h growth on thiamine-free medium at 25°C, cell length at division was determined, and found to be significantly reduced in -

S.pombe wisl protein kinase 1

:

A go

,am

Om_

BglII

PstI

(EcoRI)

XbaI

HinDIII EcoRI I I

xtbaI

B

.:1w

Fig. 4. Northern blot analysis of wisi transcription. Total RNA was prepared from strains showing various levels of wisl expression (see text for details), run on an agarose gel, and subjected to Northern blot analysis. Panel A shows hybridization to the 4.1 kb XbaI fragment from pX3 containing the entire wisl ORF. Panel B shows hybridization to a 1 kb fragment internal to the Spombe adhl gene. Lane 1, 972; Lane 2, D6X1-2; Lane 3, D6X1-3; Lane 4, INT3; Lane 5, OP2 (-thiamine); Lane 6, OP2 (+thiamine). For details of strains and growth conditions, see Materials and methods.

pwis 1-RIP integrant strains with respect to both wild type strains, and integrant strains grown in thiamine-containing medium (Table I). Northern blot analysis revealed a wisl + transcript slightly smaller than that seen in wild type strains present in pwisl-RIP strains grown in the absence of thiamine. This transcript was present at a very high level following several days growth without thiamine, and was undetectable in the presence of thiamine (Figure 4). The results of these experiments suggest that wisl + is acting in a dosage-dependent manner to induce premature mitosis and cell division. The fact that strong wisl + overexpression is not lethal suggests that a separate control mechanism is acting to set a minimum cell length, beyond which increased wisl + expression has no effect. pwisl-1 is not capable of suppressing the temperaturesensitive cdc phenotype of cdc2-33, cdc25-22 or cdc13-11 7, suggesting that high levels of wisl+ expression do not bypass the requirement for these genes. Increased wisl + copy number suppresses the phenotype of cell elongation shown by win]-l and mcs4-13 (Ogden and Fantes, 1986; Molz et al., 1989). Multiple copies of pwisl-l are also capable of suppressing the temperature-sensitive cdc phenotype resulting from the combination of mcs4-13with wee]-50 and cdc25-22. Little effect upon cell length at division was seen when pwisl-I was transformed into cdr] -34 or cdr2-69 strains, which have a phenotype of increased cell length. This suggests that both cdrl+ and cdr2+ activities are either required for, or are related to, wis] + function. Very high levels of wis] + expression had no effect upon the phenotype of decreased cell length shown by weel-SO, cdc2-Jw and cdc2-3w. This contrasts with the overexpression of another identified mitotic inducer, cdc25+, which results a lethal mitotic catastrophe phenotype when combined with wee1-SO.

Loss of wis 1 + causes a mitotic delay A one step gene disruption was performed to determine if wis] + activity is essential for cell viability. wis] deletion mutants were constructed by the technique of gene transplacement (Rothstein, 1983). A 1.2 kb region, which

PstI I

BrlII I

I

lKb

Fig. 5. wisl + gene disruption. The wild type wisi sequence is shown below, and the fragment used for deletion shown above. The filled region indicates the wisi open reading frame, and the shaded region S. cerevisiae sequences containing the LEU2 gene. See Materials and methods for details of this construct.

represents nearly two thirds of the proposed wis] + open reading frame (including most of the conserved protein kinase domain) was replaced with the S. cerevisiae LEU2 gene in a plasmid construct (pwisl-XPL9), and used to disrupt the wis] + gene (Figure 5). Sixteen leu+ diploid strains resulting from this transformation were subjected to tetrad analysis. Of these, 11 showed a phenotype of increased cell length which cosegregated with the LEU2 marker. Five strains were selected and subjected to Southern blot analysis (Figure 6). In order to confirm that wis] + sequences were deleted from these strains, genomic DNA was probed with the 1.2 kb BglI -EcoRI fragment, which represents those sequences removed in the DNA construct used for the transplacement procedure. This probe showed hybridization with the wild type DNA sample, though none with those derived from the transformants (Figure 6), showing that these sequences are entirely absent. Genomic DNA from these integrant strains was digested separately with EcoRI and HindlI, and probed with a more extensive wis] fragment, which would be expected to hybridize to sequences on each side of those replaced with the LEU2 gene. The samples digested with EcoRI show a very complex pattern of hybridizing bands (Figure 6), in contrast to the single hybridizing band from the wild type sample, suggesting that complex integration events have occurred, possibly involving concatenated fragments. Results from the samples digested with HindIII show that the two wild type HindIH fragments are replaced with a single, much larger, fragment in the integrant strains (data not shown). This indicates that the sequences containing the central HindIII site lying within the wisl + ORF has been deleted, and replaced with a much larger fragment, which is consistent with multiple integration events having taken place.

