Slm9, a Novel Nuclear Protein Involved in Mitotic Control in Fission Yeast

Corresponding author: Paul Russell, The Scripps Research Institute, MB3, 10550 North Torrey Pines Rd., La Jolla, CA 92037., prussell{at}scripps.edu (E-mail)

Communicating editor: G. R. SMITH

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In the fission yeast Schizosaccharomyces pombe, as in other eukaryotic cells, Cdc2/cyclin B complex is the key regulator of mitosis. Perhaps the most important regulation of Cdc2 is the inhibitory phosphorylation of tyrosine-15 that is catalyzed by Wee1 and Mik1. Cdc25 and Pyp3 phosphatases dephosphorylate tyrosine-15 and activate Cdc2. To isolate novel activators of Cdc2 kinase, we screened synthetic lethal mutants in a cdc25-22 background at the permissive temperature (25°). One of the genes, slm9, encodes a novel protein of 807 amino acids. Slm9 is most similar to Hir2, the histone gene regulator in budding yeast. Slm9 protein level is constant and Slm9 is localized to the nucleus throughout the cell cycle. The slm9 disruptant is delayed at the G₂-M transition as indicated by cell elongation and analysis of DNA content. Inactivation of Wee1 fully suppressed the cell elongation phenotype caused by the slm9 mutation. The slm9 mutant is defective in recovery from G₁ arrest after nitrogen starvation. The slm9 mutant is also UV sensitive, showing a defect in recovery from the cell cycle arrest after UV irradiation.

EUKARYOTIC cells sense their environment and control their growth to survive in various conditions. In fission yeast Schizosaccharomyces pombe, checkpoints prevent mitosis when DNA is damaged or DNA replication is incomplete (RHIND and RUSSELL 1998 ). When cells are starved for carbon, they stop dividing, arrest in G₂, and then enter a quiescent phase (COSTELLO et al. 1986 ). S. pombe is a useful organism for the study of G₂-M control. When the entry into mitosis is inhibited, cells become elongated without septation. In fission yeast, as in other eukaryotic cells, Cdc2/cyclin B complex controls entry into mitosis. The activity of Cdc2 protein kinase is regulated in many ways. One of the most important forms of regulation is the inhibitory phosphorylation of tyrosine-15 of Cdc2 protein (GOULD and NURSE 1989 ). Wee1 and Mik1 protein kinases phosphorylate tyrosine-15 and inactivate Cdc2 (RUSSELL and NURSE 1987 ; FEATHERSTONE and RUSSELL 1991 ; LUNDGREN et al. 1991 ; LEE et al. 1994 ). Cdc25 and Pyp3 phosphatases dephosphorylate tyrosine-15 and activate Cdc2 (MILLAR et al. 1991 , MILLAR et al. 1992 ).

Wee1 is regulated by several proteins. Nim1/Cdr1 protein kinase phosphorylates the C-terminal catalytic domain of Wee1 and thereby inhibits Wee1 activity (COLEMAN et al. 1993 ; PARKER et al. 1993 ; WU and RUSSELL 1993 ). Cdr2 interacts with and phosphorylates the N-terminal domain of Wee1 in vitro and is thought to inhibit Wee1 in vivo (BREEDING et al. 1998 ; KANOH and RUSSELL 1998 ). Swo1 protein, which is an Hsp90 homolog in fission yeast, associates with and stabilizes Wee1 protein (ALIGUE et al. 1994 ). When DNA replication is blocked by hydroxyurea (HU), the protein kinase Cds1 binds to and phosphorylates Wee1 in vitro (BODDY et al. 1998 ). Wee1 was also proposed as a target of Chk1 kinase when DNA is damaged (O'CONNELL et al. 1997 ).

