comparison of thermosensitive alleles of the cdc25 gene ... · transplacement method (winston,...

Copyright 0 1990 by the Genetics Society of America

Comparison of Thermosensitive Alleles of the CDC25 Gene Involved in the cAMP Metabolism of Saccharomyces cerevisiae

Anne Petitjean,* FranGois Hilger* and Kelly Tatchellt Faculti des Sciences Agronomiques, Laboratoire de Microbiologie, 6 Avenue Marichal Juin, 5800 Gambloux, Belgium, and

tDepartment oflnicrobiology, North Carolina State University, Raleagh, North Carolina 27695-7615 Manuscript received March 7, 1989

Accepted for publication December 8, 1989

ABSTRACT The CDC25 gene from Saccharomyces cerevisiae is an essential component of the RAS-adenylate

cyclase pathway. Genetic and biochemical evidence has led to the proposal that the gene product may act upstream of RAS, possibly as a guanine nucleotide exchange factor. We report here the cloning, sequencing and characterization of four mutations in the CDC25 gene. All four are missense mutations which reside within the carboxy-terminal quarter of the single open reading frame found within the gene. Three of the four are missense mutations in the same amino acid codon. A search of protein data bases reveals that the carboxy terminus of the putative CDC25 gene product is similar to that of LTEl, a gene required for growth at low temperature and SCD25, a suppressor of cdc25. Taken together these data indicate that the carboxy terminus of CDC25 plays a critical role in the function of the CDC25 gene product and that other proteins, such as LTEl or SCD25, may have related activities.

C YCLIC AMP is essential for cell growth in the yeast Saccharomyces cerevisiae. Temperature-sen-

sitive (ts) mutants in adenylate cyclase, coded for by the CYRl or C D C 3 5 gene, arrest as unbudded nongrowing cells at the restrictive temperature (MATSU- MOTO et al. 1982; BOUTELET, PETITJEAN and HILGER 1985). In strains that are able to take up exogenous cAMP the temperature sensitivity due to cdc35/cyrl mutations can be reversed by addition of cAMP to the media (MATSUMOTO et al. 1982). The growth defect can also be reversed by mutations in the gene coding for the regulatory subunit of CAMP-dependent protein kinase, implying that the essential role of cAMP in the cell is to activate CAMP-dependent protein kinase (MATSUMOTO et al. 1982).

In signal transduction systems that utilize cAMP as a second messenger, the binding of ligand to receptor either activates or inactivates plasma membrane adenylate cyclase via the GTP binding proteins G, or Gi, respectively (GILMAN 1984). It has been suggested that the yeast adenylate cyclase is regulated in a similar manner due to the observation that the GTP depend- ence of yeast adenylate cyclase (CASPERSON et al. 1983) is mediated through the GTP binding proteins RASl and RAS2 (BROEK et al. 1985; FIELD et al. 1987). Although abundant evidence indicates that cAMP is essential for cell growth, direct evidence that cAMP acts as a second messenger is lacking. However, indi- rect evidence suggests that cAMP may act as a second

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Genetics 124: 797-806 (April, 1990)

messenger for various stress or nutritional signals. The addition of glucose to yeast cultures growing on nonfermentable carbon sources causes a transient in- crease in cAMP (VAN DER PLAAT and VAN SOLINGEN 1974). Yeast strains bearing mutations in various steps in the cAMP pathway have defects in the normal response to starvation and heat shock (MATSUMOTO, UNO and ISHIKAWA 1983; TODA et al. 1985; CANNON, GIBBS and TATCHELL 1986).

In addition to RASl and RAS2, the C D C 2 5 gene product is required to activate adenylate cyclase. Like ts mutants in adenylate cyclase, cdc25 ts mutants arrest as nongrowing cells in GI of the cell cycle and have been reported to have low levels of GTP-stimulated adenylate cyclase activity (ROBINSON et al. 1987; BROEK et al. 1987; DANIEL 1987; MARSHALL et al. 1987). The recent observation that dominant mutations in RAS2 which eliminate the GTPase activity of RAS also bypass the requirement for C D C 2 5 function (ROBINSON et al. 1987; BROEK 1987) has led to the hypothesis that CDC25 acts as an exchange factor for RAS, similar to the role of the receptor in other signal transduction systems (GILMAN 1984). Conclusive evidence for this model has not been obtained and some reports in the literature are not consistent with this model. If the essential function of CDC25 is to activate adenylate cyclase, it would be expected that mutations that suppressed the lethality of adenylate cyclase lesions would also suppress mutations in cdc25. Muta- tions in the regulatory subunit of CAMP-dependent protein kinase (bcyllsra2) bypass the essential requirement for both adenylate cyclase (cyrZlcdc35) and

798 A. Petitjean, F. Hilger and K.Tatchell

R A S l and R A S 2 . TRIPP and PIGON (1986) report that one allele of cdc25 is not suppressed by bcyl , but cdc25-I is suppressed by numerous alleles of sral (L. ROBINSON, personal communication). Some cdc25 mutants have high intracellular cAMP concentrations at their restrictive temperature (MARTEGANI, BARONI and WANONI 1986), although others have been reported to have reduced cAMP concentrations at the same temperature (CAMONIS et al. 1986). Like ras2 mutants, some cdc25 mutants also show reduced ability to grow on nonfermentable carbon sources. This trait is not shared by all cdc25 alleles and has not been reported for mutations in adenylate cyclase. One ob- jection with these data is that the different mutants used in these experiments come from different genetic backgrounds. As a first step in an attempt to sort out the complexities of cdc25 function we have character- ized four independent temperature-sensitive cdc25 alleles. All four mutations reside within a 225-nucleotide region near the carboxy terminus of the coding sequence.

MATERIALS AND METHODS

Strains and media: The genotypes of yeast strains are given in Table 1. The ras2-530 mutation is a LEU2 disruption of R A S 2 (TATCHELL et al. 1984). Strains LR684-4C and LR833-3A are the products of six serial backcrosses of strains Be333 and Gx3261, respectively, to strain JC482 (CANNON and TATCHELL 1987). Strains AP121, AP124 and AP127 are congenic with strain JC482 and have been obtained by transplacement (WINSTON, CHUMLEY and FINK 1983) of the wild-type CDC25 gene by cdc25-IO, cdc25-1 and cdc25-5 alleles, respectively (see below and RESULTS). The diploid strain AP183 has been isolated by crossing JC482 with JC530-4B. The cdc25-113 mutation was constructed in vitro by replacing the internal PstI fragment of the CDC25 gene by LEU2 (ROBINSON et al. 1987). This haplolethal deletion was introduced by transformation (ITO et al. 1983) into the diploid strain AP183 giving rise to strain AP184 which is heterozygous for the cdc25-113::LEU2 allele.

S. cerevisiae strains were grown in minimal SD medium supplemented with required amino acids (SHERMAN, FINK and HICKS 1986) or in rich medium (YEP) which contains 1 -yeast extract (Difco) and 2% Bacto-peptone (Difco). Either 2% dextrose (YEPD), 3% glycerol (YEPG) or 3% ethanol (YEPE) was added as a carbon source. Solid media contain 2% Bacto Agar (Difco).