The plasmid construct used for the deletion procedure (pwisl-XPL9) was transformed into the strain weel-SO cdc25-22 winl-l leul-32 in order to assess its wisl + functional activity. This plasmid showed no activity in suppressing the temperature-sensitive phenotype of this strain 4295

E.Warbrick and P.A.Fantes Table II. Interactions of wisl- with other cell cycle mutations

A

Cell length/Am Mean

(SD)

13.0 23.5 9.4

(0.8) (0.4) (1.9)

weel.50* weel.50 wisl::LEU2*

7.5 8.0

(1.3) (0.9)

cdc2. I w cdc2.1w wisl::LEU2

8.7 13.6

(0.6) (0.7)

cdc2.3w cdc2.3w wisl::LEU2

9.1 13.4

(0.5) (0.7)

adh-cdc25+ adh-cdc25+ wisl::LEU2

8.4 11.2

(0.8) (0.5)

ppal::ura4+ ppal::ura4+ wisl::LEU2

12.5 18.7

(0.4) (0.7)

ppa2::ura4+ ppa2::ura4+ wisl::LEU2

9.6 16.1

(0.4) (0.6)

Strain

972 wisl::LEU2 wisl::LEU2 (pwisl-IRTU)

Fig. 6. Southen blot analysis of wisi disruptant strains. Spombe genomic DNA was prepared from wild type cells, and from five putative disruptant strains D2, D4, D5, D7 and DIO, digested with EcoRI, separated by agarose gel electrophoresis and Southern blotted. Lane 1, wild type; Lane 2, D2; Lane 3, D4; Lane 4, D5; Lane 5, D7; Lane 6, DIO. Panel A: probed with an equimolar mixture of the 4.4 kb and 3.2 kb HindIHl fragments from pwisl-l. Panel B: probed with the 1.2 kb BglII-EcoRI fragment from pwisl-1.

when grown on minimal medium, as determined by microscopic examination of the cells. The disruptant strains described here were judged to have a complete lack of

wisi + function due to the loss of 1.2 kb of functional sequence, which represents approximately two thirds of the wisi + open reading frame. Loss of wis] + function was found to result in a phenotype of increased cell length at division (Table II). This phenotype is suppressed completely by the introduction of plasmid-borne wis] + sequences into wislA strains (Table II). Growth rates of wisA and wild type strains were found to be indistinguishable: both showed a doubling time of 4.4 h when grown on minimal medium at 250C. These observations show that wislA strains show a specific cell cycle, as opposed to growth, phenotype, and that wis] + is not absolutely required for cell growth or division. Loss of wisi + activity also results in greatly reduced viability upon entry into stationary phase, in addition to the effect upon cell length. Cells were grown in minimal medium from logarithmic phase into stationary phase, and cell number and viability monitored (Figure 7). Both wis]o and wislA strains show simIlar viabilities when actively growing, though once the end of the logarithmic phase of growth is reached, wislA cells very rapidly lose viability. Several genetic backgrounds have been described which result in a reduced cell size at division. These include mutations in wee], certain alleles of cdc2, including cdc2-lw and cdc2-3w, and overexpression of cdcar5t (reviewed by Nurse, 1990). Double mutant strains were constructed combining these mutations with wislA, and the cell division

4296

Cells were grown at 25°C unless otherwise indicated. *indicates 35°C.

