pch1 , a Second Essential C-type Cyclin Gene in Schizosaccharomyces pombe *

The Schizosaccharomyces pombe gene pch1 (cid:49) ( pombe cyclin C homology) was isolated in a two-hybrid screen for proteins that interact with Cdc2. The cyclin box re- gion of Pch1 protein shares greatest sequence identity with mammalian and Drosophila C-type cyclins ( (cid:59) 33% identity). Pch1 is significantly less similar to Mcs2 (19% identity), a second member of the C-type cyclin family in S. pombe . Cdc2 co-precipitates with Pch1 in S. pombe cell lysates, although Cdc2 may not be the major catalytic partner of a Pch1 kinase in vivo . Purified Pch1- associated kinase phosphorylated myelin basic protein, histone H1, and a peptide corresponding to the carbox- yl-terminal domain repeat of RNA polymerase II. The amount of pch1 mRNA does not oscillate during the cell cycle, as is the case for mRNA transcripts of other C-type cyclin genes. (cid:68) pch1 cells are inviable, therefore S. pombe has two essential genes that encode members of the C-type cyclin family, pch1 (cid:49) and mcs2 (cid:49) . The (cid:68) pch1 mutation causes pleiotropic morphological defects and an associated growth deficiency, but loss of Pch1 activity does not result in a cdc cell cycle-arrest phenotype. In Schizosaccharomyces pombe , cell cycle progression is determined by the activation state of a single cyclin-dependent kinase (CDK), 1 Cdc2 (1, 2). Construction of the (cid:68) pch1 Allele and Germination Experiments— A plasmid carrying the pch1 (cid:49) genomic clone, pBF108, was digested with Sal I and Sma I to remove a 900-bp fragment containing the majority of the pch1 (cid:49) open reading frame. This DNA fragment was replaced with the 1.9-kb Sal I/ Sma I fragment containing his7 (cid:49) derived from pEA2 (44) to create pBF110. The 3.5-kb Eco RI fragment of pBF110 was used to replace pch1 (cid:49) with the disrupted allele ( pch1::his7 (cid:49) ) by transformation into a diploid strain generated by mating CH428 and CH429 (44). The pch1::his7 (cid:49) /pch1 (cid:49) diploid strain was induced to sporulate by inoculat- ing malt extract medium with log-phase cultures grown in yeast extract with supplements medium. After 2 days at 22 °C, asci were collected, washed twice with water, and resuspended in 0.4% glusulase. After an overnight incubation at 30 °C, spores were washed twice with water and stored at 4 °C. Spores at 0.1 A 600 were germinated in Edinburgh minimal medium 2 plus leucine, uracil, and adenine at 30 °C. At the indicated times, samples containing 10 A 600 of cells were taken for microscopic observation, Northern analysis, and kinase assays. For both Northern mRNA analysis and kinase assays, cells were harvested by filtration, washed once with ice-cold water, frozen in liquid nitrogen, and stored at (cid:50) 70 °C. For microscopic observation, cells were harvested by centrifugation, resuspended in ice-cold water, and mixed with 100% ethanol to adjust to a final concentration of 70% ethanol, and samples were stored at 4 °C overnight. After fixation, 1 (cid:109) g/ml 4 (cid:57) , 6-diamidino- 2-phenylindole or

In Schizosaccharomyces pombe, cell cycle progression is determined by the activation state of a single cyclin-dependent kinase (CDK), 1 Cdc2 (1, 2). Activation of Cdc2 requires the formation of a complex between itself and a cyclin. In S. pombe, Cdc2 has been shown to form a complex with a B-type cyclin, Cdc13 (3,4). This cyclin-kinase complex accumulates during interphase, and the cyclin component is rapidly degraded upon exit from mitosis (M-phase) (4). Although Cdc13-Cdc2 complex is required for the onset of M-phase, Cdc2 functions at both the G 1 -S-phase and G 2 -M-phase transitions (5). Thus, there must be an additional cyclin-Cdc2 complex that promotes the onset of S-phase. Recent studies have shown that fission yeast Cig2 B-type cyclin is most abundant and has the highest associated kinase activity during S-phase (6,7). In ⌬cig2 cells, S-phase is delayed, and Cdc13 becomes essential for DNA replication, suggesting that Cig2-Cdc2 normally promotes the onset of Sphase, but in a ⌬cig2 strain this function can be performed by Cdc13-Cdc2 (7,8). The