Bacterial cloning and plasmid maintenance were done in Escherichia coli strain HBlOl grown in Luria Broth (MA- NIATIS, FRITSCH and SAMBROOK 1982) and supplemented with 50 f ig of ampicillin per ml, when required for selection.

Cloning the wild-type CDC25 gene: The CDC25 structural gene was isolated, from a YCp50 library (ROSE et al. 1987), by complementing the temperature sensitivity of a cdc25-5 strain. pAP2554, one of the plasmids suppressing the ts defect, was shown by restriction mapping analysis to contain the structural gene (CAMONIS et al. 1986; BROEK et al. 1987).

Construction of plasmids pAP5410 and pAF5411: The plasmids pAP5410 and pAP5411 were both constucted by inserting the 5.5-kb CDC25 SalI-Pvu1I fragment of pAP2554, into the replicating 2~ vector YEp24 and the

integrating vector YIp5 respectively. Both YEp24 and Ylp5 vectors carry the yeast gene and confer ampicillin resistance to E. coli (PARENT, FENIMORE and BOSTIAN 1985). The SalI- PvulI fragment contained the entire coding region of the CDC25 gene as well as its 3' and 5' flanking regions (see Figure 1A). These plasmids were constructed as follows: both vectors were linearized with BamHI and their 5' overhangs were blunt ended with the Klenow fragment of DNA polymerase I. The plasmids were then digested to completion at the unique Sal1 restriction site. The resulting plasmids were ligated with the purified SalI-PvulI CDC25 fragment. In both constructions the Pvull site of CDC25 was replaced by a BamHl site (see Figure 1A).

Cloning the cdc25 ts alleles: The cdc25-IO mutant strain Be359 was isolated in a screen for new cdc mutants (Bou- TELET, PETITJEAN and HILGER 1985). The cdc2.5-I mutant strain ts321 (HARTWELL et al. 1973) was obtained from the Yeast Genetic Stock Center. Strains DlBR205, carrying the cdc25-2 mutation, and D12BR209 and OL86, both bearing the cdc25-5 mutations were received from M. JACQUET. Strains DlBR205 and D12BR209 were originally isolated in L. HARTWELL'S laboratory. These cdc25 ts strains were crossed to a u r a 3 strain (see Table 1). cdc25 ura3 segregants were transformed to Ura+ with the integrating plasmid pAP5411, linearized with KpnI. The ts mutations were cloned from thermosensitive Ura+ transformants by the eviction method (WINSTON, CHUMLEY and FINK 1983). The DNA of these transformants was extracted (SHERMAN, FINK and HICKS 1986), digested with Sal1 and ligated at a concentration of 1 r g per ml. Escherichia coli strain HBlOl was transformed to ampicillin resistance to select for the YIp5 plasmid.

Sequence determination and oligonucleotides: DNA sequencing was performed by using the dideoxynucleotide chain-termination method (SANGER, NICKLEN and COULSON 1977; SANCER and COULSON 1978). The BamHl-BamHl fragment containing the wild type and each ts allele was purified from plasmids pAP5411, -1, -5 , -2, -10 and subcloned into the BamHl site of M 13mpl8 vector. In addition to the universal primer, three synthetic oligonucleotides were designed to prime sequencing reactions for this fragment of the CDC25 gene. Their sequences correspond to the CDC25 coding sequence from nucleotide 4023 to 4038 (oliel), 4191 to 4207 (olie2) and 4455 to 4471 (olie3) (BROEK et al. 1987). Combining the use of the universal primer and the three oligonucleotides the antisense strand of the BamHI-BamHI (Puull) fragment was sequenced from nucleotide 3765 (5' BamHI) to nucleotide 4770 (TAA stop codon) of the wild type and the four ts alleles.

Construction of isogenic cdc25 strains: We used the transplacement method (WINSTON, CHUMLEY and FINK 1983) to introduce the three different cdc25 ts alleles cloned in the same genetic background. The CDC2.5 strain JC482 was transformed to Ura+ with the uncut integrating plasmids pAP5411-1, -5 and -10. After growth at 26" 5-fluorooro- tate-resistant clones (BOEKE, LACROUTE and FINK 1984) were then replicated on YEPD medium and incubated at 36" to identify which one had retained the cdc25 ts alleles. Five to ten percent of these recombinants were thermosensitive. In every case the ts mutants failed to complement cdc25-5 strains and the ts phenotype was tightly linked to the cdc25 locus. Strains AP121, AP124 and AP127 (Table 1) which carry cdc25-IO, cdc25-1 and cdc25-5 mutations, respectively, were retained for further study.

Thermosensitivity, glycogen, acid phosphatase, cAMP and adenylate cyclase assays: Thermosensitivity was stud- ied by replica plating isolated colonies onto plates followed by incubation at 26", 34" and 36". Accumulation of glyco-

cdc25 gene from S. cereuisiae 799

gen in yeast strains grown for two days on YEPD plates, was scored in situ by inverting the plates over I p crystals in a chromatography tank. Glycogen levels were measured quan- titatively in cells grown in liquid YEPD medium by using the method Of GUNJA-SMITH, PATIL and SMITH (1977). Acid phosphatase was measured in yeast strains that were grown in low Pi medium (LEGRAIN, DE WILDE and HILGER 1986) to an ODs4" of 1.3. Cells were harvested by centrifugation, washed with cold Na-acetate 50 mM (pH 4) and then sus- pended in the same buffer. The cells were broken in a "French" press (Aminco 4-3398A) at 5500 psi. Unbroken cells and large cell debris were removed by centrifugation at 12,000 X g for 15 min. Acid phosphatase activity of the supernatant was determined using the method of TOH-E et al. (1 973). Protein content in crude extracts was measured as described by LOWRY et al. (1 95 1). Levels of phosphatase activity from cells grown in high phosphate media were below the level of detection.

Membranes fractions (CASPERSON et al. 1983) were pre- pared from cultures grown at 26" to an OD540~f 0.6 to 0.7 in SD medium, supplemented with the required amino acids. In the shift experiment to 36", cells were grown to an OD54~ of 0.3 at 26" and shifted to 36" for 90 min. Spheroplasts obtained by glusulase digestion were broken with a Dounce homogenizer using pestle A in lysis buffer (50 mM sodium MES (pH 6), 0.1 mM EGTA, 0.1 mM MgCI2, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) and then centrifuged at 1000 X g for 10 min at 4" to pellet unbroken cells and large debris. The supernatant was centrifuged at 20,000 X g for 45 min at 4". Membranes pellets were resuspended gently in lysis buffer and protein content was measured as described by LOWRY et al. (1951). Mem- branes were stored frozen at -70" in 10% glycerol. Aden- ylate cyclase assays (1 00 pl reaction volume) which measure the conversion of [a-"P]ATP to [32P]cAMP were performed using 25-75 pg of protein per assay and have been previously described (CASPERSON et al. 1983). The cAMP formed was separated from reactants by sequential chromatography on Dowex AG 50W-X4 (200 to 400 mesh; Bio-Rad) and neutral alumina (Sigma, type WN-3). Guanine nucleotide, Gpp(NH)p, was purchased from Sigma. In the indicated experiments, GppNHp was preincubated in the absence of MgCI2 with the membranes at a concentration of 500 PM for 30 min at 4" in a 20-pl volume before dilution with the assay components. These conditions facilitate RAS protein- guanine nucleotide exchange (MARSHALL et al. 1987).