length of such strains determined (Table II). The results of these experiments show that all these mutations showed a strong effect upon the cell length phenotype caused by loss of wisi + function. The strongest effect is shown by wee]-50 which completely abolishes the cell length effect of wislA. Interestingly, none of these mutations had a strong effect upon the phenotype of loss of viability shown by wisl i\ strains following nutrient limitation (data not shown). Two genes (ppal + and ppa2 +) encoding type 2A phosphatase homologues have been identified in S.pombe, and while deletion of ppal appears to have little effect on the cell, deletion of ppa2 (which is responsible for the major type 2A phosphatase activity) leads to an early entry into mitosis (Kinoshita et al., 1990). Double mutant wis]A ppaA strains were constructed, and both found to show a reduced cell length at division compared to wislA ppa+ strains (Table II). Neither of the ppa deletion alleles completely suppressed the wislA cell length phenotype, though it is particularly interesting that loss of ppal function has even a partial effect, since loss of ppal function has no significant effect upon the cell length of wis] + strains (Kinoshita et al., 1990). This evidence suggests that it is a specific interaction between wis] + and ppal + functions, rather than a general effect upon cell length, that leads to the suppression of a wislA mutation by loss of ppal activity.

Discussion We report in this paper the isolation of the wis] + gene as a multicopy suppressor of the cdc defect of cdc2Ss weelJS win] strains, along with four other genes designated wis2 + - wis5+, whose properties will be described elsewhere. The wis] + gene is a dosage-dependent regulator of mitosis, in that overexpression leads to a reduction in cell size, while loss of wis] + function results in elongated cells. wis] + is not allelic with any described regulator of mitosis

S.pombe wisl protein kinase 10 9

o

f

10 8 -

7

E

10

0

10 6

,

10

0

10

20

30

4kdeterined 50

40

)O

0o 0-

:.D D

10

0

I 10

20

30

40

50

Time (hours) Fig. 7. Th e effects of nutritional limitation upon wisl + strains. Cell densities a nd viabilities were determined as described in Materials and methods. I Cell cultures were grown on minimal medium at 25°C. Open circl les indicate the wild type strain 972, and shaded circles a strain of g

;enotype wisl::LEU2 leul-32 h-.

in S.pon ibe, as judged by comparison of restriction maps, genetic rnapping, and complementation studies. The wis] + DNA se quence contains a single ORF, uninterrupted by introns, that potentially encodes a polypeptide of 605 amino acids wiith strong homology to serine/threonine protein kinases. The predicted wis] + product shows the highest protein sequence homologies to S.pombe byr] + and to S. cerevisFiae STE7 and PBS2, though the homologies do not extend oiutside the protein kinase domains (Nadin-Davis and Nasim, 1L990; Teague et al., 1986; Boguslawski and Polazzi, 1987). Increa sed wis] + expression causes a reduction in cell division size, while deletion of the genes causes cell elongation, su,ggesting that it is a dosage-dependent mitotic regulatorr. One extra copy of wis] + has a significant effect upon cell size, suggesting that the timing of entry into mitosis is highlI y sensitive to wis] + activity. Further evidence pointing to a role for wis] + in the mitotic control mechaniP sm derives from its interaction with the mitotic control Igenes wee], cdc25, win] and also with mcs4, mutationIs in which was isolated by their interaction with wee]-50 and cdc2-3w. The e ffects of either increasing or decreasing wisl + expressi(on both appear to be limited in extent: overexpressi(on does not result in a fully wee phenotype, while deletion mutants do not show a lethal cdc phenotype. One explanatiion for this is that the function performed by the wisl + piroduct is absolutely required for mitosis, but that ,

wis]+ is functionally redundant. Alternatively, it may be that wis]+ is not essential for mitosis, but rather has a purely regulatory role in modulating the activity of the central mitotic control. Loss of wisl + function results not only in an effect upon cell length at division, but also in a rapid loss of viability when the growth medium is depleted. This suggests that wis] + has cellular roles other than in the mitotic control, and supports the idea that the gene product may be involved in the integrating the nutritional status of the cell with regulation of timing of mitosis. A model for the control of entry into mitosis has been proposed in which the regulation of entry into mitosis is pb recise timing of activation of the cdc2 protein kinase (p34 ),which is dependent on the dephosphorylation of a crucial tyrosine residue, Tyrl5 (Gould and Nurse,

