The role ofSaccharomyces cerevisiae Cdc40p in DNA replication and mitotic spindle formation and/or maintenance

Successful progression through the cell cycle requires the coupling of mitotic spindle formation to DNA replication. In this report we present evidence suggesting that, inSaccharomyces cerevisiae, theCDC40 gene product is required to regulate both DNA replication and mitotic spindle formation. The deduced amino acid sequence ofCDC40 (455 amino acids) contains four copies of a β-transducin-like repeat. Cdc40p is essential only at elevated temperatures, as a complete deletion or a truncated protein (deletion of the C-terminal 217 amino acids in thecdc40-1 allele) results in normal vegetative growth at 23°C, and cell cycle arrest at 36°C. In the mitotic cell cycle Cdc40p is apparently required for at least two steps: (1) for entry into S phase (neither DNA synthesis, nor mitotic spindle formation occurs at 36°C and (2) for completion of S-phase (cdc40::LEU2 cells cannot complete the cell cycle when returned to the permissive temperature in the presence of hydroxyurea). The role of Cdc40p as a regulatory protein linking DNA synthesis, spindle assembly/maintenance, and maturation promoting factor (MPF) activity is discussed.     

Mutations in <Emphasis Type="Italic">CDC14</Emphasis> result in high sensitivity to cyclin gene dosage in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14.     

Mutations in CDC14 result in high sensitivity to cyclin gene dosage in Saccharomyces cerevisiae

We screened for mutations that resulted in lethality when the G1 cyclin Cln2p was overexpressed throughout the cell cycle in Saccharomyces cerevisiae. Mutations in five complementation groups were found to give this phenotype, and three of the mutated genes were identified as MEC1, NUP170, and CDC14. Mutations in CDC14 may have been recovered in the screen because Cdc14p may reduce the cyclin B (Clb)-associated Cdc28 kinase activity in late mitosis, and Cln2p may normally activate Clb-Cdc28 kinase activity by related mechanisms. In agreement with the idea that cdc14 mutations elevate Clb-Cdc28 kinase activity, deletion of the gene for the Clb-Cdc28 inhibitor Sic1 caused synthetic lethality with cdc14-1, as did the deletion of HCT1, which is required for proteolysis of Clb2p. Surprisingly, deletion of the gene for the major B-type cyclin, CLB2, also caused synthetic lethality with the cdc14-1 mutation. The clb2 cdc14 strains arrested with replicated but unseparated DNA and unseparated spindle pole bodies; this phenotype is distinct from the late mitotic arrest of the sic1::TRP1 cdc14-1 and the cdc14-1 hct1::LEU2 double mutants and of the cdc14 CLN2 overexpressor. We found genetic interactions between CDC14 and the replication initiator gene CDC6, extending previous observations of interactions between the late mitotic function of Cdc14p and control of DNA replication. We also describe genetic interactions between CDC28 and CDC14. Received: 24 May 1999 / Accepted: 19 October 1999     

Novel alleles ofcdc13 andcdc2 isolated as suppressors of mitotic catastrophe inSchizosaccharomyces pombe

Cell cycle control in the fission yeastSchizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including thecdc2, cdc13, cdc25, weel, andmik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Mik1 play a negative regulatory role. Here, we have screened for suppressors of the lethal premature entry into mitosis, termed mitotic catastrophe, which results from simultaneous loss of function of both Wee1 and Mik1. Through such a screen, we hoped to identify additional components of the cell cycle regulatory network, and/or G2/M-specific substrates of Cdc2. Although we did not identify such molecules, we isolated a number of alleles of bothcdc2 andcdc13, including a novel wee allele ofcdc2, cdc2-5w. Here, we characterizecdc2-5w and two alleles ofcdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.     