Cyclic AMP concentrations were determined using the cAMP assay kit of Amersham based on the assay of GILMAN (1970). Cells were collected by filtration onto nylon membrane filters, washed off the filters into 10% trichloroacetic acid and frozen on liquid nitrogen. Cells were permeabilized by repeated freezing and thawing. Trichloroacetic acid was removed by four diethyl ether extractions and the samples were lypholized prior to assaying CAMP.

RESULTS

Four cdc25 mutations reside in the 3' end of the gene: T h e mutations responsible for the temperature sensitivity in cdc25-I, cdc25-2, cdc25-5 and cdc25-10 were localized using the gap repair method of ORR- WEAVER, SZOSTAK and CARTER (1983). ts strains (Table 1) were transformed to Ura+ with the integrating plasmid pAP5411 that contains the wild-type CDC25 gene in the integrating vector YIp5 (Figure 1A). Gaps were introduced into the CDC25 coding

sequence by restriction enzyme digestion. When double-strand gapped plasmids integrate into the yeast genome, the plasmid deletion is repaired from homologous chromosomal DNA (ORR-WEAVER, SZO- STAK and CARTER 1983). Consequently, only transformants obtained by integration of a plasmid containing a double-strand gap that deletes the region of the wild-type CDC25 gene homologous to the cdc25 chromosomal mutation remain thermosensitive. All the transformants resulting from integration of plasmid pAP.54 1 1, or the same plasmid deleted within the CDC25 coding region by restriction enzyme digestion (Figure IB) were thermoresistant at 36". This indicated that the ts lesions were not in the amino-terminal half of the CDC25 coding region. In contrast, about 50% of the transformants obtained by integration of pAP5411 plasmid linearized in the carboxy-terminal part of the coding region at the KpnI site (Figure 1B) remained thermosensitive. The recovery of thermosensitivity would be predicted if exonuclease digestion from the KpnI site of the CDC25 DNA had uncovered the ts mutations before or during the integration event of the KpnI cut plasmids. Taken together, these results are consistent with localization of all four mutations in the vicinity of the KpnI site in CDC25.

cdc25-1, cdc25-2, cdc25-5 and cdc25-10 are missense mutations in the carboxy terminus of the cdc25 open reading frame: The nature and precise location of the cdc25-1, 2 , 5 and 10 was determined by sequencing the cloned mutant alleles. ts strains derived from the mapping experiments in the previous section contained an integrated YIp5 plasmid flanked by copies of the cdc25 gene (Figure 1C). Genomic Southern analysis confirmed that the predicted structure was present at the cdc25 locus in these transformants (data not shown). We isolated the mutant genes in the YIp5 plasmid from two transformants of each mutant by the "eviction" method of WINSTON, CHUMLEY and FINK (1 983). These plasmids had the same restriction pattern as parent plasmid pAP5411. The mutant plasmids were called

the second number referring to the ts allele. To prove that we had cloned the four cdc25 ts

alleles and that all four mutations are located near the KpnI site, we replaced the BamHI-BamHI (PvuII) restriction fragment of the wild-type CDC25 gene (see Figure 1 A) in the replicating plasmid pAP54 10 by the corresponding fragment of the cloned ts alleles (pAP5411-1, -2, -5 and -10). 3 hese replicating plasmids were transformed into the diploid strain AP 184, heterozygous for a cdc25-I 13::LEU2 deletion allele (ROBINSON et al. 1987). Ura+ transformants were spor- ulated and meiotic progeny analyzed for the cdc25 genotype. As expected for a haplolethal deletion, Leu+ (cdc25-113::LEU2) spores were viable only if

pAP5411-1, pAP5411-2, pAP5411-5, pAP5411-10;


TABLE 1

Yeast strains used

S. cereuisiae Strain of

Genotype Reference or source

Be333 Gx3061 Be359 ts321 DlBR205 D12BR209 OL86 Be383 LR598B-2B LR656A-4D AP101 JC482" JC530-4B" JC302-26B" LR684-4Cb LR833-3Ab API'LI" AP124" API27" AP183" APIX4"

Mata argl cdc35-IO Mata argl cdc35-13 Mata argl cdc25-IO Mata adel ade2 ural tyrl his7 lys2 gall cdc25-I Mata adel ade2 adeb ade7 arg4 thr4 ural gall cdc25-2 Mata adel ade2 arg4 his7 trpl ural cdc25-5 Mala trpl ade2 cdc25-5 Mata ura3-52 cdc25-10 Mata ade2 his4 leu2 ura3-52 cdc25-1 Mata his4 leu2 ura3-52 cdc25-2 Mata ade2 ura3-52 cdc25-5 Mata leu2 ura3-52 his4 Mata leu2 ura3-52 his4 Mata leu2 ura3-52 his4 rad-530 Mata leu2 ura3-52 his4 cdc35-IO Mala leu2 ura3-52 his4 cdc35-13 Mata leu2 ura3-52 his4 cdc25-IO Mata leu2 ura3-52 his4 cdc25-I Mata leu2 ura3-52 his4 cdc25-5 MatalMata leu2/leu2 ura3-52/ura3-52 his41his4 MatalMata leu2/leu2 ura3-52/ura3-52 his4/his4 CDC25/cdc25-113::LEU2

BOUTELET et al. (1 985) BOUTELET et al. (1985) BOUTELET et al. (1 985) Yeast stock center M . JACQUET

M. JACQUET

M. JACQUET This study L. ROBINSON L. ROBINSON This study CANNON and TATCHELL (1 987) J. CANNON CANNON, GIBBS and TATCHELL (1 986) L. RORINSON L. ROBINSON This study This study This study This study This study

These strains are congenic (see RESULTS and MATERIALS AND METHODS). These strains are the product of the sixth serial backcross to JC482.

they contained the CDC25 (Ura') plasmid. Those containing pAP54 10 were thermoresistant while all those from diploids transformed with pAP5410-1, -2, -5 and -10 were thermosensitive. These results confirmed that we had cloned the four cdc25 ts alleles and that each is located in the BamHI-BamHI (PvuII) fragment of these genes.

The BamHl-BamHI fragment from plasmids pAP5411-1,-2,-5and-lOwereclonedintoM13mpl8 for DNA sequencing. We found that the coding sequence of each fragment from plasmids pAP5411-1, -2, -5 and -10 was identical to the published sequence (CAMONIS et al. 1986; BROEK et al. 1987) except for one nucleotide change in each ts allele (Figure 2).

Three nucleotide changes were located in the GAA codon at residue 1328 of the CDC25 open reading frame, very close to the KpnI site. In both cdc25-2 and cdc25-5 alleles, the first codon base G is replaced by an A and leads to the replacement of the original glutamic acid by a lysine. In the cdc25-IO allele, the second base A of the same codon is replaced by a T resulting in replacement by valine. The cdc25-1 mutation lies closer to the 3' end of the coding region and replaces the second base C of the 1403 GCT codon with a T . This new codon specifies a valine instead of an alanine.