1989). The cdc25 phosphatase activates

p34Cdc2 by

dephosphorylating this tyrosine residue, and the wee] protein kinase inhibits p34cdc2 activity by phosphorylation (reviewed by MacNeill et al., 1991). The activity of the wee] protein kinase is proposed to be regulated in turn by the nimllcdr] protein kinase (Feilotter et al., 1991; Russell and Nurse, 1987b). One possible mode of action of the wis] + protein kinase is to regulate p34Cdc2 activity by acting through either the wee]+ or cdc25+ gene products. If wisl+ acted through only one of these elements, then differential interactions of various wee mutations would be expected with wis] deletion alleles. For example, if wisl+ acted solely through the wee] pathway, then cdc2-]- w mutations, which are relatively

*

* -

insensitive to wee] expression, or mutations in the wee] gene itself, would be expected to suppress the phenotype of wis] - mutants, while cdc2-3w mutations, or overexpression

of cdc25+ should have no effect. However, we have shown that wee mutations in either the wee] pathway (wee], cdc2-1w) or the cdc25 pathway (overexpression of cdc25+, cdc2-3w) at least partly suppress the elongated cell phenotype of wis]A, so it is impossible to place wisl+ on either pathway. Preliminary data suggest that the elongation of cdrl-34 cells is unaffected by multiple copies of wis] +, indicating that the functions of wis] + and niml +/cdr] + may be related. However, overexpression of wis] +, in contrast to niml +lcdrl+,] does not suppress cdc25's mutations, as would be expected if both acted through wee]+. On the basis of the results described above, it seems likely that wis] + affects p34cdc2 activity either by regulating both wee] + and cdc25+, or by some third pathway. In many eukaryotes it has been demonstrated that p34Cdc2 activation is inhibited by type 2A phosphatase activity, and it seems likely that this inhibition is the result of threonine dephosphorylation of p34Cdc2 (Picard et al., 1989; Felix et al., 1990; Solomon et al., 1990; Yamashita et al., 1990; Lee et al., 1991). In S.pombe, as in many higher eukaryotes, p34Cdc2 is phosphorylated on threonine 167 and this phosphorylation appears to be essential for p34Cdc2 activity (Gould et al., 199 1). Two genes (ppal and ppa2) encoding type 2A phosphatase homologues have been identified in S.pombe, and while deletion of ppal appears to have little effect on the cell, deletion ofppa2 (which is responsible for the major type 2A phosphatase activity in the cell) leads to an early entry into mitosis (Kinoshita et al., 1990). We show here that loss of either ppal or ppa2 activity has the effect 4297

E.Warbrick and P.A.Fantes

of suppressing the phenotype of increased cell length resulting from a wislA allele; loss ofppal to a lesser extent, while loss of ppa2 activity has a stronger effect. A highly speculative model could be proposed in which wisl + regulates p34cdc2 activity by increasing the level of Thrl67 phosphorylation. In such a model, the level of p34cdc2 threonine phosphorylation would be decreased in wisi strains, an effect which could be partly compensated for by a decrease in type 2A phosphatase activity.