role of B-type cyclins at both the G 1 -Sphase and G 2 -M-phase transitions seems to be a common feature between the budding yeast Saccharomyces cerevisiae and S. pombe (9 -11). Three additional S. pombe cyclin genes have been previously identified, although their function in the cell cycle is unclear. The first of the genes, cig1 ϩ , encodes a B-type cyclin that is most abundant and has the highest associated kinase activity during M-phase (12). Disruption of cig1 ϩ causes no obvious phenotype (13)(14)(15)(16). The second gene, puc1 ϩ , which encodes an unusual type of cyclin, was initially proposed to function as a G 1 cyclin, although more recent studies have suggested that it might have a role in delaying G 1 arrest in nitrogen-starved cells (16,17). The third cyclin gene, mcs2 ϩ , encodes an essential C-type cyclin that shares some of the characteristics of pch1 ϩ , the cyclin gene described in this study (18). Recent studies have shown that Mcs2 is the cyclin partner of Mcs6 kinase, which is also known as Crk1 or Mop1 (19,20). The Mcs2-Mcs6 kinase can carry out the activating threonine 167 phosphorylation of Cdc2 in vitro, although it is unknown whether it performs this function in vivo.
Human and Drosophila cyclin C cDNA clones were initially identified by virtue of their ability to rescue the cell cycle-arrest defect of a S. cerevisiae strain lacking G 1 cyclins CLN1-3 (21)(22)(23). Human G 1 cyclins, cyclin D1 and cyclin E, were also obtained in the same screen (21). Unlike the G 1 functions of cyclin D1 and cyclin E, which are well characterized, the function of cyclin C is unknown. Cyclins with significant homology to the cyclin box region of cyclin C seem to be involved in the initiation of RNA polymerase II transcription and also in the activation of other CDKs through phosphorylation. Cyclin H, a human C-type cyclin, is the regulatory subunit of a mammalian kinase known as CDK-activating kinase that carries out the activating phosphorylation of Cdc2 and Cdk2 kinase in vitro (24). The cyclin H complex is also part of the general transcription factor IIK, a subunit of transcription factor IIH, which is essential for initiation of transcription (25)(26)(27). In addition to activating CDKs, the cyclin H complexes can phosphorylate the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II, which consists of heptapeptide repeats (YSPTSPS) (28). Phosphorylation of the CTD seems to be important for the regulation of transcription (29,30). In S. cerevisiae, the SRB10 and SRB11 genes, which encode a kinase and C-type cyclin, respectively, were isolated as high copy suppressors of a CTD truncation mutant (31). Independently, SRB10 (SSN3) and SRB11 (SSN8) were isolated as suppressors of a snf1 mutation (32,33). Snf1, a serine/threonine kinase, is involved in the regulation of glucose-repressed genes (34). SRB10 and SRB11 were shown to be involved in transcriptional repression and activation of a variety of genes in S. cerevisiae (33). CDK8, the associated kinase of cyclin C, shares significant homology with Srb10, although the functional homology between the two complexes is unknown (35,36). Another S. cerevisiae C-type cyclin and kinase pair, Ccl1 and Kin28, form a complex that phosphorylates CTD but not the mitotic CDK Cdc28 in vitro. Loss of Kin28 activity results in a decrease in the abundance of all mRNA species, suggesting that Kin28 kinase activity is essential for all transcription catalyzed by RNA polymerase II (37). Recently, a gene expressing the C-type cyclin subunit of a CTDK1 (CTD kinase) activity, CTK2, was cloned in S. cerevisiae (38). This cyclin-kinase pair, Ctk2-Ctk1, co-purifies with CTDK1 activity, although the in vivo role of this kinase in transcription is unknown.
We report the isolation of pch1 ϩ , a S. pombe gene that encodes a cyclin that is most closely related to the C-type cyclin genes of higher eukaryotes. We demonstrate that pch1 ϩ is an essential gene, showing that there are at least two essential cyclin C genes in fission yeast.

MATERIALS AND METHODS
Media and General Methods-Methods and media used for general genetics and biochemical procedures with fission yeast have been described (39,40).