Functional analysis of theDrosophila CDC2 Dm gene in fission yeast

Thecdc2 ⁺ gene product (p34^cdc2) is a protein kinase that regulates entry into mitosis in all eukaryotic cells. The role that p34^cdc2 plays in the cell cycle has been extensively investigated in a number of organisms, including the fission yeastSchizosaccharomyces pombe. To study the degree of functional conservation among evolutionarily distant p34^cdc2 proteins, we have constructed aS. pombe strain in which the yeastcdc2 ⁺ gene has been replaced by itsDrosophila homologue CDC2Dm (theCDC2Dm strain). ThisCDC2Dm S. pombe strain is viable, capable of mating and producing four viable meiotic products, indicating that the fly p34^CDC2Dm recognizes all the essentialS. pombe cdc2 ⁺ substrates, and that it is recognized by cyclin partners and other elements required for its activity. The p34^CDC2Dm protein yields a lethal phenotype in combination with the mutant B-type cyclin p56^cdc13-117, suggesting that thisS. pombe cyclin might interact less efficiently with theDrosophila protein than with its native p34^cdc2 counterpart. ThisCDC2Dm strain also responds to nutritional starvation and to incomplete DNA synthesis, indicating that proteins involved in these signal transduction pathways, interact properly with p34^CDC2Dm (and/or that p34^cdc2-independent pathways are used). TheCDC2Dm gene produces a ‘wee’ phenotype, and it is largely insensitive to the action of theS. pombe weel ⁺ mitotic inhibitor, suggesting thatDrosophila weel ⁺ homologue might not be functionally conserved. ThisCDC2Dm strain is hypersensitive to UV irradiation, to the same degree asweel-deficient mutants. A strain which co-expresses theDrosophila and yeastcdc2⁺ genes shows a dominantwee phenotype, but displays a wild-type sensitivity to UV irradiation, suggesting that p34^cdc2 triggers mitosis and influences the UV sensitivity by independent mechanisms. Communicated by B. J. Kilbey     

Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase

In excised pith parenchyma from Nicotiana tabacum L. cv. Wisconsin Havana 38, auxin (naphthalene-1-acetic acid) together with cytokinin (6-benzylaminopurine) induced a greater than 40-fold increase in a p34^cdc2-like protein, recoverable in the p13^suc1-binding fraction, that had high H1 histone kinase activity, but enzyme induced without cytokinin was inactive. In suspension-cultured N. plumbaginifolia Viv., cytokinin (kinetin) was stringently required only in late G2 phase of the cell division cycle (cdc) and cells lacking kinetin arrested in G2 phase with inactive p34^cdc2-like H1 histone kinase. Control of the Cdc2 kinase by inhibitory tyrosine phosphorylation was indicated by high phosphotyrosine in the inactive enzyme of arrested pith and suspension cells. Yeast cdc25 phosphatase, which is specific for removal of phosphate from tyrosine at the active site of p34^cdc2 enzyme, was expressed in bacteria and caused extensive in-vitro activation of p13^suc1-purified enzyme from pith and suspension cells cultured without cytokinin. Cytokinin stimulated the removal of phosphate, activation of the enzyme and rapid synchronous entry into mitosis. Therefore, plants can control cell division by tyrosine phosphorylation of Cdc2 but differ from somatic animal cells in coupling this mitotic control to hormonal signals.Abbreviations BAP 6-benzylaminopurine - BrdUrd 5-bromo-2-deoxyuridine - cdc cell division cycle - Cdc25 cdc phospho-protein phosphatase - CKI cyclin dependent kinase inhibitor - 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6 diamidino-2-phenylindole - GST-cdc25 glutathione sulfur transferase-truncated cdc25 fusion - MS Murashige and Skoog (1962) - NAA naphthalene-1-acetic acid - p34^cdc2 34-kDa product of the cdc2 gene     

CDC44: a putative nucleotide-binding protein required for cell cycle progression that has homology to subunits of replication factor C.

