SDC25, A Dispensable Ras Guanine Nucleotide Exchange Factor of ...

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Molecular Biology of the Cell Vol. 7, 529-539, April 1996

SDC25, A Dispensable Ras Guanine Nucleotide Exchange Factor of Saccharomyces cerevisiae Differs from CDC25 by Its Regulation Emmanuelle Boy-Marcotte,* Pranvera Ikonomi, and Michel Jacquet Institut de Genetique et Microbiologie, URA Centre National de la Recherche Scientifique D1354, Universite Paris XI, Batiment 400, 91405 Orsay Cedex, France Submitted November 13, 1995; Accepted February 1, 1996 Monitoring Editor: Michael H. Wigler

The SDC25 gene of Saccharomyces cerevisiae is homologous to CDC25. Its 3' domain encodes a guanine nucleotide exchange factor (GEF) for Ras. Nevertheless, the GEF encoded by CDC25 is determinant for the Ras/cAMP pathway activation in growth. We demonstrate that the SDC25 gene product is a functional GEF for Ras: the complete SDC25 gene functionally replaces CDC25 when overexpressed or when transcribed under CDC25 transcriptional control at the CDC25 locus. Chimeric proteins between Sdc25p and Cdc25p are also functional GEFs for Ras. We also show that the two genes are differentially regulated: SDC25 is not transcribed at a detectable level in growth conditions when glucose is the carbon source. It is transcribed at the end of growth when nutrients are depleted and in cells grown on nonfermentable carbon sources. In contrast, CDC25 accumulation is slightly reduced when glucose is replaced by a nonfermentable carbon source. INTRODUCTION The 3' half of the SDC25 gene from the yeast Saccharomyces cerevisiae has been shown to encode the first guanine nucleotide exchange factor (GEF)V found to act in vitro on Ras proteins (Crechet et al., 1990). It was selected as a multicopy suppressor of mutations in the essential CDC25 gene of S. cerevisiae, which encodes the Ras GEF required to activate the Ras/cAMP pathway (Boy-Marcotte et al., 1989). When transfected into mammalian cells, it activates a Ras-dependent pathway (Rey et al., 1991), overcomes the dominant negative phenotype of H-rasN17 (Schweighoffer et al., 1993), and acts as an oncogene (Barlat et al., 1993a). The ability to make chimeric genes between the 3' part of CDC25 and SDC25 that encode active chimeric GEF on Ras was suggestive of a close relationship between the two genes (Boy-Marcotte et al., 1993). Nevertheless, the role of the complete SDC25 gene as a GEF for Ras in yeast was questionable because the deletion of SDC25 leads to no detectable phenotype, and the over* Corresponding author. Abbreviations used: GEF, guanine nucleotide exchange factor; PCR, polymerase chain reaction.

© 1996 by The American Society for Cell Biology

expression of the complete gene was not able to rescue the growth of cdc25 mutants (Damak et al., 1991). In this paper we have revisited the activity of the complete SDC25 gene and found that the version used in the previous experiments was cloned from a strain in which the 5' part contained an inactivating deletion. The existence of multiple GEFs for Ras has already been described in mammalian cells. They share in common a conserved catalytic domain of 250 amino acids (Ras GEF domain) similar to the catalytic

domain of the two GEFs of S. cerevisiae Cdc25p and Sdc25p (Boguski and McCormick, 1993). The best characterized are the two related GEFsoSl and GEFS0S2, which are the products of two distinct genes and are expressed ubiquitously in fetal and adult cell lines. These GEFs have a C-terminal extension able to bind the SH3 domains of the adaptators Grb2 and Shc. Their amino terminal regions contain a PH domain and a dbl-related sequence. These GEFs are thought to be recruited as a GEF 5-Grb2 complex close to Ras in the plasma membrane through the SH2 domain of the Grb2 adaptator, when growth factor tyrosine-kinase receptors are activated (Bowtell et al., 1992; Chardin et