A mitogen-activated protein (MAP) kinase pathway in fission yeast links cell cycle control to changes in the extracellular environment that affect cell physiology. The Spc1/Sty1 MAP kinase cascade is activated by many forms of stress, such as high osmolarity, heat shock, oxidative stress, and nutrient limitation (MILLAR et al. 1995 ; DEGOLS et al. 1996 ; SHIOZAKI and RUSSELL 1996 ; DEGOLS and RUSSELL 1997 ). The spc1 mutant is delayed at G₂-M and displays a cell elongation phenotype that is exacerbated by stress. Genetic analysis indicated that Spc1 is able to influence mitotic control independently of Cdc25 and Wee1 (SHIOZAKI and RUSSELL 1995 ), although these findings do not exclude possible regulation of Cdc25 or Wee1 by Spc1. The available data suggest that Spc1 regulates Cdc2 activity indirectly.

In this study, we used cdc25-22, a temperature-sensitive mutant allele of cdc25, to identify novel regulators of mitosis. At the restrictive temperature of 35°, cdc25-22 mutant cells arrest in late G₂ phase and become highly elongated (RUSSELL and NURSE 1986 ). At the permissive temperature of 25°, cells are moderately elongated, because Cdc25 protein is partially inactivated and the G₂-M transition is delayed. When other mutations that show similar phenotypes, such as nim1/cdr1, cdr2, spc1, or cdc13-117 (cdc13⁺ encodes the major B-type cyclin in fission yeast), are combined with cdc25-22, the double mutant cells become highly elongated and arrest in G₂ at 25° (YOUNG and FANTES 1987 ; SHIOZAKI and RUSSELL 1995 ; BREEDING et al. 1998 ; KANOH and RUSSELL 1998 ). We screened for "slm" mutations that exhibit synthetic lethality in a cdc25-22 background at 25° in order to identify new genes that control the timing of the entry into mitosis (KANOH and RUSSELL 1998 ). In this article, we describe slm9, one of the genes identified in the screen. Genetic analyses are consistent with a model in which Slm9 regulates Cdc2 activity through Wee1. The slm9 mutant is sensitive to UV irradiation and heat shock and is defective in recovery from nitrogen starvation. Our data suggest that Slm9 is involved in multiple signal transduction pathways that affect cell growth.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains and general techniques:
The S. pombe strains used in this study are listed in Table 1. Yeast extract medium YES and synthetic minimal medium EMM2 were used for growing cells. Growth media and basic genetic and biochemical techniques for fission yeast have been described (ALFA et al. 1993 ). Immunoblot methods were performed as described (KANOH and RUSSELL 1998 ).

Cloning and nucleotide sequence determination of slm9:
The slm9-1 cdc25-22 wee1-50 mutant strain was transformed with a S. pombe genomic library constructed in pDB248' (BEACH et al. 1982 ; MAEDA et al. 1994 ). Transformants were grown on EMM2 plates at 25° for 7 days, and then were replica-plated onto new EMM2 plates and incubated at 35°. Three fast growing colonies were isolated among ~10,000 transformants, and they showed plasmid-dependent suppression of slow growth of the host strain at 35°. The plasmids were recovered from each transformant and were shown to be identical by restriction enzyme mapping and Southern blotting. The sequence of the insert fragment of pB29-4c was determined.

Gene disruption of the slm9 gene:
One-step gene disruption of slm9⁺ was carried out as follows (ROTHSTEIN 1983 ). A 1.1-kb SalI-ClaI DNA fragment was cloned into pBlueScript SK (Stratagene, La Jolla, CA) and digested by StuI, and then a 1.8-kb fragment of the ura4⁺ cassette was inserted (GRIMM et al. 1988 ). The resultant plasmid was digested by SalI and ClaI, and the slm9::ura4⁺ fragment was used to transform diploid cells made by crossing PR1319 with PR1320.