To investigate the means by which a cell regulates the progression of the mitotic cell cycle, we characterized cdc44, a mutation that causes Saccharomyces cerevisiae cells to arrest before mitosis. CDC44 encodes a 96-kDa basic protein with significant homology to a human protein that binds DNA (PO-GA) and to three subunits of human replication factor C (also called activator 1). The hypothesis that Cdc44p is involved in DNA metabolism is supported by the observations that (i) levels of mitotic recombination suggest elevated rates of DNA damage in cdc44 mutants and (ii) the cell cycle arrest observed in cdc44 mutants is alleviated by the DNA damage checkpoint mutations rad9, mec1, and mec2. The predicted amino acid sequence of Cdc44p contains GTPase consensus sites, and mutations in these regions cause a conditional cell cycle arrest. Taken together, these observations suggest that the essential CDC44 gene may encode the large subunit of yeast replication factor C.     

Dominant mutant alleles of yeast protein kinase geneCDC15 suppress thelte1 defect in termination of M phase and genetically interact withCDC14

LTE1 encodes a homolog of GDP-GTP exchange factors for the Ras superfamily and is required at low temperatures for cell cycle progression at the stage of the termination of M phase inSaccharomyces cerevisiae. We isolated extragenic suppressors which suppress the cold sensitivity oflte1 cells and confer a temperature-sensitive phenotype on cells. Cells mutant for the suppressor alone were arrested at telophase at non-permissive temperatures and the terminal phenotype was almost identical to that oflte1 cells at non-permissive temperatures. Genetic analysis revealed that the suppressor is allelic toCDC15, which encodes a protein kinase. Thecdc15 mutations thus isolated were recessive with regard to the temperature-sensitive phenotype and were dominant with respect to suppression oflte1. We isolatedCDC14 as a low-copy-number suppressor ofcdc15-rlt1.CDC14 encodes a phosphotyrosine phosphatase (PTPase) and is essential for termination of M phase. An extra copy ofCDC14 suppressed the temperature sensitivity ofcdc15-rlt1 cells, but not that ofcdc15-1 cells. In addition, some residues that are essential for the Cdc14 PTPase activity were found to be non-essential for the suppression. These results strongly indicate that Cdc14 possesses dual functions; PTPase activity is needed for one function but not for the other. We postulate that the cooperative action of Cdc14 and Cdc15 plays an essential role in the termination of M phase.     

Electron microscopic studies onNosema mesnili Paillot (Microsporidia: Nosematidae) infecting the Malpighian tubules ofPieris canidia larva

Summary An entire coding region of theCDC24/CLS4 gene and its truncated derivatives were overexpressed in yeast cells under the control of theGAL1 promoter. Western blotting analysis of the yeast cell lysates showed that the CDC24/CLS4 protein (Cdc24p) was induced to reach its maximum level after 9 h incubation of the cells in galactose medium. Overexpression of Cdc24p within the cells caused the morphological change, accumulating large spherical unbudded cells which exhibited actin cytoskeleton disturbed, chitin delocalized on the cell surface, and cell viability decreased. Multiple nuclei were observed in these cells, indicating that only budding cycle but not nuclear division cycle is blocked by the overproduction of Cdc24p. In order to identify the region of Cdc24p responsible for the growth inhibition, several truncatedCDC24 genes were expressed. Surprisingly, overexpression of fragments either containing the C-terminal 76 amino acid residues or deleting the same region inhibited cellular growth. This suggests that Cdc24p contains multiple functional domains for its tasks, likely cooperating signals of bud positioning and bud timing.     

Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing

The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export. Received: 9 April 1998 / Accepted: 10 August 1998     

A novel yeast mutant that is defective in regulation of the Anaphase-Promoting Complex by the spindle damage checkpoint