al., 1993; Li et al., 1993). The second class of GEF 529

E. Boy-Marcotte et al.

present in mammals (CDC25 Mm, Ras-GRF, and HGRF) has been found by structural and functional homology with Cdc25p. They do not have the Cterminal extension found in GEFsos, but possess in their N-terminal region a PH domain and dbl-related sequence. These GEFs are mostly expressed in brain tissue (Martegani et al., 1992; Shou et al., 1992; Schweighoffer et al., 1993b). Recently an activation of the Ras-GRF has been described that occurs through a distinct non-tyrosine-kinase pathway, in response to Ca2+ influx and mediated by calmodulin (Farnsworth et al., 1995). A third type of Ras GEF named C3G was found in mammalian cells by its ability to bind the SH3 domain of the adaptator Crk-1 by its N-terminal domain. As GEFsos, it is expressed ubiquitously (Tanaka et al., 1994). This diversity of mammalian GEF points to the existence of various signalization pathways to Ras, which remain to be elucidated. In the yeast S. cerevisiae, although SDC25 and CDC25 are two related genes, the CDC25 gene product appears to be the determinant GEF for growth because mutations lead to a Gl arrest (Hartwell et al., 1973). The SDC25 gene is dispensable under usual growth conditions and the absence of a detectable phenotype associated with deletion of the gene did not reveal its biological function (Damak et al., 1991). Nevertheless, its conservation through the evolution suggests a biological role still to be elucidated. To progress in the understanding of the SDC25 physiological role, we asked for the ability of the complete SDC25 gene to functionally replace CDC25, and we investigated its transcriptional regulation. MATERIALS AND METHODS Strains, Plasmids, Media, and Yeast Methods Strains and plasmids used in this study are described in Table 1. Rich medium is 2% bactopeptone and 1% yeast extract with either 2% glucose (YPD), 2% ethanol (YPE), 2% glycerol (YPG), or 2% potassium acetate (YPA). Minimal medium (YNB) is 0.17% yeast nitrogen base without ammonium sulfate and amino acids, 0.5% ammonium sulfate, and 2% glucose, supplemented with the required amino acids and purine/pyrimidines at the indicated concentration (Rose et al., 1990). Sporulation medium was 0.5% yeast extract, 0.5% bacto peptone, and 2% potassium acetate. The lithium acetate method was used for yeast transformation (Rose et al., 1990). The cell concentration in the culture was estimated by turbidity at 710 nm with a Jenway 6061 colorimeter.

Oligonucleotides, DNA Preparation, DNA Amplification, and DNA Sequences The oligonucleotides used in this study are presented in Table 2. Genomic and plasmid DNA were prepared from yeast cells as described (Hoffman and Winston, 1987). Usually, amplification was done with 1 jig of genomic DNA or 10 ng of plasmid DNA by 1.5 ,u Taq-polymerase (Appligene, Heidleberg, Germany) in conditions described by the supplier, in a Perkin-Elmer 480 thermo-cycler apparatus (Norwalk, CT). Nucleotide sequences were determined on double-stranded DNA by automated DNA sequencing, as previously described (Demolis et al., 1993), using the "Prism Kit" 530

(Applied Biosystems, Foster City, CA). The sequence of the 5' part of SDC25 gene on the pRG3 plasmid and on genomic DNA from strains OL136, W303, and FY1679 was done on amplimers generated by polymerase chain reaction (PCR) with the primers olSDC1648 and olSDC2220. The 19-bp deletion was assessed by PCR with the primers olSDC1752 and olSDC2220 on the genomic DNA from OL129-1A, OL120-2A, and OL124-2A strains.

Northern Blotting Total RNA was prepared from yeast cells as previously described (Schmitt et al., 1990). Poly A' RNA was purified in one step on oligo-dT cellulose. Northem blotting was carried out with glyoxalated RNA as described (Sambrook et al., 1989) except DMSO was omitted. When DNA probes were used, hybridization was performed at 65°C in "Church" buffer (Church and Gilbert, 1984); washing was performed at 55'C in 0.2x SSC, 0.1% SDS. When riboprobes were used, hybridization was done in 0.75 M NaCl, 0.15 M Tris, pH 8, 10 mM EDTA, 0.2 M phosphate buffer, pH 6.8, 12% formamide; washing was performed at 65°C in 0.1 x SSC, 0.1% SDS. Double-stranded probes were radiolabeled with [a32P]dCTP either by random priming using the Ready to Go DNA labeling kit (Pharmacia, Piscataway, NJ) or by PCR amplification with 100 ng DNA template, 40 nM primers, and 30 ,uCi [Ia32P]dCTP (800Ci/ mmol or 3000 Ci/mmol) with 1.5 ,t Taq polymerase (Appligene) with buffer indicated by the supplier for 10 cycles. The riboprobes were synthesized with T7 RNA poymerase with an in vitro run off transcription reaction using the protocol described by Promega, which is a modification of the one described by Melton (Melton et al., 1984).