Chromosomal integration of slm9-HA6H and slm9-GFP:
To tag genomic slm9⁺ with a sequence encoding two copies of HA epitope and hexahistidine at the carboxyl terminus, the slm9⁺ open reading frame (ORF) was amplified by PCR with primers jk71 (5'-TCCTCCCCCGGGCGATGCACATTTTTGTGCCTAAG-3'; SmaI site in boldface type) and jk123 (5'-AAATATGCGGCCGCATAAAAGTGCAGATCGTCGTAATA-3'; NotI site in boldface type). The PCR product was cloned into pRIP42-HA6H (SHIOZAKI and RUSSELL 1997 ). After the nmt1 promoter was excised from the vector, the resultant plasmid was linearized at the XbaI site in slm9⁺ and used for transformation of wild-type (PR109) or cdc25-22 (JK1864) strains. To tag genomic slm9⁺ with a sequence encoding green fluorescent protein (GFP) at the carboxyl terminus (CHALFIE et al. 1994 ), the slm9⁺ ORF was amplified by PCR with primers jk94 (5'-GCGCGCCTGCAGTTCCCTCACCCCACAACGA-3'; PstI site in boldface type) and jk123. The PCR product was cloned into pRIP-GFP. The resultant plasmid was linearized at the XbaI site in slm9⁺ and used for transformation of wild type (PR109). Stable integration and tagging were confirmed by Southern and Western blotting. The functions of Slm9-HA6H and Slm9-GFP were confirmed by analysis of cell morphology.

Microscopy:
For the study of Slm9-GFP protein localization, cells were grown at 30° to log phase, washed with water, and observed with a Nikon Eclipse E800 microscope equipped with a Photometrics Quantix CCD camera. Images were acquired with IPLab Spectrum software (Signal Analytics Corp., Vienna, VA). For the study of Cdc25-12myc protein localization, cells were fixed with a 3.7% formaldehyde solution for 1 hr at 30° and washed with PEM buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgSO₄, pH 6.9). The cell wall was digested with 0.5 mg/ml of Zymolyase 100T (Seikagaku, Rockville, MD) at 37° for 40 min in PEMS buffer (PEM buffer supplemented with 1 M sorbitol), followed by permeabilization with 1% Triton X-100. After washes, cells were blocked in PEMBAL buffer (PEM buffer supplemented with 1% BSA, 0.1% NaN₃, 100 mM L-lysine monohydrochloride) for 1 hr at room temperature. Cells were incubated with anti-Myc antibodies (9E10, BabCo) overnight at room temperature. After washes, cells were incubated with FITC-conjugated goat anti-mouse IgG antibodies overnight at room temperature followed by washes. Samples were suspended in PBS containing 4',6-diamidino-2-phenylindole (DAPI). Photographs were taken as described above.

Northern blot analysis:
S. pombe cells were harvested and lysed by vortexing with glass beads in a buffer containing Tris-HCl (pH 7.5), 0.5 M NaCl, 0.01 M EDTA, and 1% SDS. After repeated extraction with phenol-chloroform, total RNA was precipitated by ethanol. A total of 8 µg of RNA of each sample was denatured with formamide, separated by formaldehyde gel electrophoresis, and blotted to a membrane. A DNA fragment of the H2A.1⁺ ORF was amplified by PCR using primers jk79 (5'-CGTCATGTCTGGAGGTAAATCTG-3') and jk80 (5'-GACGACTGACTTTACAGCTCC-3') and was labeled with [-³²P]dCTP by the random priming method.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Cloning of the slm9 gene:
To identify novel mitotic genes, we screened for synthetic lethal mutations in a cdc25-22 background at the permissive temperature of 25° (KANOH and RUSSELL 1998 ). In this article, we describe one of these genes, slm9. We cloned slm9 from a S. pombe genomic library by complementation of the slow growth of the slm9-1 cdc25-22 wee1-50 triple mutant at 35° (see below). A restriction enzyme map of the insert in the original clone, pB29-4, is shown in Fig 1A. The region essential for the complementation was delimited to a 4.0-kb HindIII fragment by subcloning (Fig 1A). Nucleotide sequence analysis of this fragment in pB29-4c revealed an ORF of 807 amino acids. The deduced amino acid sequence of Slm9 was compared with the databases. The ORF of the slm9 gene was found to be identical to AL031349, an ORF in c15D4, a cosmid sequenced as part of the S. pombe genome sequencing project at the Sanger Centre (Cambridge, UK). Slm9 showed the highest similarity to Hir2, a histone gene regulator of Saccharomyces cerevisiae (Fig 1B; SHERWOOD et al. 1993 ; SPECTOR et al. 1997 ). Slm9 and Hir2 share 30% sequence identity in a 606-amino-acid region (Fig 1B). Hir2 protein interacts with Hir1 in vivo, and they appear to function as transcriptional corepressors of histone genes.