The accurate segregation of sister chromatids at the metaphase to anaphase transition in Saccharomyces cerevisiae is regulated by the activity of the anaphase-promoting complex or cyclosome (APC/C). In the event of spindle damage or monopolar spindle attachment, the spindle checkpoint is activated and inhibits APC/C activity towards the anaphase inhibitor Pds1p, resulting in a cell cycle arrest at metaphase. We have identified a novel allele of a gene for an APC/C subunit, cdc16-183 , in S. cerevisiae. cdc16-183 mutants arrest at metaphase at 37°C, and are supersensitive to the spindle-damaging agent nocodazole, which activates the spindle checkpoint, at lower temperatures. This supersensitivity to nocodazole cannot be explained by impairment of the spindle checkpoint pathway, as cells respond normally to spindle damage with a stable metaphase arrest and high levels of Pds1p. Despite showing metaphase arrest at G2/M at 37°C, cdc16-183 mutants are able to perform tested G1 functions normally at this temperature. This is the first demonstration that a mutation in a core APC/C subunit can result in a MAD2-dependent arrest at the restrictive temperature. Our results suggest that the cdc16-183 mutant may have a novel APC/C defect(s) that mimics or activates the spindle checkpoint pathway.Communicated by C. P. Hollenberg     

“Reductional anaphase” in replication-defective cells is caused by ubiquitin-conjugating enzyme Cdc34-mediated deregulation of the spindle

Equal partitioning of the duplicated chromosomes into two daughter cells during cell division is a coordinated process and is initiated only after completion of DNA synthesis. However, this strict order of execution breaks down in CDC6-deficient cells. Cdc6, an evolutionarily conserved protein, is required for the assembly of pre-replicative complexes (pre-RCs) and is essential for the initiation of DNA replication. Yeast cells lacking Cdc6 function, though unable to initiate DNA replication, proceed to undergo “reductional anaphase” by partitioning the unreplicated chromosomes and lose viability rapidly. This extreme form of genomic instability in cdc6 cells is thought to be due to inactivation of a pre-RC based, Cdc6-dependent checkpoint mechanism that, during normal cell cycle, inhibits premature onset of mitosis until pre-RC is assembled. Here, we show that chromosome segregation in cdc6 mutant is caused not by precocious initiation of mitosis in the absence of a checkpoint, but by the deregulation of spindle dynamics induced via a regulatory network involving the ubiquitin-conjugating enzyme Cdc34, microtubule-associated proteins (MAPs) and the anaphase-promoting complex (APC) activator Cdh1. This regulatory circuit governs spindle behavior in the early part of the division cycle and precipitates catastrophic chromosome segregation in the absence of DNA replication.     

Fission yeast sta mutations that stabilize an unstable minichromosome are novel cdc2-interacting suppressors and are involved in regulation of spindle dynamics

Cytological observations have shown that the presence of unstable minichromosomes can delay progression through the early stages of mitosis in fission yeast (Schizosaccharomyces pombe), suggesting that such minichromosomes may provide a useful tool for examining the system that regulates the coordinated segregation of chromosomes. One such unstable minichromosome is a large circular minichromosome. We previously showed that the mitotic instability of this minichromosome is probably due to the frequent occurrence of catenated forms of DNA after replication. To identify genes involved in the regulation of chromosome behavior in mitosis, we isolated mutants which stabilized this minichromosome. Three loci (stal, sta2, and sta3) were identified. Two of them were found to be suppressors of temperature-sensitive mutations in cdc2, which encodes the catalytic subunit of muturation promoting factor (MPF). They show no linkage to, and are thus different from, sucl, and cdc13, previously identified as genes that interact with cdc2. The other mutation mapped to a gene previously identified as being required for the correct formation of the mitotic spindle. Data provided in this study suggest that the sta genes are involved in the regulation of spindle dynamics to ensure proper chromosome segregation during mitosis.     

The identification of a Caenorhabditis elegans homolog of p34cdc2 kinase

We have identified a Caenorhabditis elegans homolog of p34^cdc2 kinase. The C. elegans homolog, ncc-1, is -60% identical to p34^cdc2 of Homo sapiens. When expressed from a constitutive yeast promoter, ncc-1 is capable of complementing a conditional lethal mutation in the CDC28 gene of Saccharomyces cerevisiae, indicating that this C. elegans homolog can properly regulate the cell cycle.     