RESULTS SDC25 and CDC25 Genes Are under Different Transcriptional Regulation The level of accumulation of both CDC25 and SDC25 mRNA has been compared by Northern blot analysis under different growth conditions. As shown in Figure 1, CDC25 was expressed during exponential growth in rich medium containing glucose. In contrast, SDC25 mRNA remained undetectable during this period. At the end of the growth phase, when glucose is exhausted, SDC25 mRNA started to be accumulated and later in stationary phase it reached a much higher level. In contrast, the CDC25 mRNA level was reduced when growth was arrested and remained low as already described (Russel et al., 1993). As an anabolic control, the level of TRP1 mRNA was drastically reduced in late stationary phase. Induction of the SDC25 mRNA accumulation was also observed at the end of growth on minimal medium when the limiting nutrient is not glucose as in rich medium but an unknown component (our unpublished results). The expression of SDC25 was also followed on different carbon sources. SDC25 mRNA was expressed when cells were shifted from glucose to ethanol (Figure 2) and continued to be accumulated with ethanol as a carbon source. During the transition, a gene encoding a ribosomal protein URPI (Demolis et al., 1995) was transiently repressed and re-expressed to a lower level than on glucose, which is the behavior described for many genes encoding ribosomal proteins (WoolMolecular Biology of the Cell

Regulation and Function of the SDC25 Gene Table 1. S. cerevisiae strains and plasmids used in this study

Strains

Genotype

Source/references

JCL300-3A OL1 OL97 OL97.1-11B OL120-2A OL124-2A OL129-1Aa

Mata cdc25::HIS3, (YEpCDC25, LEU2) ade2, his3, leu2, lysl, trpl Mata leu2, his3, ura3 Mata, cdc25-5, ade2, arg4, his7 MATa, cdc25-5, leu2, his3, his7, ura3 MATa, cdc25-5, adel, ural, rcal MATa cdc35-10 ural, his, arg4, rcal MATa, cdc25-5, cdc35-10, his7, ural, rcal MATa cdc25-5, cdc35-10, his7, ural, rcal, ICYI MATa ade2, ade8, his3, leu2, trpl, ura3 MATa/MATa, cdc25-5/cdc25-5, ade-(?)/ADE arg4/ARG4, leu2/leu2, ura3/ura3

(Van Aelst et al., 1990) (Boy-Marcotte and Jacquet, 1982) M. Jacquet collection (Camonis and Jacquet, 1988) M. Jacquet collection M. Jacquet collection M. Jacquet collection (Boy-Marcotte et al., 1989) (Nasmyth, 1985) M. Jacquet collection (Thierry et al., 1995)

OL136b W303-1B DOL97

FY1679C

Mata/Mata, his3A200/HIS3, leu2Al/LEU2, trplA63/TRPI, ura3-52/ura3-52

Plasmids

Inserts/Comments

pKS, pSK YEp352 pRS316

BlueScriptIIKS+ and Blue Script IISK Episomique URA3 shuttle vector YCp URA3 shuttle vector URPI (860 bp) in pKS 570 bp ClaI fragment of ACTI from pYA301 in pSK 1.45 kb EcoRI fragment containing the TRP1 gene from YRP7 (Tschumper and Carbon, 1980) in pKS CDC25 5' part in pKS The whole CDC25 ORF in pKS CDC25 5' part (SALI-BamHI) from pL25a (Camonis et al., 1986) in YEp352 CDC25 deleted from BglII fragment The SalI-NruI SDC25 sequence in YEp352 SaIlI-Sacl fragment from pRG3 that contains the SDC25 ORF in pKS SDC25 3' part (XbaI-EcoRI) in pKS SDC25 wild type in YEp352 CDC25-SDC25 chimeric sequence in YEp352 SDC25-CDC25 chimeric sequence in YEp352 CDC25-SDC25-CDC25 chimeric sequence in YEp352

p129 pSK-ACT1 pKS-TRP1 pTl pT2

pBM1 pPIlABglII pRG3 pKS2 pKS-CT

pFC1 pCSl pSC2 pCS3

Stratagene (Hill et al., 1986) (Sikorski and Hieter, 1989) (D6molis et al., 1995) This work This work