A probable secondary structure of the CDC25 protein was predicted by the algorithm of GARNIER, OC- SUTHORPE and ROBSON (1 978) (data not shown). Ex- amination of alterations due to cdc25 mutations

showed that both substitutions in the 1328 GAA codon eliminate the probability of having an a-helix in this region of the protein. The nucleotide change in the 1403 GCT codon of cdc25-1 does not affect the predicted structure.

Phenotypic characterization of the cdc25 ts mutants: The CDC25 gene product has been implicated in the regulation of CAMP metabolism and has been proposed to modulate the stimulation of adenylate cyclase (CDC35) by the RAS proteins (ROBINSON et al. 1987; BROEK et al. 1987; MARSHALL et al. 1987). If this model is correct, one would predict that cdc25 mutants would have the same phenotype as ras2 and cdc?5/cyrl mutants. Yeast strains lacking the RAS2 protein hyperaccumulate the storage carbohydrates glycogen and trehalose and show a growth defect on the nonfermentable carbon sources ethanol and glycerol (TATCHELL, ROBINSON and BREITENBACH 1985; TODA et al. 1985; FRAENKEL 1985; CANNON, GIBBS and TATCHELL 1986). We have observed that some but not all alleles of cdc25 and cdc35 have similar traits (unpublished data).

In order to accurately compare the three different alleles of cdc25 with ras2 and cdc35 mutations we constructed a set of congenic strains differing only at the cdc25 locus (Table 1, MATERIALS AND METHODS). The wild-type uC482) and congenic strains were grown on rich medium (YEPD) in parallel with a ras2- 530 mutant and two different cdc?5 mutants. The plates were replicated onto media containing various

cdc25 gene from S. cereuisiae 80 1

1 hb +3972 + 4 0 1 4

A - ACT ATG TTT GAA TGC TTG GAT AGG GCA TGG [-lAAG TAT

S H S c HBg B K Thr Met Phe Glu Cys Leu Asp Arg Ala Trp Gly Thr LYS Tyr

1338 ...

1325 ...

GTA

CDC25 Val cdc25-10

* AAA Lys cdc25-2, cdc25-5

c s s

Ylo5 s FY

t c c- CDC2S URA3 CDC2S

FIGURE I.-Mapping and cloning of the cdc25 ts alleles. A) Restriction map of the CDC25 Sall-PvuII fragment subcloned in plasmids pAP54 10 and pAP5411. Note that in these constructions the 3' PvuII site of CDC25 is replaced by a BarnHI site as indicated by Pv (B) on the figure. The CDC25 open reading frame is indicated by hatching. B) Mapping by gap repair. Deletions and double strand breaks introduced within the CDC25 coding sequence of plasmid pAP541 I are represented by the-interrupted lines. C) Cloning of the cdc25 ts alleles. Structure of the cdc25 locus in stable Ura+ cdc25 ts transformants after integration of plasmid pAP5411 linearized with KpnI. The integrated YIp5 plasmid sequences are represented by the thick line separating the tandem cdc25 genes. Arrows indicate the direction of transcription. Abbreviations used are as follows: B, BamHI; Bg, BgllI; H, HpaI; K, KpnI; Pv, PvuI1; S, SalI; Sc, SacI.

carbon sources and their growth and glycogen storage characteristics were scored at different temperatures (Figure 3). On glucose medium (YEPD) all the cdc25 mutants grew as well as the wild-type strain JC482 at the permissive temperature of 26 " . Furthermore, no difference was observed in growth rate in liquid cul- ture, although all mutant cells were smaller in size than wild-type cells in stationary phase. Also, as shown previously, all had a longer lag phase when diluted into fresh liquid YEPD medium (IIDA and YAHARA 1984). Allelic differences were clearly observed for thermosensitivity. The growth of the cdc25-5 mutant was already impaired at 34" and totally blocked at 36" while both the cdc25-10 mutant and the cdc25-1 mutant required a higher temperature to fully arrest (Figure 3).

Like the ras2-530::LEU2 mutant, the cdc25 and cdc35 mutants have a defect for growth on the nonfermentable carbon sources glycerol and ethanol (Fig- ure 3). The severity and thermosensitivity of the defect are allele-specific. ras2-530::LEU2, cdc25-l and

+4186 t 4 2 2 7

TCA ATG ACT GCC ATA GTG TCC GCT TTG TAT TCC TCC CCA ATC Ser Met Thr Ala Ile Val Ser Ala Leu Tyr Ser Ser Pro Ile ...

1396 ... 1409

Val cdcZ5-1 GTT

FIGURE 2,"Sequence of the cdcZ5 ts alleles. The upper line is the nucleotide sequence of the wild-type CDC25 coding strand. Nucleotides and amino acid residues are numbered from the ATG initiator codon according to the sequence of BROEK et al. (1987). The sequence in bold letters is the KpnI recognition site. The lower line is the deduced amino acid sequence. The positions of the nucleotide changes in the four cdc25 ts alleles are indicated by an asterisk. The nucleotides and amino acid substitutions corresponding to the ts mutations are shown below the wild-type sequence.

cdc35-13 mutants show a reduction in growth rate at 26" whereas this trait becomes apparent only at the semipermissive temperature 30" for the other mutants (Figure 3). Even at 30°, the growth of the cdc25- 5 mutant is totally inhibited, while the cdc25-10 and cdc35-10 mutants show only a slight reduction of growth.

cdc25, cdc35 and ras2-530::LEU2 mutants grown on YEPD glucose medium accumulated more glycogen than the wild-type strain JC482 (Figure 3). Direct measurement of glycogen levels in cells grown to stationary phase in liquid YEPD medium at 26" (Fig- ure 4A) confirmed our qualitative observations. Like the ras2-530 mutant, all the cdc25 and the cdc35 mutants accumulate more glycogen than the wild-type strain JC482. In exponential phase, the glycogen content of cdc25 mutants cells was already much higher than that of the wild-type cells (data not shown).

Acid phosphatase levels were also measured in the mutants. MATSUMOTO, UNO and ISHIKAWA (1984a) showed that cyrllcdc35 mutants fail to derepress the acid phosphatase encoded by p H 0 5 under conditions of phosphate starvation and that de novo synthesis of this enzyme is controlled by CAMP-dependent phos- phorylation. We assayed the level of pH05 derepression in crude extracts of strains grown in low phosphate medium at 26". As shown in Figure 4B, the level of acid phosphatase derepression is always lower in the cdc25, cdc35 and ras2-530 cells than in the wild- type JC482 strain. The lack of derepression closely parallels the level of glycogen hyperaccumulation and


JC482

AP121

AP124

AP127

JC302-26B

LR684-4C

LR833-3A

0 0

temperature sensitivity. For example, at 26" the cdc25-I mutant has the highest content of glycogen, the lowest level of p H 0 5 activity and the slowest growth rate on nonfermentable carbon sources.

Adenylate cyclase activities: The CYR I lCDC35 gene encodes the catalytic subunit of yeast adenylate cyclase (MATSUMOTO, UNO and ISHIKAWA 1984b; BOUTELET, PETITJEAN and HILCER 1985; KATAOKA, BROEK and WICLER 1986) and evidence indicates that the RAS proteins mediate adenylate cyclase regulation by guanine nucleotides (TODA et al. 1985; BROEK et al. 1985; FIELD et al. 1987). It has been proposed that CDC25 participates in this regulation by modulating the exchange of the guanine nucleotides bound to the RAS proteins (ROBINSON et al. 1987; BROEK et al. 1987; DANIEL et al. 1987). We further analyzed this hypothesis by testing how the three different cdc25 alleles affect the adenylate cyclase activity.