View larger version (99K): In this window In a new window Download PPT slide   Figure 1. Structure of the slm9 gene. (A) Restriction map of slm9+. The insert DNA in the plasmid pB29-4 is shown. Restriction sites: B, BamHI; C, ClaI; H, HindIII; Sa, SalI; St, StuI; Xb, XbaI. Restriction fragments of the insert were subcloned in pDB248' as shown, and their ability to complement the poor growth of the slm9 cdc25-22 wee1-50 mutant (JK2245) at 35° is indicated. The structure of the linear fragment used to disrupt the slm9 gene is shown at the foot of the figure. (B) Comparison of amino acid sequences of Slm9 and S. cerevisiae Hir2. Identical amino acids between two proteins are shown in white on black, and conservative amino acids are shown in white on gray. The nucleotide sequence data of slm9+ will appear in the GenBank/EMBL/DDBJ nucleotide sequence databases (accession no. AL031349).

Disruption of slm9:
One-step gene disruption of slm9 was carried out by insertion of a S. pombe ura4⁺ gene cassette in diploid cells by homologous recombination (Fig 1A). Proper integration of the disruption construct was confirmed by Southern blot analysis (data not shown). The resulting heterozygous Ura⁺ diploid cells were sporulated and most of the asci gave four viable spores, two of which were Ura⁺. The phenotypes of the disruptant and the original slm9-1 mutant were indistinguishable. That is, cells grew normally but were elongated compared with wild-type cells (see Fig 5). The haploid disruptant was crossed with the slm9-1 original mutant and was found to be tightly linked. The slm9 disruptant was crossed with the cdc25-22 mutant. The presumptive double mutant spores germinated and showed a cdc-arrest phenotype at 25° (data not shown). These data supported the conclusion that the cloned gene was slm9. Overproduction of Slm9 from an nmt1:slm9⁺ construct caused no obvious phenotype (data not shown).

View larger version (25K): In this window In a new window Download PPT slide   Figure 2. The amount of Slm9 protein is constant during the cell cycle. Strain JK2365 (h- slm9HA6H cdc25-22) was grown in EMM2 medium to log phase at 25°, and cells were arrested in late G2 phase by a temperature shift to 35.5° for 4 hr, and then released from the arrest by a temperature shift to 25°. Samples were taken every 20 min after the shift to permissive temperature. The percentage of cells with septa was determined by counting ~200 cells at each time point. Whole-cell extracts were prepared and immunoblotting was performed with anti-HA antibodies for Slm9-HA protein and with anti-PSTAIRE antibodies for Cdc2 protein (KANOH and RUSSELL 1998 ).

View larger version (38K): In this window In a new window Download PPT slide   Figure 3. Slm9-GFP is localized to the nucleus throughout the cell cycle. Wild-type (PR109) and slm9-GFP (JK2360) strains were grown in YES medium to log phase. Living cells in the medium were observed without fixation.

View larger version (35K): In this window In a new window Download PPT slide   Figure 4. Slm9 does not regulate the expression of histone H2A.1 gene in fission yeast. Wild-type (PR109) or slm9 (JK2246) cells were synchronized in early G2 by centrifugal elutriation and reinoculated into fresh YES medium. Samples were collected every 20 min. The septation index was determined by counting ~200 cells at each point (top). The samples were analyzed by the H2A.1 probe. Ethidium bromide staining of rRNA is shown below for the control of equal amount of loading.