(Kaplon and Jacquet, 1995) (Kaplon and Jacquet, 1995) This work This workd (Damak et al., 1991) This work This work This work This work This work This work

a OL129-1A is a meiotic product from the cross OL120-2A x OL124-2A. OL136 is a thermoresistant revertant derived from OL129-1A. c FY1679 strain was the source of DNA for sequencing in the Yeast Genome Project. d The CDC25 sequence present in the pPIl plasmid (Camus et al., 1994) was deleted from intemal BgiII restriction fragment (nucleotides 1059 to 2628).

b

ford and Warner, 1991). Interestingly, during the same period, the CDC25 mRNA was expressed at a lower level than during growth on glucose. The expression of SDC25 was not only observed on ethanol but also on other nonfermentable carbon sources such as acetate and glycerol. The higher level of expression was observed on acetate medium. The SDC25 ORF Is Able to Suppress cdc25 Mutations We have previously reported that although the 3' part of SDC25 was able to supress cdc25 mutations, the complete reconstituted gene, even on a multicopy plasmid, was not (Damak et al., 1991). A possible explanation for this paradoxical result was that the 5' part of the gene cloned from the strain OL136 was untranslatable. Indeed, we have found that the reconstituted gene in the plasmid pRG3 contains a frameVol. 7, April 1996

shift in the 5' moiety of the gene. This frameshift was already present in the genomic DNA from the strain OL136 from which the SDC25 DNA was originally cloned. The sequence comparison with DNA from other strains revealed that this frameshift was due to a deletion of 19 bp from nucleotide position 1751 to 1769, which occurred in the strain 0L129-1A from which was derived the strain OL136. Indeed, the wildtype sequence was present in the parental strains of OL129-1A (OL120-2A and OL124-2A) as well as in commonly used strains W303-1B and FY1679 (see MATERIALS AND METHODS for experimental details). Therefore, the wild-type version of SDC25 contains 1253 codons (EMBL accession number: M26647). The replacement of the deleted sequence in the cloned SDC25 gene was performed directly in yeast by homologous recombination between a PCR-amplified fragment from the genomic DNA of the strain 531

E. Boy-Marcotte et al.

Table 2. Oligonucleotides used in this study

sense/reverse

Name

Gene

Oligonucleotide sequence

olSDC-633 olSDC132

SDC25 id id id id id id id CDC25 id id CDC25/SDC25 CDC25/SDC25

CGGAATTCTTTTACAATTAA TTTATTCCGTGATTTTGTAA TGGACGGGAGTAGGTGA CGTTTGAAGAAAGAGAAGCC TTTGACGAAGATGTCGC ATATTTTCATGATGTAGTAGTGAA ACGGAATTGCTATTGCGCGG GGACCCATAGCCTTCTTCCCCGCGC AAGCGATGTCAGAATGC GATGACCTGTCTCTTAAAGG ACCTCAAATGTAACTCCTGT -AAGCGGGTGGATATTGGATAGTTGTATC-ATGAGTTGCACTGCGTC ATCGGCATAAAATGTTGGATCGATAACTTAAC-CGATGGTCGCTACG

olSDC451 olSDC914 olSDC1648 olSDC1740a olSDC2152 olSDC2199 olCDC-693 olCDC389 olCDC5192

olCSlb olCS2b

sense reverse

reverse sense sense sense sense reverse sense reverse reverse sense reverse

The numbers of the oligonucleotides used in this study are the position of their 5' ends with regard to the first basepair of the SDC25 ORF or the CDC25 ORF. a The 12 nucleotides on the 3' side of this oligonucleotide are specific for the wild type SDC25 sequence, which is deleted in the strains OL129-1A, OL136, and the plasmid pRG3. b The SDC25 nucleotides are typed in italic. In olCS1 sens, the nucleotides -29 to - 1 of CDC25 are fused to nucleotides + 1 to + 17 of SDC25. In olCS2 reverse, the nucleotides +5037 to +5002 of CDC25 are fused to nucleotides +4002 to +3990 of SDC25.