To compare the relative proportion of active GTP- bound RAS proteins present i n vivo in the cdc25 mutants we measured the adenylate cyclase activities in the presence of Mg2+ without the addition of guanine nucleotide (CASPERSON et al. 1983; MARSHALL et al. 1987; FIELD et al. 1987). We further assayed the ability of guanine nucleotide to maximally stimulate the Mg2+-dependent adenylate cyclase activity in the same mutants by adding the hydrolysis resistant GTP analog, guanosine P,y-imidotriphosphate [Gpp(NH)p] (CASPERSON et al. 1983; FIELD et al. 1987). The adenylate cyclase catalytic activity was measured in the presence of Mn2+. All activities were assayed on membrane preparations from exponentially growing cells on SD-glucose medium at 26". The results are pre- sented in Table 2. The RAS-dependent adenylate cyclase activity, assayed with Mg'+, was markedly re-

#b W

'r ', ,_

Wild-Type

cdc25- 10

cdc25- 1

cdc25-5

ras2-530

cdc35- 10

cdc35- 13

FIGURE 3.-Phenotypic characterization of isogenic wild-type, cdc25. cdc35 and ras2 strains. The wild-type strain JC482 and the congenic mutants were patched on a master plate that was replica plated to the various media shown. The plates are (from left to right): YEP- glucose at the permissive temperature 26". the semipermissive temperature 30" and the restrictive temperature 36": YEP-ethanol at 26" and 30": YEP-glycerol at 26" and SO": and iodine vapor-stained cells grown on YEP-glucose at 26" and 30". All plates were incubated i n parallel for two days at the stated temperature.

duced from the wild-type in all cdc25 mutants. The allelic variation of the cdc25 mutations, apparent from the phenotypic analysis, was also observed for the adenylate cyclase activity. While the cdc25-1 and cdc25-5 mutations strongly affected both the basal (Mg2+) and the stimulated [Mg'+ + Gpp(NH)p] RAS- dependent adenylate cyclase activity, the cdc25-10 mutation had a relatively weaker defect. In fact, the Gpp(NH)p ability to stimulate the cyclase in the cdc25- 10 mutant membranes was comparable to wild-type since the ratio between their catalytic activity (Mn2+) and their stimulated RAS-dependent activity [Mg2+ + Gpp(NH)p was about the same. However, in all the cdc25 mutants, the catalytic activity assayed in the presence of Mn2+ was impaired.

The cdc25 mutations have a more dramatic effect on the adenylate cyclase activity in membranes pre- pared from strains grown exponentially at 26" and shifted to 36". As shown in Table 2, the adenylate cyclase activities of the wild-type strain remain un- changed whereas the temperature shift causes a de- crease of activity in all cdc25 mutants. This in v ivo thermosensitivity is particularly significant in the cdc25-10 mutant which lost almost all its basal and Gpp(NH)p stimulated RAS-dependent activity. The impaired activities measured in the cdc25 mutants in the presence of Mg2+ confirmed that the CDC25 protein modulates the RAS-dependent adenylate cyclase activity. Nevertheless, the cdc25 mutations also affect the ability of Gpp(NH)p to stimulate adenylate cyclase in vitro (Table 2) since addition of guanine nucleotides is carried out under conditions that allow free exchange of guanine nucleotides bound to the RAS proteins (MARSHALL et d . 1987; FIELD et d . 1987).

cdc25 gene from S. cerevisiae 803

DISCUSSION

FIGURE 4.-Glycogen accumulation and pH05 acid phosphatase derepression in cells of isogenic wild-type, cdc25, cdc35 and rus2 strains grown at 26". A) Glycogen levels measured in cells grown to stationary phase in liquid YEP-glucose medium at 26". B) Acid phosphatase derepression in cells grown in low Pi medium to late exponential phase at 26". The pH05 activity is expressed as mi- cromoles p-nitrophenol formed using p-nitrophenylphosphate as the substrate. Comparable phosphatase derepression has been observed for cdc25-IO and cdc35-10 mutants in their original genetic background isogenic to BeGX2. In both graphs, the results are the average of two determinations made with two independent cultures of each strain.

It has been reported that a cdc25-1 strain does not show a reduction in intracellular cAMP levels when the strain is shifted to the restrictive temperature (MARTEGANI, BARONI and WANONI 1986). In contrast, cAMP levels in a cdc25-5 strain have been reported to fall at the restrictive temperature (CAMONIS et al. 1986). A cdc25 gene disruption also has low intracellular cAMP levels (BROEK et al. 1987). We have measured cAMP levels in our strains at permissive and restrictive temperatures and observe a drop in cAMP levels within 10-30 min of shifting cells to 36". A similar drop was observed in all cdc25 mutants (data not shown).

The CDC25 gene contains a 1587 amino acid open reading frame coding for a protein of 180 kD (CA- MONIS et al. 1986; BROEK et U L . 1987). Our observation that four missense mutations in cdc25 reside in this open reading frame confirms the assignment of the gene product to this frame. These mutations are tightly clustered in the carboxy terminus of the protein. cdc25-2, cdc25-5 and CDC25-10 all are missense mutations in glutamic acid codon 1328 while the cdc25-I missense mutation is in alanine codon 1403. The close proximity of these four mutations under- scores the importance of the carboxy terminus of the CDC25 protein in biological function. Previous stud- ies have shown that a fragment encoding only the C- terminal 7 12 amino acids is required to complement cdc25 mutations, albeit at high gene dosage (CAMONIS et al. 1986; MARTECANI et al. 1986; BROEK et al. 1987). Some gene disruptions that leave the 3' end of the CDC25 gene intact are also partially functional (MUNDER, MINK and KUNTZEL 1988; L. C. ROBINSON and A. PETITJEAN, unpublished observations).

In this study we find no unique traits associate with RAS or CDC25 that cannot be found for adenylate cyclase mutants. The growth deficiency on nonfermentable carbon sources described for ras2 mutants (TATCHELL, ROBINSON and BREITENBACH 1985; FRAENKEL 1985) is also found in cdc25 and cdc35 mutants and the failure of cyrllcdc35 mutants to express acid phosphatase (MATSUMOTO, UNO and ISHI- KAWA 1984a) is also observed for cdc25 and ras2 mutants.

Based on analysis of disrupted and deletion alleles of cdc25, MUNDER, MINK and KUNTZEL (1988) have proposed that the CDC25 protein consists of three functional domains, the amino-terminal of which is required for growth on nonfermentable carbon sources. We have shown here that missense mutations in the carboxy terminus of CDC25 convey a condi- tional lethal phenotype and also result in the failure to grow on nonfermentable carbon sources at the permissive or semipermissive temperature. The fact that the gluconeogenic growth defect for cdc25 and ras2 alleles is shared by some alleles in the adenylate cyclase gene suggests to us that the defect is due to impairment of adenylate cyclase activity and not nec- essarily to a separate function for cdc25 or ras2.