View larger version (62K): In this window In a new window Download PPT slide   Figure 5. Cellular localization of Cdc25 is unaffected by slm9 mutation. Strains OM1730 (cdc25-12myc) and JK2320 (slm9 cdc25-12myc) were grown in YES medium, stained with anti-Myc antibodies, and processed for indirect immunofluorescence microscopy.

Detection of Slm9 protein:
To determine whether the level of Slm9 protein is regulated during the cell cycle, the single chromosomal copy of slm9⁺ was tagged with a sequence encoding two copies of the HA epitope and six consecutive histidine residues. Cells were synchronized by a cdc25-22 block and release protocol, and whole-cell extracts of each sample were used for the detection of Slm9-HA protein by immunoblotting. Slm9-HA protein was detected as a band of ~85 kD. Slm9 protein was present throughout the cell cycle, with no significant change in abundance and mobility (Fig 2).

Slm9 protein is localized in the nucleus throughout the cell cycle:
To examine the localization of Slm9 protein, the single chromosomal copy of slm9⁺ was tagged with a sequence encoding GFP. In control wild-type cells, faint green fluorescence was observed mainly in the cytoplasm. In contrast, strong green fluorescence was observed in nuclei of cells expressing Slm9-GFP protein (Fig 3). The strength and localization of green fluorescence did not vary among cells at different stages in the cell cycle.

Slm9 does not regulate expression of H2A.1 histone in S. pombe:
The sequence homology between Slm9 and several regulators of histone genes suggested that Slm9 might regulate the expression of histone genes in S. pombe. Cells were synchronized by elutriation and the abundance of mRNA of a histone gene, H2A.1, was examined by Northern blot analysis (CHOE et al. 1985 ; MATSUMOTO and YANAGIDA 1985 ). Expression of H2A.1 was increased in S phase in both wild-type and slm9 cells (Fig 4). No significant difference in the amount of H2A.1 mRNA between the two strains was observed. Histone gene expression is thought to be coordinately regulated in fission yeast (AVES et al. 1985 ). These data suggested that Slm9 does not regulate expression of histone genes, unlike Hir2 in budding yeast (SHERWOOD et al. 1993 ; SPECTOR et al. 1997 ).

Cdc25 overproduction suppresses slm9 mitotic delay phenotype:
The slm9 disruptant cells were elongated at division compared with wild-type cells. This elongation occurred in both rich YES and synthetic EMM2 media (Table 2 and Table 3). These haploid cells had a 2C DNA content with a single nucleus, indicating a G₂ cell cycle delay (see Fig 6). We transformed slm9 cells with pREP3-cdc25. Overexpression of cdc25⁺ complemented the cell elongation phenotype of slm9 (data not shown). Furthermore, the wee1-50 mutation suppressed the synthetic lethality of the slm9 cdc25-22 strain (data not shown). These data indicate that slm9 regulates the activity of Cdc2 directly or indirectly.

View larger version (23K): In this window In a new window Download PPT slide   Figure 6. The slm9 disruptant cells have a defect in recovery from nitrogen starvation. (A) slm9 cells arrest in G1 after nitrogen starvation. Flow cytometric analyses of DNA content of wild-type (PR109), slm9 (JK2246), or nim1 (LW1817) cells. Cells were grown in EMM2 medium to log phase and then shifted to nitrogen-free EMM2 medium at 30°. Cells were harvested at indicated intervals after nitrogen starvation and subjected to flow cytometry. (B) The slm9 cells cannot restart the cell cycle normally after arresting in G1. Cells of wild-type (PR109) or slm9 (JK2246) strains were grown in EMM2 medium to log phase, shifted to nitrogen-free EMM2 medium, and incubated for 12 hr at 30°. Cells were then shifted to EMM2 medium with nitrogen and harvested at indicated intervals after adding nitrogen back to the medium. Each sample was subjected to flow cytometry.