W303-lb and the deleted linearized plasmid pRG3 as described in Figure 3. The resulting plasmid pFC1, containing the reconstituted wild-type SDC25 gene, was able to suppress the thermosensitivity of the cdc25-5 strain DOL97. The cdc25-deleted strain JCL300-3A rescued by the CDC25-containing plasmid pCDC25-LEU2 was transformed with pFC1. After growth on nonselective glucose medium, 24 clones of 50 had lost pCDC25-LEU2 and 14 pFC1. These results demonstrate that the wild-type gene SDC25 on a multicopy plasmid is able to functionally replace CDC25 with the same efficiency. The similarity between SDC25 and CDC25 at the nucleotide level allowed us to use homologous recombination as already described (Mezard et al., 1992; Boy-Marcotte et al., 1993) to construct two chimeric genes between SDC25 and CDC25 as shown on Figure 3. In the plasmid pCS1 the 5' part of CDC25 (codons 1 to 1031) has been fused to the 3' part of SDC25 (codons 704 to 1253). In pCS2, the 5' part of SDC25 (codons 1 to 717) has been fused to the 3' part of CDC25 (codons 1046 to 1589). These two chimeric sequences present on these plasmids were able to suppress the cdc25-5 mutation. This result confirms the interchangeability of the Ras GEF domains of Cdc25p and Sdc25p and provides further indication of the structural and functional conservation between them.

The SDC25 ORF Replaces CDC25 when Expressed at the CDC25 Locus To assess whether the lack of functional redundancy between CDC25 and SDC25 in wild-type cells is due to the different expression of these two genes or to 532

more complex reasons, we constructed a yeast strain in which the CDC25 ORF has been replaced by the SDC25 ORF and looked for functional complementation. To obtain this replacement, the homozygous thermosensitive diploid cdc25-5/cdc25-5 strain DOL97 was cotransformed with the Sall-Sacl DNA fragment from pCS3 (Figure 3) and a selectable URA3 plasmid. This restriction fragment contains the SDC25 ORF inserted between the 5' and the 3' flanking regions of the CDC25 ORF (312 bp and 324 bp, respectively). The CDC25 flanking sequences on each side of this fragment were aimed to promote homologous recombination at the CDC25 locus. If the SDC25 ORF functionally replaces CDC25 at its locus, then the cells should become thermoresistant. To test this possibility, thermoresistance of the selected Ura+ transformants was assessed. Thermoresistant clones were then further analyzed by PCR to test the replacement of the CDC25 ORF by the SDC25 ORF. Two thermoresistant clones were found (DOL97.6 and DOL97.7) in which the replacement had occurred. For both cases, tetrad analysis after meiosis of, respectively, 12 and 15 asci, revealed that the four spores were viable. It gave a 2:2 thermosensitivity segregation pattern, indicating that thermoresistance is linked to a single locus. In the case of DOL97.7, linkage with CDC25 locus was further confirmed by analyzing progeny of thermoresistant haploids (a OL97.7-1A and a OL97.71B) crossed with a wild-type strain (a W303-1B and a W303-1B): no thermosensitive progeny were observed at 36°C. The location of the SDC25 ORF at the CDC25 locus was demonstrated by PCR. The results Molecular Biology of the Cell

.