The cdc25-5 and cdc25-IO mutations are missense mutations in GAA codon 1328. The glutamic acid codon in the wild type is replaced with valine in cdc25- 10 or lysine in cdc25-5. Strains that possess these mutations behave as ts mutants. At the permissive temperature they exhibit close to a wild-type phenotype and nearly normal adenylate cyclase activity while at the restricitve temperature they rapidly arrest and possess low adenylate cyclase activity. In contrast,

804 A. Petitjean, F. Hilger and K.Tatchel1

TABLE 2

Adenylate cyclase activities in membrane preparations from wild-type, cdc25, cdc35 and ras2 mutants ~ ~ ~

Cells grown at 26" Cells shifted at 36"

Mg" + Mg2+ + Strain Relevant genotype Mn'+ Mg2+ GPP(NH)P Mn2+ Mg'+ Gpp(NH)p

~ ~ ~

JC482 CDC25 RASZ CDC35 40.0f 4.4 12.8 f 1.6 29.7 f 4.4 43.1 f 3.1 14.5 f 0.7 28.9 & 0.8 AP121 cdc25-10 RAS2 CDC35 21.8 f 1.9 5.7 f 0.6 20.6 f 3.0 12.2 f 1.2 1.3 f 0.1 2.2 f 0.3 AP124 cdc25-I R A S Z CDC35 13.5 2 1.1 0.7 f 0.2 2.4 f 0.7 0.3 f 0.2 0.6 2 0.1 AP127 cdc25-5 RAS2 CDC35 14.9 f 1.4 0.6 & 0.1 2.6 f 0.4 11.3 & 1.4 0 . 4 f 0.1 0.7 f 0.04

~~

JC302-26B CDC25 ras2-530 CDC35 24.3 f 2.9 6.1 f 0.7 15.9 f 1.8 18.8 f 0.9 2.8 f 0.3 6.7 2 0.5 LR684-4C CDC25 RAS2 cdc35-IO 8.6 f 1.0 1.5 & 0.4 2.4 f 0.3 0.1 f 0.01 0.2 f 0.1

Enzyme activities are expressed in pMoles of CAMP formed per minute per mg of membrane protein. The reaction mixture contains 1 mM Mn'+ or 3.5 n1M M$+ or 3.5 mM M$+ and 100 p~ Gpp(NH)p and incubated 30 min at 26". All assays were run in duplicate with two different amounts of membrane protein. The results are the average of the activities measured in the same experiment, for two independent membrane preparations of each strain.

cdc25-I is not strictly a ts mutant. It is defective at all temperatures and has greatly impaired adenylate cyclase activity at 26'.

The CDC25 protein may provide GTP-GDP exchange for RASl and RAS2. Evidence for such a model is that only the GTP-bound forms of RASl or RAS2 stimulate adenylate cyclase (FIELD et al. 1987, 1988) and that the essential requirement for CDC25 function is eliminated in strains that express RAS genes with impaired GTPase activity (ROBINSON et al. 1987; BROEK et al. 1987) or reduced affinity for guanine nucleotides (CAMONIS and JACQUET 1988). POWERS, O'NEILL and WICLER (1989) have shown that the mutant phenotype caused by certain dominant alleles of R A S 2 can be reversed by the overex- pression of CDC25. All these results point to a model in which RAS and CDC25 interact directly.

Although the favored hypothesis for CDC25 is one to facilitate GTP/GDP exchange, not all our data is strictly consistent with this model. Adenylate cyclase activity measured in the presence of Mn2+ has been proposed to represent full catalytic activity (CASPER- SON et al. 1983), yet this activity is impaired in all three mutants (Table 2). Second, if CDC25 plays the role of a guanine nucleotide exchange factor for RAS, the addition of a nonhydrolyzable GTP analog [Gpp(NH)p] to membrane preparations should fully restore Mg*+-dependent activity. Gpp(NH)p fails to stimulate Mg2+-dependent adenylate cyclase at 26" and 37" in cdc25-1 and cdc25-5. DANIEL et al. (1987) have also observed a loss of Gpp(NH)p stimulated activity after membrane preparations from strains were incubated at the restrictive temperature. In cdc25-IO at 26", Gpp(NH)p stimulates adenylate cyclase to the level of Mn2+ but the catalytic activity is only half that of wild type. BROEK et al. (1987) have not observed a similar reduction in adenylate cyclase activities in their cdc25 deficient strains. Differences in strain background or biochemical methods might account for the differences. These data clearly do not

eliminate the possibility that CDC25 is an exchange factor for RAS but clearly suggest that other models must be considered. For example, CDC25 could serve a structural role by facilitating the association between RAS and adenylate cyclase or serve to direct adenylate cyclase or RAS to the proper subcellular location. The low levels of adenylate cyclase activity we observe in cdc25 mutants could simply be due to low concentration of adenylate cyclase present in our membrane preparations. In any case, if such a direct interaction does occur it is likely that such an interaction is with the carboxy terminal domain of CDC25.

A growing number "RAS related" genes have been identified in yeast: YPT (GALLWITZ, DONATH and SANDER 1983), RHO1 and RH02 (MADAULE, AXEL and MYERS 1987), and SEC4 (SALMINEN and NOVICK 1988). The gene products have guanine nucleotide binding properties similar to RAS but clearly function in different pathways. Is it possible that these other guanine nucleotide binding proteins require a function similar to that played by CDC25 for RASl and RAS2? In this regard it is of interest that two yeast genes have been found that are strikingly similar to CDC25. In a comparison with a protein data base the presumptive product of the yeast LTEl gene was found to be (WICKNER et al. 1987) similar to the carboxy-terminal portion of CDC25 (Figure 5). Over 60% of the LTEl open reading frame can be aligned with the carboxy-terminal part of the CDC25 protein. There is 22% identity between LTEl and CDC25, 38% with conservative replacements. The greatest homology with CDC25 is in the region containing the ts mutations. In the optimal alignment between CDC25 and LTEl the amino acid that is altered by the cdc25-1 mutation (alanine 1403) is also alanine in LTEl (alanine 172). SCD25, a gene that complements cdc25 when present on a multicopy plasmid (BOY- MARCOTTE et al. 1989), is even more similar to CDC25 than LTEl. As with LTEl , the similarity is confined to the carboxy terminus of CDC25 and the location

cdc25 gene from S. cerevisiae 805

&25-2, 5, 10 U

c~c25 aa 1240 QKINEKLINENEKEPVDPKQQDSVSAWQTTKRDNKSPIHMSSSSLPSSASSAFFRLKKLKLLDIDPYTYATQLTVLEHD----LYLRITMFECLD~WG * * I * I * I * * I l l * I I I I * I * 1 I I I * * I

L E I aa8 DQSEKEGFTSSSPEKIDVSANvDVAVaGEIQELIGQYRIHDSRLMISNNESHVPFILMYDSLSVAQQMTLIEKEILGEIDWKDLLDLKMKHEGPQVISWL

cdc25-1 II

13% TKYCNMGGSPNITKFIANANTLTNFVSHTIVKQADVKTRSKLTQYFVTVAQHCKELNNFSSMTAIVSALYSSPIYRLKKTWDLVSTESKDLLKNLNNLMD * * I I * I *** I * I * * I I * I I * I I * * I I * * I * I I I I * * I I * * * I *