View this table: In this window In a new window   Table 2. The slm9 mutation causes cell elongation in wild-type and cdc25 cdc2-3w backgrounds

View this table: In this window In a new window   Table 3. Inactivation of Wee1 fully suppresses the slm9 cell elongation phenotype

Inactivation of Wee1 fully suppresses cell elongation phenotype of slm9:
Experiments suggested that Slm9 regulates the activity of Cdc2 directly or indirectly at G₂-M. We examined whether Slm9 affects the abundance of the B-type cyclin encoded by cdc13⁺, because the cdc13-117 cdc25-22 mutant is synthetically lethal at 25° (our unpublished data). Cdc13 is required for the activation of Cdc2, whereas destruction of Cdc13 protein is required for the exit from mitosis (YAMANO et al. 1996 ). The protein level of Cdc13 in the slm9 cells was similar to that in wild-type cells (data not shown). These data indicate that Cdc13 is not the primary target of Slm9.

We tested the possibility of Cdc25 as a target of Slm9. We examined the phenotype of the slm9 cdc2-3w cdc25 strain. The cdc2-3w mutation activates Cdc2, thereby bypassing the requirement for Cdc25 (FANTES 1979 ). The slm9 cdc2-3w cdc25 cells were elongated relative to cdc2-3w cdc25 cells, indicating that slm9 regulates cell size in the absence of Cdc25 (Table 2). Cdc25 accumulates in the nucleus during late G₂ and M phase (LOPEZ-GIRONA et al. 1999 ). Deletion of slm9⁺ had no obvious effect on the amount or localization of Cdc25 protein (data not shown; Fig 5). These data indicate that Cdc25 is not the primary target of Slm9.

We examined whether the slm9 cell elongation phenotype was suppressed by the temperature-sensitive wee1-50 mutation. The slm9 wee1-50 cells were slightly elongated at permissive temperature of 20° and became the same size as wee1-50 cells at the restrictive temperature of 35° (Table 3). In contrast, spc1 wee1-50 cells were longer than wee1-50 cells at both temperatures (Table 3). Thus, inactivation of Wee1 fully suppressed the cell elongation phenotype of slm9. These data suggested that Slm9 might regulate Wee1.

Slm9 influences mitotic entry independently of Nim1/Cdr1 and Cdr2:
The data described above suggested that Slm9 might regulate Wee1 by controlling Nim1/Cdr1 or Cdr2 activity. To test this possibility, the phenotypes of slm9 nim1 and slm9 cdr2 strains were examined. The slm9 nim1 and slm9 cdr2 double mutants were longer than either single mutant (data not shown). This finding indicated that Slm9 acts independently of Nim1 or Cdr2.

Slm9 role in recovery from nitrogen starvation:
To investigate whether slm9 cells have a defect in monitoring nutritional conditions, as proposed for some other mutants that show a G₂ delay (YOUNG and FANTES 1987 ; SHIOZAKI and RUSSELL 1996 ; WU and RUSSELL 1997 ; BREEDING et al. 1998 ; KANOH and RUSSELL 1998 ), we examined the response of slm9 cells to nitrogen starvation. Arrest of slm9 cells with a 1C DNA content was delayed in response to nitrogen starvation, but most of the cells eventually arrested in G₁ phase after 24 hr of starvation (Fig 6A). When nitrogen was added back to the medium, wild-type cells resumed cell cycle progression by entering S phase. The first round of DNA synthesis was completed by 4 hr (Fig 6B). In contrast, the first round of DNA replication was substantially delayed in slm9 cells. A large fraction of cells remained with a 1C DNA content even 9 hr after addition of nitrogen (Fig 6B). These data suggested that Slm9 is required for the proper recovery from nitrogen starvation.