Regulation and Function of the SDC25 Gene

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Exp.2

Exp.1 b c

a

b

a

E

c

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0 1

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12 16 h o u rs

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2

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IQ A C T 1-i

ACT

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*

Figure 1. SDC25 mRNA is induced in stationary phase. (A) OL971-11B strain was grown in YPD at 26°C. RNA was prepared from cells withdrawn in exponential growth phase (OD710nm 3.5, lane a), in early stationary phase (OD710nm 15, lane b), and in late stationary phase 42 h after inoculating (OD710nm 35, lane c). Five micrograms of PolyA+ RNA prepared from each sample was analyzed by Northern blot in two experiments. Probes were labeled by random priming. In one experiment (Exp. 1) the SDC25 probe (the 3.2-kb EcoRI DNA fragment inside the ORF, prepared from pKS2) and the TRP1 probe (the 1.45-kb EcoRI DNA fragment including the TRP1 gene, prepared from pKS-TRP1) were used. In the other experiment (Exp. 2) the CDC25 probe (the 5.3-kb XhoI-NotI DNA fragment including the whole ORF, prepared from pT2) and the TRP1 probe were used. (B) OL97-1-11B was grown in YNB, histidine, leucine, and uracil at 26°C, samples (1-5) were withdrawn at times indicated by arrows on growth curve. (C) Northem blotting was done with 40 ,gg of total RNA from each sample (lanes 1-5). Hybridization was performed with the riboprobe SDC25 (transcribed from the T7 promoter on the plasmid pKS2 linearized at the XbaI site). The membrane was rehybridized with an ACTI probe prepared by random priming from a ClaI DNA restriction fragment of 570 bp from pSK-ACT1, with 160 Ci/mmol [a-32P]dCTP. The respective sizes of mRNAs were 4 kb for SDC25, 5.3 kb for CDC25, 0.9 kb for TRP1, and 1.45 kb for ACTI.

presented in Figure 4 have been obtained with the thermoresistant diploid DOL97.7 and the four meiotic products from a single tetrad (OL97.7-1A, B, C, and D). Similar experimental data have been obtained with DOL97-6 (our unpublished results). Vol. 7, April 1996

6 8 10 12 1 4 h ou rs

Glu.

C

1

4

Ex p. 1 3 2

B E

t

2

Eth.

1

Exp.2 2 3

-CDC25

Ac.

I.. .

Figure 2. SDC25 mRNA is induced in stationary phase and on nonfermentable carbon sources. (A) DOL97 was grown in YPD at 26°C. At zero time, the cells were centrifuged and then resuspended either in glucose (YPD, black squares) or ethanol (YPE, empty squares) containing medium. At the times indicated by arrows, samples (1-3) were withdrawn and total RNA was prepared. (B) Two Northern blots were performed with 40 [kg of total RNA of each sample. In one experiment (Exp. 1) the riboprobe SDC25 (see Figure 1) was used. In the other experiment (Exp. 2) the riboprobe CDC25 (prepared by transcription from the T7 promoter on the plasmid pTl linearized at the XhoI site) was used. The two Northern blots were rehybridized with the DNA probe URP1 (DNA fragment of 860 bp synthesized by PCR with the M13 universal sequencing and M13 reverse sequence primers [Pharmacia] from the plasmid p129). (C) Forty micrograms of total RNA prepared from cells grown at 26°C for at least two generations in YPD (Glu.), YPE (Eth.), and YPA (Ac.) were analyzed by Northern blot. SDC25 DNA probe (1515 bp) and ACTI probe (570 bp) were synthesized by PCR from pKS-CT and pSK-ACT1 with the M13 universal sequencing and M13 reverse sequence primers. In the case of ACTI probe, the specific radioactivity of [ca-32P]dCTP was 80 Ci/mmol.

From this analysis it was concluded that thermoresistance at 36°C was associated with the allele cdc25::SDC25. The expression of the cdc25::SDC25 gene was analyzed by Northern blot as shown in Figure 4D. In the diploid DOL97-7, the SDC25 mRNA was expressed during growth in glucose-containing medium at the 533