Cloning and Sequencing of the pch1 ϩ Gene-A 1,147-bp pch1 cDNA in pACT2 was obtained in a two-hybrid screen of 2 ϫ 10 7 S. pombe cDNA clones using pAS1-Cdc2 as the bait (41). The cDNA was sequenced in both orientations and found to encode the entire Pch1 protein. The pch1 cDNA was used to probe filters containing an ordered array of cosmid and P1 genomic clones of S. pombe to determine the chromosomal location of pch1 ϩ (42,43). A 4.5-kb genomic clone of pch1 ϩ was obtained by colony hybridization of XhoI/XbaI fragments from P1 clone 7B10p inserted between the XhoI and XbaI sites in pBluescript II (pBF108). Sequence analysis of the genomic clone was performed in both orientations upstream and downstream of the pch1 ϩ coding region.
Construction of the ⌬pch1 Allele and Germination Experiments-A plasmid carrying the pch1 ϩ genomic clone, pBF108, was digested with SalI and SmaI to remove a 900-bp fragment containing the majority of the pch1 ϩ open reading frame. This DNA fragment was replaced with the 1.9-kb SalI/SmaI fragment containing his7 ϩ derived from pEA2 (44) to create pBF110. The 3.5-kb EcoRI fragment of pBF110 was used to replace pch1 ϩ with the disrupted allele (pch1::his7 ϩ ) by transformation into a diploid strain generated by mating CH428 and CH429 (44). The pch1::his7 ϩ /pch1 ϩ diploid strain was induced to sporulate by inoculating malt extract medium with log-phase cultures grown in yeast extract with supplements medium. After 2 days at 22°C, asci were collected, washed twice with water, and resuspended in 0.4% glusulase. After an overnight incubation at 30°C, spores were washed twice with water and stored at 4°C. Spores at 0.1 A 600 were germinated in Edinburgh minimal medium 2 plus leucine, uracil, and adenine at 30°C. At the indicated times, samples containing 10 A 600 of cells were taken for microscopic observation, Northern analysis, and kinase assays. For both Northern mRNA analysis and kinase assays, cells were harvested by filtration, washed once with ice-cold water, frozen in liquid nitrogen, and stored at Ϫ70°C. For microscopic observation, cells were harvested by centrifugation, resuspended in ice-cold water, and mixed with 100% ethanol to adjust to a final concentration of 70% ethanol, and samples were stored at 4°C overnight. After fixation, 1 g/ml 4Ј, 6-diamidino-2-phenylindole or 50 ng/ml Calcofluor was added to samples that were resuspended in PBS.
Northern (RNA) Blot Analysis-Total yeast RNA from a 10 A 600 cell pellet was extracted as described (39). Ten g of the total RNA preparation was resolved on a 1% formaldehyde agarose gel and transferred to nitrocellulose. The filters were hybridized with random-primed 32 Plabeled probes (Prime-It II; Stratagene) in 50% formamide, 1ϫ Denhardt's solution, 0.15% SDS, 5ϫ SSC, 10% dextran sulfate, and 100 g/ml denatured salmon sperm. After an overnight incubation at 42°C, the filters were washed at 42°C twice with 2ϫ SSC/0.1% SDS and once with 0.2ϫ SSC/0.1% SDS and then subjected to autoradiography or detection using a Molecular Dynamics PhosphorImager.
Expression of GST-Pch1 and Antibody Production-The 1,147-bp XhoI fragment from pACT-Pch1 was cloned into the SalI site of pGEX-KG (45) in frame with GST to create pBF103. Expression of the GST-Pch1 fusion protein was performed as described (46). Fresh overnight cultures of Escherichia coli BL21(DE3)pLysS transformed with pBF103 were diluted 1:20 in LB medium containing both chloramphenicol (34 g/ml) and ampicillin (50 g/ml). After 2 h in a 37°C shaker, isopropyl-1-thio-␤-D-galactopyranoside was added to a final concentration of 0.1 mM, and the incubation was continued for an additional 2 h. The culture was harvested by centrifugation at 2,500 ϫ g for 10 min at 4°C, resuspended in ice-cold 1ϫ PBS, and stored at Ϫ70°C. The sample was lysed by rapid thawing at 37°C and mild sonication. After centrifugation at 10,000 ϫ g for 15 min at 4°C, GST-Pch1 was recovered from the supernatant by incubation with glutathione-Sepharose 4B beads (Pharmacia) for 15 min at 4°C. The glutathione-Sepharose beads complexed with the fusion protein were washed three times with ice-cold PBS. GST-Pch1 was eluted from the beads at room temperature for 15 min in 20 mM glutathione, 100 mM Tris (pH 8.0), and 120 mM NaCl.