UV sensitivity of slm9 cells:
Fig 6 showed that the slm9 mutant has a defect in recovery from G₁ arrest after nitrogen starvation, a finding which suggested that slm9 cells might have a similar defect in recovery from other forms of stress. We tested the sensitivity of the slm9 mutant to UV and HU treatment. The slm9 mutant was not abnormally sensitive to HU (data not shown). On the other hand, slm9 cells were sensitive to UV treatment (Fig 7A). The sensitivity of the slm9 mutant was similar to that of the spc1 mutant, which is sensitive to various forms of stress, such as high osmolarity, heat shock, oxidative stress, and nutritional limitation. Next, we examined whether other mutants that show a G₂ delay were also sensitive to UV. The nim1 and cdr2 mutants were not sensitive to UV (Fig 7B). At 24 hr postirradiation (200 J/m²), most of the slm9 cells were elongated without a septum (data not shown). These cells appeared to be arrested in interphase (data not shown). Furthermore, deletion of rad3⁺, which encodes an essential component of both DNA damage and DNA replication checkpoint (ENOCH and NURSE 1990 ; BENTLEY et al. 1996 ), did not fully suppress the cell elongation phenotype of the slm9 mutant (data not shown). This observation indicated that the G₂ delay caused by the slm9 mutation was not due to a defect in DNA damage checkpoint. These data suggested that Slm9 is required for recovery from cell cycle arrest after UV irradiation.

View larger version (15K): In this window In a new window Download PPT slide   Figure 7. The slm9 disruptant is UV and heat-shock sensitive. (A) Wild-type (PR109), slm9 (JK2246), spc1 (KS1366), or rad24 (KS1455) strains were grown to log phase, plated on YES medium, and then exposed to the indicated doses of UV irradiation at 254 nM using Bio-Rad Genelinker (Richmond, CA). Survival was measured by counting the number of colonies formed on YES plates. (B) Wild-type (PR109), slm9 (JK2246), nim1 (LW1817), or cdr2 (JK2240) strains were exposed to UV irradiation. (C) The slm9 disruptant is sensitive to heat shock. Wild-type (PR109) or slm9 (JK2246) strains were grown to the log phase in YES medium at 25° and were shifted to 48°. After each interval, an aliquot was taken and diluted with ice-cold YES medium and then plated onto YES plates. The numbers of colonies were counted after 6 days of incubation at 25°.

The slm9 mutant is sensitive to heat shock:
As the slm9-1 cdc25-22 wee1-50 mutant grew slowly at 35°, we examined whether slm9 cells were sensitive to heat shock. Log phase cells were grown in YES medium at 25° and were shifted to 48°. After the heat treatment, survival of wild-type and slm9 cells was examined. The sensitivity of slm9 cells was higher than wild type (Fig 7C). After 15 min of incubation at 48°, only 0.9% of the slm9 cells survived, whereas 31.3% of the wild-type cells were viable. These data suggested that Slm9 is important for survival of heat shock.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have described the cloning and initial analysis of slm9⁺, a novel gene that appears to be involved in regulation of the onset of mitosis in fission yeast. The slm9 disruptant cells are moderately elongated with a 2C DNA content and the slm9 cdc25-22 mutant is synthetically lethal when Cdc25 is partially inactivated at the permissive temperature. The effect of slm9 mutation could be seen in the absence of Cdc25, and the cell elongation phenotype of the slm9 mutant was fully suppressed by inactivation of Wee1. These findings are most consistent with a model in which Slm9 regulates the onset of mitosis by affecting Wee1 function. The double mutants, slm9 nim1 and slm9 cdr2, have an additive effect on cell length. Therefore, Slm9 appears to influence Wee1 function in a different manner from Nim1 or Cdr2.

The amino acid sequence of Slm9 protein is similar to that of corepressors of histone gene transcription in budding yeast. S. cerevisiae Hir1 and Hir2 are thought to interact indirectly with DNA because they have no obvious DNA-binding motifs (SHERWOOD et al. 1993 ; SPECTOR et al. 1997 ). Thus, it is thought that Hir1 and Hir2 are targeted to specific histone gene promoters by association with other DNA-binding proteins. Slm9 protein is localized in the nucleus throughout the cell cycle, but it does not bind to DNA in vitro (our unpublished data). These observations suggest that Slm9 might regulate the gene transcription in a manner similar to Hir1 and Hir2, although expression of the histone gene H2A.1 appears normal in slm9 cells. A molecular understanding of the functions of Hir1 and Hir2 is currently lacking; however, progress has been made recently with the demonstration that Hir1 and Hir2 form a complex in vivo with components of the Swi/Snf complex of proteins that regulate transcription of a subset of genes in budding yeast (DIMOVA et al. 1999 ).