LEI aa QLLVRNETLSGIDLAISRFNLTVDWIISEILLTKSS~K~IQRFIHVADHCRTFQNFNT~EIIIALSSSWQFKTDAWRLIEPGDLLTWEELKKIPS

CDC25 aa 14% SKRNFVKYRELLRSVTD-VACVPFFGVYLSDLTFTFVGNPDFLHNSTNIINFSKRTKIANIVEEIISFKRFHYKLKRLDDIQTVIEASLENVPHIEKQYQL l I * I * I I 1 1 I * I I I I l l l l l l * I * * * I * I * I t * ) * * * * I I

L E 1 208 LDRNYSTIRNLLNSVNPLVGCVPFIWYLSDLSANAEKKDWILED--KWNYNKFDTNVQIVKNFIQRVQWSKFYTFKVNHELLSKCVYISTLTQEEINEL

FIGURE 5.-Similarity between LTEl and CDC.25. The amino acid sequence of the LTEl and CDC25 proteins are aligned using the algorithm Of WILBUR and LIPMAN (1983). Lines between amino acids designate identities and stars designate conservative amino acid changes. Conservative amino acid changes are defined by the following groups of amino acids: (L, I, V, M), (A, G, P, S, T), (Q, D, E, N), (R, K, H), (F, Y , W). The location of the cdc25 missense mutations are located with arrows above the CDC25 sequence. The amino acid coordinate system of BROEK et al. ( 1 987) is used.

of the cdc25 missense mutations are within the con- served region. Could LTEl and SCD25 carry out a “CDC25-like” activity for other guanine nucleotide binding proteins such as YPTl , RHOl , RH02 and SEC4? Little is known about the biological function of LTEl and SCD25; only that a disruption of LTEl results in the inability to grow at low temperature (WICKNER et al. 1987). Additional genetic and biochemical analysis of these genes will be necessary to answer this question.

We thank L. ROBINSON for useful discussions during the course of this work. We thank MICHEL WERNER, LUCY ROBINSON and SANDRA THOMPSON-JAEGER for critically reading the manuscript and B. HORION for technical assistance with the glycogen determinations. We thank LUCY ROBINSON and MICHEL JACQUET for gen- erously providing strains and plasmids. We also appreciate M. WERNER’S assistance with DNA sequencing and J. DUMONT’S advice for homology searches. A.P. is a Senior Research Assistant of the Belgian F.N.R.S.. This work was supported in part by U.S. Public Health Service grant CA37702 from the National Cancer Institute awarded to K.T. This is paper #I 1843 of the Journal Series of the North Carolina Agriculture Research Service, Raleigh, North Car- olina.

LITERATURE CITED

BOEKE, J. D., F. LACROUTE and G. R. FINK, 1984 A positive selection for mutants lacking orotidine-5’-phosphate decarbox- ylase activity in yeast: 5-fluoro-orotic acid resistance. Mol. Gen. Genet. 197: 345-346.

BOUTELET, F., A. PETITJEAN and F. HILGER, 1985 Yeast cdc35 mutants are defective in adenylate cyclase and are allelic with cyrl mutants while CASI, a new gene, is involved in the regulation of adenylate cyclase. EMBO J. 4: 2635-2641.

BOY-MARCOTTE, E., F. DAMAK, J. CAMONIS, H. GARREAU and M. JACQUET, 1989 The C-terminal part of a gene partially homologous to CDC25 gene suppresses the cdc25-5 mutation in Saccharomyces cereuisiae. Gene 77: 2 1-30.

BROEK, D., 0. SAMIY, 0. FASANO, A. FUJIYAMA, F. TAMANOI, J. NORTHUP and M. WIGLER, 1985 Differential activation of yeast adenylate cyclase by wild-type and mutant RAS proteins. Cell 41: 763-769.

BROEK, D., T . TODA, T. MICHAELI, L. LEVIN, C. BIRCHMEIER, M.

ZOLLER, S. POWERS and M. WIGLER, 1987 The S. cereuisiae CDC25 gene product regulates the RAS/adenylate cyclase pathway. Cell 48: 789-799.

CAMONIS, J. H., and M. JACQUET, 1988 A new mutation that suppresses the CDC.25 gene requirement for growth of Saccha- romyces cerevisiae. Mol. Cell. Biol. 8: 2980-2983.

CAMONIS, J. H., M. KAKELINE, G. BERNARD, H. GARREAU, E. BOY- MARCORE and M. JACQUET, 1986 Characterization, cloning and sequence analysis of the CDC25 gene which controls the cyclic AMP level of Saccharomyces cereuisiae. EMBO J. 5 375- 380.

CANNON, J. F., J. G. GIBBS and K. TATCHELL, 1986 Suppressors of the ras2 mutation of Saccharomyces cereuisiae. Genetics 113:

CANNON, J. F., and K. TATCHELL, 1987 Characterization of Sac- charomyces cereuisiae genes encoding subunits of cyclic AMP- dependent protein kinase. Mol. Cell. Biol. 7: 2653-2663.

CASPERSON, G., N. WALKER, A. BRASIER and H. BOURNE, 1983 A guanine nucleotide-sensitive adenylate cyclase in the yeast Sac- charomyces cereuisiae. J. Biol. Chem. 258: 7931-7914.

DANIEL, J., J. M. BECKER, E. ENARI and A. LEVITZKI, 1987 The activation of adenylate cyclase by guanyl nucleotides in Saccha- romyces cereuisiae is controlled by the CDC25 start gene product. Mol. Cell. Biol. 7: 3857-3861.

FIELD, J., D. BROEK, T. KATAOKA and M. WIGLER, 1987 Guanine nucleotide activation of, and competition between, RAS proteins from Saccharomyces cereuisiae. Mol. Cell. Biol. 7: 2128- 2133.

FIELD, J., J. NIKAWA, D. BROEK, B. MACDONALD, L. RODGERS, I. A. WILSON, R. A. LERNER and M. WIGLER, 1988 Purification of a RAS-responsive adenylyl cyclase complex from Saccharo- myces cereuisiae by use of an epitope addition method. Mol. Cell. Biol. 8: 2 159-2 165.

FRAENKEL, D. G., 1985 On ras gene function in yeast. Proc. Natl. Acad. Sci. USA 82: 4740-4744.

GALLWITZ, D., C. DONATH and C. SANDER, 1983 A yeast gene encoding a protein homologous to the human c-haslbas proto- oncogene product. Nature 306 704-707.

GARNIER, J., D. J. OSGUTHORPE and B. ROBSON, 1978 Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J. Mol. Biol. 120 97-120.

GILMAN, A. G., 1970 A protein binding assay for adenosine 3’,5’- monophosphate. Proc. Natl. Acad. Sci. USA 67: 305-312.

GILMAN, A. G. , 1984 G proteins and dual control of adenylate cyclase. Cell 3 6 577-579.

247-264.

806 A. Petitjean, F. Hilger and K.Tatchel1

GUNJA-SMITH, Z . , N. B. PATIL and E. E. SMITH, 1977 Two pools of glycogen in Saccharomyces. J. Bacteriol. 130: 818-825.