Purified GST-Pch1 protein was used to immunize rabbit #0645. The polyclonal anti-Pch1 antibodies from rabbit serum were purified first by subtraction against GST on nitrocellulose blocked with 2.5% fetal calf serum, 0.1% Tween 20, and TBS and then absorbed to GST-Pch1 on nitrocellulose blocked with 2.5% fetal calf serum. After a 1-h incubation at 4°C, the nitrocellulose with anti-Pch1 antibodies was washed twice with 0.1% Tween 20 in TBS at 4°C. Antibodies were eluted in 1 ml of 100 mM glycine (pH 2.2) for 10 min at 4°C followed by neutralization with 0.1 ml of 1 M Tris (pH 8.0).
GST-Pch1 was expressed in S. pombe from the nmt1 promoter in the pREPGST construct (41). pBF123 was constructed by polymerase chain reaction (PCR) amplification of the coding region of pch1 from pBF108 with the following primers: 5ЈPch-BamHI, 5Ј-CGCGGATCCAATGAGT-GAAGTAATAAAATCTGTACC-3Ј; and 3ЈPch-BamHI, 5Ј-CATGGATC-CTTATGAAGCTTCCGTCTC-3Ј. Both the 5Ј and the 3Ј primers contain a BamHI site (underlined) adjacent to a initiation codon or the stop codon, respectively. PCR reactions and PCR product purification were performed as described (47). The isolated PCR-amplified DNA was digested with BamHI for 2 h and subjected to gel electrophoresis through a 1% agarose gel. The PCR fragments were isolated from agarose by using the QIAquick gel extraction kit (Qiagen) as specified by the manufacturer and ligated into the BamHI site of pREPGST, which had been treated with calf intestinal phosphatase (Promega). The resulting construct, pBF123, was transformed into a pch1::his7 ϩ /pch1 ϩ diploid strain and selected on Edinburgh minimal medium 2. A transformed diploid strain was induced to sporulate on malt extract medium, and spores were collected and processed as indicated above. A pch1::his7 ϩ haploid strain isolated on Edinburgh minimal medium 2 was rescued by pBF123.
Preparation of Cell Extracts and Immunochemical Assays-Frozen cell pellets containing 10 A 600 of cells in 1.5-ml microcentrifuge tubes were thawed and resuspended in 0.5 ml of lysis buffer containing 50 mM Tris (pH 8.0), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 50 mM NaF, 0.1 mM sodium orthovanadate supplemented with 1 mM phenylmethylsulfonyl fluoride, 1 M microcystin, and 5 g/ml aprotinin, leupeptin, and pepstatin. Cells were lysed by vortexing with glass beads for 5 min at 4°C. Debris was removed by centrifugation at 14,000 ϫ g for 15 min at 4°C, and cell extracts were normalized by the addition of lysis buffer based on protein concentrations determined by a Bradford assay (Bio-Rad).
Cyclin-kinase complexes were isolated from cell extracts by glutathione-Sepharose (Pharmacia), p13 suc1 -Sepharose (48), or protein A-Sepharose (Pharmacia) prebound to GST-Pch1 antibodies or preimmune sera from the same rabbit. Precipitation of cyclin complexes was carried out on a rocker at 4°C for 1-2 h. Isolated complexes were washed three times with lysis buffer.
For Western blot analysis, a fraction of each co-precipitation was boiled in Laemmli sample buffer and loaded onto SDS-polyacrylamide gels. After electrophoresis, the separated proteins were transferred to nitrocellulose as described (49). The blots were blocked for at least 1 h in 5% (w/v) nonfat dry milk dissolved in TBS/0.1% Tween 20. The blots were then incubated with a 1:1,000 dilution of either purified polyclonal anti-Pch1 (#0645) or polyclonal anti-Cdc2 antibodies (#9808) for 1 h at room temperature. After washing with TBS/0.1% Tween 20, the blots were incubated with a 1:3,000 dilution of horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Promega), washed, and developed by enhanced chemiluminescence (Amersham) according to the manufacturer's specifications.