Although our data suggest that Slm9 might regulate Wee1 function, the slm9 mutation did not cause a significant change in the abundance of Wee1 protein (data not shown). Thus, Slm9 might be involved in regulation of genes which affect Wee1 function. Wee1 protein is predominantly localized to the nucleus (WU et al. 1996 ). It is possible that Slm9 forms a complex with Wee1 and other proteins to regulate Wee1 activity. Identification of Slm9-binding proteins would be helpful to clarify the putative signal cascade in which Slm9 participates.

We found that the slm9 mutant is sensitive to heat shock and UV irradiation. The terminal phenotype of the slm9 mutant after UV irradiation was almost the same as the spc1 mutant (DEGOLS and RUSSELL 1997 ), a finding which suggests that Slm9 and Spc1 might share some functions in vivo. However, it is clear that they do not function completely in the same pathway, because there are several differences between slm9 and spc1 mutations. First, the cell elongation phenotype of the spc1 mutant is not fully suppressed by inactivation of Wee1. Second, the spc1 mutant cells are more elongated in synthetic EMM2 medium than in complete YES medium (SHIOZAKI and RUSSELL 1995 ), while the cell length of the slm9 mutant is not significantly affected by nutritional condition (Table 2 and Table 3). Third, the spc1 mutant cannot arrest in G₁ after nitrogen starvation (SHIOZAKI and RUSSELL 1996 ). On the other hand, the slm9 mutant can arrest in G₁, but cannot normally recover from the arrest after addition of nitrogen to the medium. Fourth, the spc1 mutant is highly sensitive to high osmolarity (SHIOZAKI and RUSSELL 1995 ), while the slm9 mutant is not sensitive to high osmolarity (our unpublished data).

In summary, Slm9 appears to be important for many cellular functions: regulation of mitosis, recovery from G₁ arrest caused by nitrogen starvation, heat shock, and UV irradiation. Genetic studies are consistent with a model in which Slm9 regulates Wee1 function, although this regulation appears not to involve any of the proteins that are known or suspected to regulate Wee1. The other phenotypes caused by slm9 mutations are not easily explained by regulation of Wee1, nor do there appear to be mutants that share the same spectrum of phenotypes. Although the Slm9 sequence is most similar to Hir2 protein in budding yeast, the slm9 and hir2 phenotypes have little in common. Hir2 appears to be a transcriptional corepressor in budding yeast (SHERWOOD et al. 1993 ; SPECTOR et al. 1997 ), but very little is known about the molecular function of Hir2 protein. The studies described in this report lay the foundation for future analyses of an important but poorly understood class of proteins represented by Slm9 and Hir2. A fission yeast gene (accession no. P87314) that encodes a protein with 27% (197/722) identity to Slm9 has been discovered recently in the fission yeast genome sequence project. Future studies will be aimed at determining if Slm9 and this new protein have functional overlap.

	ACKNOWLEDGMENTS

We are grateful to the members of the Scripps Cell Cycle Group for helpful advice and support. We especially thank Miguel Rodriguez for discussion. We also thank Masayuki Yamamoto and Yuichi Iino for a S. pombe genomic library, Mitsuhiro Yanagida for plasmids of S. pombe histone genes, and Kathy Gould for anti-Cdc13 antibodies. Junko Kanoh was supported by a long-term fellowship of the Human Frontier Science Program. This work was supported by National Institutes of Health grant GM41281.

Manuscript received October 14, 1999; Accepted for publication March 2, 2000.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ALFA, C., P. FANTES, J. HYAMS, M. MCLEOD and E. WARBRICK, 1993 Experiments with Fission Yeast: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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