HARTWELL, L. H., R. K. MORTIMER, J. CULOTTI and M. CULOTTI, 1973 Genetic control of the cell division cycle in yeast: V. Genetic analysis of cdc mutants. Genetics 74: 267-286.

IIDA, H., and I . YAHARA, 1984 Specific early-Gl blocks accom- panied with stringent response in Saccharomyces cerevisiae lead to growth arrest in resting state similar to the GO of higher eucaryotes. J. Cell Biol. 98: 1185-1 193.

ITO, H., Y. FUKUDA, K. MURATA and A. KIMURA, 1983 Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 153: 163-168.

KATAOKA, T., D. BROEK and M. WIGLER, 1986 DNA sequence and characterization of the S. cerevisiae gene encoding adenylate cyclase. Cell 43: 493-505.

LECRAIN, M., M. DE WILDE and F. HILGER, 1986 Isolation, phys- ical characterization and expression analysis of the Saccharo- myces cerevisiae positive regulatory gene P H 0 4 . Nucleic Acids Res. 14: 3059-3073.

LOWRY, 0. H., N. J. ROSEBROUGH, A. C. FARR and R. J. RANDALL, 1951 Protein measurement with Fohn reagent. J. Biol. Chem. 193: 265-275.

MADAULE, P., R. AXEL and A. M. MYERS, 1987 Characterization of two members of the rho gene family from the yeast Saccha- romyces cerevisiae. Proc. Natl. Acad. Sci. USA 84: 779-783.

MANIATIS, T., E. F. FRITSCH and J. SAMBROOK, 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

MARSHALL, M., J. B. GIBBS, E. M. SCOLNICK and I. SIGAL, 1987 Regulatory function of the Saccharomyces cereuisiae RAS C-terminus. Mol. Cell. Biol. 7: 2309-2315.

MARTEGANI, E., M. BARON] and M. WANONI, 1986 Interaction of cAMP with the CDC25-mediated step in the cell cycle of bud- ding yeast. Exp. Cell Res. 162: 544-548.

MARTECANI, E., M. D. BARONI, G. FRASCOTTI and L. ALBERGHINA, 1986 Molecular cloning and transcriptional analysis of the start gene CDC25 of Saccharomyces cereuisiae. EMBO J. 5: 2363- 2369.

MATSUMOTO, K., I . UNO and T. ISHIKAWA, 1983 Initiation of meiosis in yeast mutants defective in adenylate cyclase and cyclic AMP-dependent protein kinase. Cell 32: 417-423.

MATSUMOTO, K., I . UNO and T . ISHIKAWA, 1984a Regulation of repressible acid phosphatase by cyclic AMP in Saccharomyces cerevisiae. Genetics 108: 53-66.

MATSUMOTO, K., I . UNO and T. ISHIKAWA, 1984b Identification of the structural gene and non-sense alleles for adenylate cyclase in Saccharomyces cerevisiae. J. Bacteriol. 157: 277-282.

MATSUMOTO, K., I . UNO, Y. OSHIMA and T. ISHIKAWA, 1982 Isolation and characterization of yeast mutants deficient in adenylate cyclase and CAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 79: 2355-2359.

MUNDER, T., M. MINK and H. KUNTZEL, 1988 Domains of the Saccharomyces cerevisiae CDC25 gene controlling mitosis and meiosis. Mol. Gen. Genet. 214 271-277.

ORR-WEAVER, T. L., J . W. SZOSTAK and B. L. CARTER, 1983 Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol. 101: 228-245.

PARENT, S. A., C. M. FENIMORE and K. A. BOSTIAN, 1985 Vector systems for the expression, analysis and cloning of DNA sequences in S. cerevisiae. Yeast 1: 83-138.

POWERS, S., K. O’NEILL and M. WIGLER, 1989 Dominant yeast and mammalian RAS mutants that interfere with the CDC25- dependent activation of wild-type RAS in Saccharomyces cereu- isae. Mol. Cell Biol. 9 390-395.

ROBINSON, L. C., J. B. GIBBS, M. S. MARSHALL, I. SIGAL and K. TATCHELL, 1987 CDC25: a component of the RAS-adenylate cyclase pathway in Saccharomyces cerevisiae. Science 235: 12 18- 1221.

ROSE, M. D., P. NOVICK, J. H. THOMAS, D. BOTSTEIN and G. FINK, 1987 A Saccharomyces cereuisiae genomic plasmid bank based on a centromere-containing vector. Gene 60: 237-243.

SALMINEN, A,, and P. J. NOVICK, 1987 A ras-like protein is required for a post-Golgi event in yeast secretion. Cell 49: 527- 528.

SANGER, F., and A. R. COULSON, 1978 The use of thin acrylamide gels for DNA sequencing. FEBS Lett. 87: 107-1 10.

SANGER, F., S. NICKLEN and A. R. COULSON, 1977 DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74: 5463-5469.

SHERMAN, F., G. R. FINK and J. B. HICKS, 1986 Methods in Yeast Genetics. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.

TATCHELL, K., D. T. CHALEFF, D. DEFEO-JONES and E. M. SCOL- NICK, 1984 Requirement of either of a pair of ras-related genes of Saccharomyces cereuisiae for spore viability. Nature 3 0 9

TATCHELL, K., L. C. ROBINSON and M. BREITENBACH, 1985 RASP of Saccharomyces cerevisiae is required for gluconeogenic growth and proper response to nutrient limitation. Proc. Natl. Acad. Sci. USA 82: 3785-3789.

TODA, T., I . UNO, T. ISHIKAWA, S. POWERS, T. KATAOKA, D. BROEK, S. CAMERON, J. BROACH, K. MATSUMOTO and M. WKG- LER, 1985 In yeast, RAS proteins are controlling elements of adenylate cyclase. Cell 40: 27-36.

and characterization of acid phosphatase mutants in Saccharo- myces cereuisiae. J. Bacteriol. 113: 727-738.

TRIPP, M. L., and R. PIGON, 1986 Control of the cAMP pathway by the cell cycle start function, CDC25, in Saccharomyces cerevisiae. J. Gen. Microbiol. 132: 1143-1151.

VAN DER PLAAT, J. B., and P. VAN SOLINGEN, 1974 Cyclic 3’,5’- adenosine monophosphate stimulates trehalose degradation in Baker’s yeast. Biochem. Biophys. Res. Commun. 5 6 580-587.

WICKNER, W. J., T . J. KOH, J. C. CROWLEY, J. O’NEIL and D. B. KABACK, 1987 Molecular cloning of chromosome 1 DNA from Saccharomyces cereuisiae: isolation of the gene and analysis of an adjacent gene essential for growth at low temperatures. Yeast 3: 51-57.

WILBUR, W. J., and D. LIPMAN, 1983 Rapid similarity searches of nucleic acid and protein data banks. Proc. Natl. Acad. Sci. USA 80: 726-730.

WINSTON, F., F. CHUMLEY and G. R. FINK, 1983 Eviction and transplacement of mutants genes in yeast. Methods Enzymol. 101: 211-228.

523-527.

TOH-E, A,, Y. WEDA, S. KAKIMoTOand Y. OSHIMA, 1973 Isolation

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