Kinase Assays-Protein A-and glutathione-Sepharose precipitates were washed three times with lysis buffer as described above followed by three washes with ice-cold kinase assay buffer (50 mM Tris (pH 7.4) and 10 mM MgCl 2 ). The bound complexes were resuspended in 50 l of kinase assay buffer containing 50 Ci of [␥-32 P]ATP, 100 M ATP, and either 50 g of histone H1 (Boehringer Mannheim), 6.25 g of myelin basic protein (MBP) (Boehringer Mannheim), or 2 g of CTD peptide (a gift from Geoffrey Laff and Mark Solomon, New Haven, CT). The assays containing MBP and CTD were incubated at room temperature for 5 and 30 min, respectively; the samples containing histone H1 were incubated at 30°C for 15 min. After the incubation, 50 l of Laemmli sample buffer was added, and the samples were boiled for 2 min. Half of each reaction was resolved on either a SDS-12% polyacrylamide gel for histone H1 or a SDS-15% polyacrylamide gel for MBP and CTD. Dried gels were subjected to quantitation (Molecular Dynamics Phos-phorImager) and autoradiography.
Nucleotide Sequence Accession Number-The GenBank TM accession number for pch1 ϩ is U92879.

RESULTS
Cloning of pch1 ϩ in a Cdc2 Two-Hybrid Screen-A yeast two-hybrid screen was used to identify proteins that interact with Cdc2 (50). A hybrid protein containing the Gal4 DNAbinding domain (Gal4 1-147 ) fused to Cdc2 was co-expressed with the hybrid proteins from the library containing the Gal4 activation domain fused to S. pombe cDNA clones in the S. cerevisiae strain Y190. Protein-protein interactions were detected by transcriptional activation of both HIS3 and lacZ reporter genes. In this screen, 2 ϫ 10 7 transformants were screened and yielded 112 positive cDNA clones representing 7 genes. Two of these genes have been described previously, orp2 ϩ (41) and suc1 ϩ (51). One gene for which 10 independent isolates were obtained is represented by the positive clone pACT-JL21, which interacted specifically with the Gal4 (1-147)Cdc2 fusion protein in the presence of 25 mM 3-aminotriazole (Fig. 1).
The nucleotide sequence of the longest (1,147 bp) cDNA insert from the clones that cross-hybridized with pACT-JL21 was found to encode a cyclin-like protein. The gene was named pch1 ϩ for pombe cyclin C homology. The chromosomal location of pch1 ϩ , determined by hybridization of the cDNA insert to an ordered array of cosmid and P1 clones (42,43), is on chromosome II between top1 ϩ and cdc10 ϩ . A genomic clone was obtained by the cloning of a 4.5-kb XbaI/XhoI fragment from P1 clone 7B10p between the XbaI and XhoI sites of pBluescript II. Sequence analysis of the genomic clone confirmed that the cDNA was full-length, and the open reading frame is encoded by a single exon (Fig. 2A). The encoded Pch1 protein contains 342 amino acids and has a predicted M r of 38,000.
Pch1 is a Member of the Cyclin C Family-The cyclin box region of Pch1 was used in a data base homology search. Drosophila and human cyclin C showed the highest identity to the cyclin box region of Pch1 (35 and 33%, respectively), especially within regions 1, 2, and 4 (Fig. 2B). Pch1 is more distantly related to human cyclin H (52), S. cerevisiae Srb11 (31, 33), S. pombe Mcs2 (18), S. cerevisiae Ccl1 (53), and S. cerevisiae Ctk2 (38) in which the sequence identity ranges from 17-24% (Table  I). These cyclin C family members have been implicated in the regulation of RNA polymerase II transcription and in the activating phosphorylation of cyclin-dependent kinases. Pch1 is only very weakly related to A-type and B-type cyclins, for example S. pombe Cdc13, a B-type cyclin that is required for mitosis, shares only 13% identity with Pch1 in the cyclin box domain.