Materials and methods Media and general techniques General molecular techniques were performed as described by Maniatis et al. (1982). Media for the propagation of S.pombe were as described by Moreno et al. (1990). The standard genetical procedures of Gutz et al. (1974) and Kohli et al. (1977) were followed. The dye Phloxin B (Sigma) was added at a concentration of 20 mg/l to facilitate the analysis of colony viability. All strains were derived from the two wild type strains 972 and 975. Diploid S.pombe strains were constructed using the complementing ade6 alleles, ade6-210 and ade6-216. Cell length measurements were made upon cells grown to a density of 1.0-5.0 x 106 cells/ml in minimal medium. The lengths of at least 24 septated cells were measured using an eyepiece graticule calibrated against a micrometer slide on a Zeiss photomicroscope using a x40 objective. Yeast transformation was performed essentially as described by Beach and Nurse (1981). Plasmids were recovered from S.pombe into Ecoli JA226 (recBC leuB6 trpE5 hsdR- hsdm+ lacY c6(X) as described by Hagan et al. (1988). Cell number per ml of liquid culture was determined from a sample fixed in a 0.1% formaldehyde/0. 1% sodium chloride solution. Following sonication, cells were counted electronically with a Coulter counter. In order to assess the viability of Spombe cells in stationary phase, cells were grown in EMM at 25°C, suitable aliquots plated onto YE plates following various incubation times, and the number of colonies formed following 3-4 days growth at 25°C counted. DNA and RNA manipulations Routine cloning procedures were performed according to Maniatis et al. (1982). Yeast RNA was prepared as described by Kaufer et al. (1985) and yeast DNA as described by Beach et al. (1982). Southern and Northern blot analysis was carried out using Gene Screen Plus (NEN) following the manufacturer's suggested protocols. DNA probes were labelled with [32P]dCTP using the random oligonucleotide labelling procedure of Feinberg and Vogelstein (1983). Transposon mutagenesis E.coli strain 554 (araDl89A 7697A lacX74 galG-gulK-hsn-hsm-strA recA13) containing pwisl-l was infected with aX phage isolate containing the transposon TnO, and kanamycin resistant plasmid clones isolated. This was done under conditions where the phage can neither lysogenize (c1857 at 37°C) nor replicate (oam in a supo background) so Km colonies reflect transposition events. Restriction mapping was used to determine the position of TnO integration events within plasmid clones, and a selection of such clones used to transform a weel-50 cdc25-22 winl-J strain to assess their ability to suppress the temperature sensitive phenotype of this strain. wis 1 sequence analysis The sequence of wisi was determined by a combination of the phagemid system devised by Vieira and Messing (1987) and the chain termination sequencing method of Sanger et al. (1977). Nested deletions were prepared from subclones of pwisl-1 contained within the plasmids pTZ18R/19R (Pharmacia), using the method of Henikoff (1987), using the Pharmacia Nested Deletion kit. Dideoxy sequencing reactions using T7 DNA polymerase were performed with the Pharmacia T7 Sequencing kit, according to the manufacturers instructions. The sequence of a 3.0 kb fragment containing the wisi ORF was determined on both strands. Analysis and homology searches were carried out using the UWGCG package available through the Seqnet VAX facility at Daresbury, UK. Isolation of complementing plasmids from gene libraries in S.pombe All gene libraries consisted of random Spombe genomic fragments resulting from eitherpartial Sau3A or partial Hindm digestion, in replicating S.pombe vectors pDB262 (Wright et al., 1986), pDB248 (Durkacz et al., 1985) or pWH5 (Wright et al., 1986) which all contain the S.cerevisiae LEU2 gene.