E. Boy-Marcotte et al. Recombinant plasmid

Recombination tI Avrll

-L vX

pFC 1

Xbal

Psti

1

P1l

V2

+

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1253 *

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704

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/ _ ~~~~~~~p1031 BamH1

F 2

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Sall

NdcI

Sail 717

V3 X

x

F3 Pstl

Sail

+

pCS3

+

Nhcl

12S3

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pSC2

1589

EcoOl09 1

Clall

. 1 p

1046

PstI EcoO109 I

+

: //////////// 4

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Figure 3. Reconstruction of the wild-type SDC25 gene and chimeric genes between CDC25 and SDC25. The recombinant plasmids pFC1, pCS1, pSC2, and pCS3 have been obtained by homologous recombination between a recipient linearized plasmid and a DNA fragment after cotransformation in the strain DOL97. In the case of pFC1, the recipient plasmid (V1) was pRG3 digested by AvrII and XbaI lacking 1119 bp within the SDC25 sequence, and the DNA fragment (Fl) was an amplified DNA fragment of 1285 bp generated by PCR from the SDC25 gene in the W303-1B genomic DNA with the oligonucleotides olSDC914 and olSDC2199. In the case of pCS1, the recipient plasmid (V2) was pRG3 digested by PstI and XbaI leading to the deletion of the SDC25 sequence before the XbaI site, and the DNA fragment (F2) was a NdeI-BamHI restriction fragment from the plasmid pBM1 containing the 5' CDC25 sequence until the BamHI site. In the case of pSC2, the recipient plasmid (V3) was pPIl digested by SalI and ApaI leading to the deletion of the CDC25 sequence until the ApaI site, and the DNA fragment (F3) was a EcoO109I restriction fragment from the plasmid pFCl containing the 3' SDC25 sequence from PstI to EcoO109I. To obtain the recombinant plasmid pCS3, the recipient plasmid (V4) was pPIlABglII digested by Clal and NheI leading to the deletion of 3562 bp inside the CDC25 ORF, and the DNA fragment (F4) was generated by PCR from the plasmid pFCl with the two CDC25/SDC25 hybrid oligonucleotides olCS1 and olCS2, which contain the SDC25 ORF of 3759 bp followed by 230 bp (which normally follows the SDC25 ORF), flanked on each side by CDC25 sequences of 28 and 35 bp, respectively. In each case the strain DOL97 was cotransformed with 200 ng of Vl, V2, V3, or V4 and 200 ng of Fl, F2, F3, or F4. Ura+ transformants were selected. The recombinant plasmids were prepared from these transformants. In the case of pFC1, the presence of the wild-type sequence was checked by PCR using the oligonucleotides olSDC1740 and oISDC2220. In the case of pCS1 and pSC2, the junction between the CDC25 and SDC25, resulting from the recombination event, was determined by nucleotide sequencing. In the case of pCS3, the restriction profile with various restriction enzymes confirmed the replacement of the CDC25 ORF by the SDC25 ORF. The presence of the right junction between the CDC25 upstream sequence and the ATG of the SDC25 ORF was checked by sequencing pCS3 with the primer olSDC132. The large line corresponds to YEp352 vector sequence, the empty box to SDC25, the hatched box to CDC25, and the black box to the nucleotide sequence of SDC25 ORF, which was deleted in the pRG3 plasmid. Crossed restriction sites have been lost by subcloning; italic numbers are codon numbers in SDC25 and CDC25 ORF, and arrows mark the position of the first and the last codon of the SDC25 and CDC25 ORF. *The plasmids pFC1, pCS1, pSC2, and pCS3 have been introduced by transformation in the cdc25-5, ura3 strain DOL97; the Ura+ transformants have been selected at 26°C and then replicated at 36°C.

same level as CDC25 mRNA. It was also expressed in the modified haploid OL97-1A where CDC25 mRNA was undetectable. SDC25 mRNA remained undetectable in the nonmodified haploid strain 0L97-1C.

Phenotype of the cdc25:.SDC25 Replacement When the GEF for Ras is SDC25p instead of Cdc25p as in haploid cdc25::SDC25 and in diploid cdc25::

SDC25/cdc25::SDC25, growth 534

occurs on

glucose

as

well as on ethanol at 26°C (our unpublished results). This Sdc25p-dependent growth also occurs at 37°C but is thermosensitive at 38.5°C on glucose (Figure 5A) and on ethanol (our unpublished results). A greater thermosensitivity is observed on ethanol because the heterozygote cdc25-5/cdc25::SDC25 does not grow even at 37°C (Figure 5A). These phenotypes are not observed in the heterozygotes cdc25::SDC25/CDC25, thus it is a recessive trait. This thermosensitivity is suppressed when CDC25 or Molecular Biology of the Cell

Regulation and Function of the SDC25 Gene

A

locus CDC25

Sall ATG

TAA

locus SDC25

ATG PstlLI 1-

Pv u I1

SD C25s

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b

ail A

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4- c

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TAA

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D

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