Co-purification of Cdc2 and Pch1 from S. pombe Cells-Experiments were carried out to characterize the strength of the in vivo interactions involving Cdc2 and Pch1. These experiments used a plasmid construct that expressed GST-Pch1 from the nmt1 promoter in S. pombe. This construct fully complemented the ⌬pch1 mutation (see below), indicating that the GST-Pch1 protein was functional. The rescued strain was used to isolate either GST-Pch1-associated proteins using glutathione-Sepharose beads or Cdc2-associated proteins using p13 Suc1 -Sepharose. A wild-type strain expressing unfused GST served as a control. Western blot analysis was performed in duplicate with an affinity-purified polyclonal ␣-Pch1 antibody and with a polyclonal ␣-Cdc2 antibody (Fig. 3). A Cdc2 signal was detected in the lane containing proteins that associate with GST-Pch1. This signal was absent in the lane containing proteins that associate with GST, indicating that the association specifically involved Cdc2 and Pch1. The Cdc2 signal in the GST-Pch1 sample was much less than that detected with p13 Suc1 -Sepharose A, suggesting that the Pch1 co-precipitates with only a very small fraction of Cdc2. This finding was underscored by the failure to detect GST-Pch1 in the mixture of proteins that precipitate with p13 Suc1 -Sepharose A (Fig. 3), although it may be that Suc1 and Pch1 associations with Cdc2 are mutually exclusive.
Pch1-associated Kinase Phosphorylates Various Substrates-To assay for Pch1 associated-kinase activity, Pch1 and associating proteins were immunoprecipitated from a wild-type cell extract with ␣-Pch1 sera or glutathione-Sepharose beads were used to purify GST-Pch1 from S. pombe cells transformed with a plasmid that expressed GST-Pch1 from the nmt1 promoter. Pch1 associated-kinase activity was detected using the following exogenously supplied substrates: MBP, a peptide of the CTD from RNA polymerase II, and histone H1 (Fig. 4). The phosphorylation of the three substrates was rather poor, with myelin basic protein seeming to be the best substrate (Fig. 4,  lane 4). To show that the kinase activity was specific to Pch1, protein A-Sepharose beads either alone (lane 2) or with preimmune sera (lane 3) were used in the assay. Kinase activity was minimal in both controls. Glutathione-Sepharose beads precipitated a Pch1-associated kinase activity that was detected in cell extracts from cells expressing GST-Pch1 (lane 7), and as expected this kinase activity was absent in extracts from cells that did not express GST-Pch1 (lane 8). Cdc2 kinase precipitated with p13 suc1 -Sepharose was used as a positive control for all three substrates (lane 9). The results of preliminary experiments suggest that the MBP kinase activity of Pch1 kinase does not oscillate during the cell cycle, 2 although definitive studies await the identification of a better substrate. perature of 26°C. pch1 mRNA was detected at all time points (Fig. 5). There was an approximate 2-fold difference between some samples, but there seemed to be no periodic change in the Pch1 mRNA signal during the cell cycle (Fig. 5). In contrast, the abundance of cdc22 mRNA underwent a highly periodic change during the two cell cycles, in agreement with previous studies (54). pch1 ϩ is an Essential Gene-The function of Pch1 protein was investigated using a pch1 null mutation. This mutation was constructed by replacing the SmaI/SalI fragment (codons 11-321) with the S. pombe his7 ϩ gene (44) (Fig. 6A). The EcoRI fragment containing the disrupted gene was used to replace one copy of pch1 ϩ in a diploid strain. Two independent gene disruption transformants were confirmed by Southern hybridization analysis (data not shown). These pch1 ϩ /pch1::his7 ϩ diploids were sporulated and analyzed by tetrad dissection. All 36 tetrads gave rise to 2 viable and 2 inviable segregants (Fig.  6B). The inviable segregants formed microcolonies consisting of approximately 100 cells; their phenotypes are described in more detail below. All viable segregants were his7 Ϫ , thus the inviable segregants carried the pch1::his7 ϩ marker. The lethal phenotype of ⌬pch1 segregants was rescued by plasmids carrying the pch1 ϩ genomic clone or the pch1 ϩ cDNA expressed from the nmt1 promoter in pREP1 (55). Thus, the lethality of the pch1::his7 ϩ haploid strain was a direct result of the loss of pch1 ϩ .