4298

S.pombe genomic DNA was prepared from the wild type strain 972 h-. Following transformation of a strain of genotype weel-50 cdc25-22 winl-l leul-32 h-, protoplasts were allowed to regenerate at 28°C for 3-5 days. Cells were then washed off the sorbitol-containing medium in a small volume of sterile water, and replated at a density of - 104 cells/plate on EMM containing Phloxin B. The cells were then incubated at 35°C to identify transformants containing plasmids capable of suppressing the temperature sensitive phenotype of this strain. All colonies formed at 35°C were picked for further analysis. The replating procedure was adopted because the transfer of large numbers of cells by standard plate replication techniques leads to a high level of background growth at 35°C in this strain. For each gene library, a total of 5-10 x I0 transformants were screened in two separate experiments. In each experiment, a number of cells greater than ten times the number of transformants was replated. Construction of strains containing two and three copies of the wis 1 + gene Plasmid pXl consists of the 4.1 kb XbaI fragment from pwisl-l (Figure 1) subcloned into pTZ18R (Pharmacia). The plasmid pD6X1 was constructed by subcloning the 2.2 kb Sall -XhoI fragment containing the S. cerevisiae LEU2 functional sequence from pYepl3 (Broach et al., 1980), into the polylinker derived SalI site in plasmid pX 1. pD6X I was used to transform an Spombe strain of genotype leul-32, and stable transformants, D6X1-2 and D6X1-3, selected for further analysis. Southern blot analysis (not shown) showed that these strains contained one and two integrated copies of pD6X 1, respectively. Strain INT3 contains multiple integrated copies of pwisl-1, and was derived by the transformation of a strain of genotype leul-32 with pwisl-l linearized within the Spombe insert sequences. Southern blot analysis of INT3 showed that five to ten copies of pwisl-l had integrated by homologous recombination, as judged by relative band intensities. Overexpression of wis 1+ in S.pombe The wisl open reading frame was fused to the nmtl promoter (Maundrell, 1990) in a plasmid construct, pwisl-RIP. The nmtl promoter sequences are capable of conferring thiamine-regulated transcription upon heterologous genes. In the absence of thiamine, expression is greatly increased, and in the presence of thiamine transcription is strongly repressed (Maundrell, 1990). In these experiments, thiamine was added to a concentration of 2 /AM when required. The wisi sequences used for the nmtl - wisi construct were taken from a plasmid (pE2) generated in the preparation of Exonuclease III deletions for sequencing. pE2 consists of a 2.4kb insert in pTZ18R, starting 15 bases upstream of the proposed start of translation (Figure 2). These sequences were subcloned into the Sall-SstI sites in the plasmid pRIP4/s (K.Maundrell, unpublished results) to give pwisl-RIP. Cells of genotype leul-32 ura4-294 h- were transformed with pwisl-RIP linearized at the unique BgilI site within the wisi sequences, and stable ura+ transformants selected. Construction of the wis 1 deletion The plasmid pTZR-4 was constructed by digestion of pTZ19R with EcoRI, treatment with Klenow enzyme and dNTPs to produce blunt ends, and subsequent religation. The 3.5 kb PstI-XbaI fragment from pwisl-l (Figure 1) was subcloned into the PstI-XbaI sites within the polylinker of pTZR4 to give the plasmid pwisl-XP. pwisl-XP was digested with EcoRI, treated with Klenow enzyme and dNTPs to give blunt ends, and then digested with Bgll. A DNA fragment was then subcloned into these sites which consisted of the BgllI-XhoI LEU2-containing fragment from pYepl3 (Broach et al., 1980), which had been treated in a similar way to that described for pXP-9 to give a Bgfll-blunt end fragment. The resulting plasmid was termed pwisl-XPL9, in which the 1.2 kb BgSll-EcoRI fragment within the wisi ORF has been replaced with a fragment containing the LEU2 gene (Figure 5). Diploid cells of the genotype ade6-210/ade6-216 leul-32/leul-32 ura4-D18/ura4-D18 h+/h- were transformed with purified PstI-XbaI fragment from pwisl-XPL (Figure 5), and leu+ transformants selected. The plasmid containing wisl + sequences used to transform disruptant strains (pwisl-IRTU) consisted of the 3.5 kb PstI -XbaI fragment from pwisl-l subcloned into plasmid pIRTU which contains the Spombe ura4+ gene. The DNA sequence reported here has been deposited with the EMBL sequence database under the accession number X62631.

Acknowledgements We are grateful to Paul Young, Paul Nurse, Maureen McLeod and David Beach for gene libraries and plasmids, to Mitsuhiro Yanagida for the ppalA and ppa2A strains, to Kinsey Maundrell for the nmtl promoter plasmid, and to Mike Stark for the X bacteriophage containing Tn5. Thanks

S.pombe wisl protein kinase are extended to all our colleagues, particularly to Paul Nurse, for helpful discussions, and to Joan Davidson for excellent technical assistance. This work was supported by the Cancer Research Campaign.

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