To more accurately characterize the ⌬pch1 phenotype, a pch1/pch1::his7 ϩ diploid was induced to sporulate, and then spores were inoculated into liquid minimal medium. This medium lacked histidine, thus only the cells that contained the pch1::his7 ϩ gene germinated. Every 4 h, aliquots of cells were taken for cell number determination via Coulter counter and for ethanol fixation for microscopic analysis. Fixed cells were stained with DNA dye diamidinophenylindole (4Ј, 6-diamidino-2-phenylindole) to observe the nuclear structure and with Calcofluor to visualize septum formation (Fig. 7). Both wild-type and ⌬pch1 spores germinated approximately 8 h after inoculation into media. However, ⌬pch1 cells arrested after 4 -5 cell divisions with a variety of morphological defects, whereas the wild-type cells continued to divide normally. At 12 h after inoculation, the ⌬pch1 cells seem fairly normal, however, although a small population of cells had off-centered nuclei. The 24-h time point revealed a large amount of septal material in a portion of the cells as well as a population of anucleate cells. A combination 4Ј,6-diamidino-2-phenylindole and Calcofluor staining revealed improper nuclear segregation in some ⌬pch1 cells, resulting in one daughter cell with two nuclei and the other with none (data not shown). A similar pattern was also observed in ⌬pch1 cells fixed 36 h after inoculation.

DISCUSSION
In this report we have described the cloning and characterization of the pch1 ϩ gene of S. pombe. pch1 ϩ was isolated based on the ability of Pch1 protein to interact with a hybrid protein composed of the Gal4 DNA-binding domain and Cdc2. This protein-protein interaction activates the transcription of both lacZ and HIS3 in the S. cerevisiae strain Y190. Sequence analysis indicates that Pch1 protein shares significant homology with members of the cyclin C family. Pch1 is 35% identical and 60% similar to Drosophila cyclin C in the cyclin box region. The Drosophila and human cyclin C genes share 72% identity and were cloned by complementation of a S. cerevisiae strain lacking G 1 cyclins CLN1-3 (21,23). Although expression of pch1 ϩ in S. cerevisiae does not complement the triple CLN disruption (data not shown), we do not believe this reflects a fundamental difference between Pch1 and other cyclin C proteins. At this point we cannot speculate as to whether the Pch1 is a functional homolog of C-type cyclins in higher eukaryotes. Cyclin C  Cyclin C  100  33  36  31  18  26  22  18  Pch1  33  100  20  24  19  17  21  13  Srb11  36  20  100  24  21  20  10  16  Cyclin H  31  24  24  100  35  29  14  18  Mcs2  18  19  21  35  100  37  14  17  Ccl1  26  17  20  29  37  100  17  17  Cdc13  18  13  16  18  17  17 18 100 a Sequence identity in the cyclin box was determined by a pairwise alignment (Drosophila cyclin C (22); S. pombe pch1; S. cerevisiae Srb11 (Ssn8) (31,33); Human cyclin H (52); S. pombe mcs2 (18); S. cerevisiae Ccl1 (53); and S. pombe cdc13 (57) . As a positive control, Cdc2 kinase activity was isolated from wild-type cells with p13 suc1 -Sepharose (lane 9); as a negative control, p13 suc1 -Sepharose was incubated in the absence of cell lysate (lane 8). Lane 1 contains substrates alone. Phosphorylated proteins were resolved using SDS-12% polyacrylamide gel electrophoresis and exposed to film for the same amount of time to compare substrate specificity. The upper panel shows the MBP kinase assay, the middle panel shows the CTD kinase assay, and the lower panel shows the histone H1 kinase assay. complementation of the triple CLN mutant requires high level overexpression and might result from cross-reactivity between the cdc28 kinase and a class of cyclins normally dedicated to transcription. We propose that a similar cross-reaction between highly overexpressed proteins accounts for our recovery of pch1 in the screen for cdc2 interacting factors.
Like mcs2 ϩ , another C-type cyclin gene in S. pombe, pch1 ϩ is an essential gene (18). Spores that carry a pch1 null allele germinate normally and proceed to form microcolonies of inviable cells arrested with heterogeneous phenotypes. ⌬pch1 cells seem swollen and display pleiotropic morphological defects such as abnormal septal formation, apparent aggregation of mitochondria, and misplacement or absence of a nucleus. ⌬pch1 cells do not display a cdc phenotype, indicating that Pch1 is not required for a progression through a specific phase of the cell cycle. Although an association involving Pch1 and Cdc2 was detected by both two-hybrid and co-precipitation assays, we think it unlikely that Cdc2 is the major catalytic partner of Pch1 in vivo. The protein-protein interactions detected between Cdc2 and Pch1 in the two-hybrid system could be accounted for by significant similarity between Cdc2 and the true catalytic partner of Pch1. Cyclin-dependent kinases are similar in overall structure, but some CDKs contain amino acid differences within the region responsible for cyclin binding. The cyclin binding region in Cdc2 and its homologs contains the PSTAIRE motif, whereas the C-type cyclin-associated kinases do not have the PSTAIRE motif, but instead, many embody the sequence SACRE (31,33,36). Western analysis of GST-Pch1 affinity-purified proteins with ␣-PSTAIRE antibody indicates that the Pch1-associated kinase does not contain a PSTAIRE motif. 3 Furthermore, overproduction of Pch1 at an intermediate temperature (32°C) in strains having temperature-sensitive alleles of cdc2 (cdc2-33 and cdc2-L7) had very little effect, although it did cause the cells to be slightly more elongated at division. 3 This finding suggests that Pch1 is not a major partner of Cdc2 in vivo.
Another possibility to account for the Cdc2 and Pch1 interaction is that Cdc2 may be a substrate of the Pch1-associated kinase. Cyclin H is a C-type cyclin that is the regulatory subunit of a human kinase that is able to carry out the activating phosphorylation of cyclin-dependent kinases in vitro (24). In S. pombe, high expression of a mutant form of Cdc2 that has a nonphosphorylatable alanine residue substituted for threonine 167 results in a typical cdc Ϫ arrested phenotype, in which cells continue to grow but are unable to divide (56). Spore germination of ⌬pch1 cells results in a multitude of morphological defects instead of a cdc Ϫ arrested phenotype, suggesting that Pch1 function is not primarily involved in the activation of Cdc2. Recent studies have shown that Mcs2-Mcs6 kinase exhibits a CDK-activating kinase-like activity in vitro, although it is unknown whether Mcs2-Mcs6 kinase contributes to the activation of Cdc2 in vivo (19,20). ⌬mcs2, ⌬mcs6, and ⌬pch1 mutations cause somewhat similar phenotypes in that all three 3 B. A. Furnari, P. Russell, and J. Leatherwood, unpublished data.
FIG. 5. pch1 ؉ mRNA is constant during the cell cycle. Cells of a cdc25-22 strain were arrested in late G 2 by a temperature shift to 36°C for 4 h; the cells were then released from the arrest by a temperature shift to 26°C. An aliquot for the preparation of total RNA was taken every hour during the block and every 20 min after the shift to permissive temperature. The percentage of cells with a septa was determined by counting approximately 200 cells/time point. Northern analysis of pch1, cdc22, and adh1 mRNA was performed on 10 g of total RNA. The pch1 mRNA signal varied up to 2-fold between samples, but variations did not correlate with any cell cycle stage. Approximately 80% of the pch1 ϩ open reading frame was deleted and replaced by the his7 ϩ gene. This construct containing the his7 gene was used to disrupt pch1 ϩ in a diploid strain. B, tetrad analysis was performed on 36 asci derived from diploids heterozygous for the pch1 disruption. A photograph of 9 of the 36 dissected asci is shown. Each tetrad gave rise to two his7 Ϫ wild-type colonies and two microcolonies of inviable cells containing the ⌬pch1 allele.
types of mutant spores germinate to form small microcolonies of inviable cells that are not highly elongated, but the majority contain septa (18 -20). It is possible that Mcs2-and Pch1associated kinases share the responsibility for carrying out the activating phosphorylation of Cdc2, which could account for the fact that the loss of either cyclin does not cause a cell cyclearrest phenotype similar to that of cdc mutants. However, it is clear that Mcs2 and Pch1 have distinct essential functions that do not seem to be directly involved in promoting cell cycle progression. As such, the in vivo functions of both Mcs2 and Pch1 remain obscure, as is the case with most members of the C-type cyclin family. Additional genetic and biochemical studies of fission yeast have the potential to provide important insights into this enigmatic class of cyclins.