Yeast Srp1, a nuclear protein related toDrosophila and mouse pendulin, is required for normal migration, division, and integrity of nuclei during mitosis

This paper describes genes from yeast and mouse with significant sequence similarities to aDrosophila gene that encodes the blood cell tumor suppressor pendulin. The protein encoded by the yeast gene, Srp1p, and mouse pendulin share 42% and 51% amino acid identity withDrosophila pendulin, respectively. All three proteins consist of 10.5 degenerate tandem repeats of 42 amino acids each. Similar repeats occur in a superfamily of proteins that includes theDrosophila Armadillo protein. All three proteins contain a consensus sequence for a bipartite nuclear localization signal (NLS) in the N-terminal domain, which is not part of the repeat structure. Confocal microscopic analysis of yeast cells stained with antibodies against Srp1p reveals that this protein is intranuclear throughout the cell cycle. Targeted gene disruption shows thatSRP1 is an essential gene. Despite their sequence similarities,Drosophila and mouse pendulin are unable to rescue the lethality of anSRP1 disruption. We demonstrate that yeast cells depleted of Srp1p arrest in mitosis with a G2 content of DNA. Arrested cells display abnormal structures and orientations of the mitotic spindles, aberrant segregation of the chromatin and the nuclei, and threads of chromatin emanating from the bulk of nuclear DNA. This phenotype suggests that Srplp is required for the normal function of microtubules and the spindle pole bodies, as well as for nuclear integrity. We suggest that Srp1p interacts with multiple components of the cell nucleus that are required for mitosis and discuss its functional similarities to, and differences fromDrosophila pendulin.     

Isolation of acdc28 mutation that abrogates the dependence of S phase on completion of M phase of the budding yeast cell cycle

We have isolated a mutation in the budding yeastSaccharomyces cerevisisae CDC28 gene that allowscdc13 cells, carrying damaged DNA, to continue with the cell division cycle. Whilecdc13 mutant cells are arrested as largebudded cells at the nonpermissive temperature 37‡C, thecdc13 cdc28 double mutant culture showed cells with one or more buds, most of which showed apical growth. The additional buds emerged without the intervening steps of nuclear division and cell separation. We suggest that thecdc28 mutation abrogates a checkpoint function and allows cells with damaged or incompletely replicated DNA an entry to another round of cell cycle and bypasses the mitotic phase of the cell cycle.     

Cloning of the polyketide synthase gene atX from Aspergillus terreus and its identification as the 6-Methylsalicylic acid synthase gene by heterologous expression

The geneCAL1 (also known asCDC43) ofSaccharomyces cerevisiae encodes the subunit of geranylgeranyl transferase I (GGTase I), which modifies several small GTPases. Biochemical analyses of the mutant enzymes encoded bycall-1, andcdc43-2 tocdc43-7, expressed in bacteria, have shown that all of the mutant enzymes possess reduced activity, and that none shows temperature-sensitive enzymatic activities. Nonetheless, all of thecall/cdc43 mutants show temperature-sensitive growth phenotypes. Increase in soluble pools of the small GTPases was observed in the yeast mutant cells at the restrictive temperature in vivo, suggesting that the yeast prenylation pathway itself is temperature sensitive. Thecall-1 mutation, located most proximal to the C-terminus of the protein, differs from the othercdc43 mutations in several respects. An increase in soluble Rholp was observed in thecall-1 strain grown at the restrictive temperature. The temperature-sensitive phenotype ofcall-1 is most efficiently suppressed by overproduction of Rholp. Overproduction of the other essential target, Cdc42p, in contrast, is deleterious incall-1 cells, but not in othercdc43 mutants or the wild-type strains. Thecdc43-5 mutant cells accumulate Cdc42p in soluble pools andcdc43-5 is suppressed by overproduction of Cdc42p. Thus, several phenotypic differences are observed among thecall/cdc43 mutations, possibly due to alterations in substrate specificity caused by the mutations.     

Multiple cyclin-dependent kinase complexes and phosphatases control G2/M progression in alfalfa cells

Reversible phosphorylation of proteins by kinases and phosphatases plays a key regulatory role in several eukaryotic cellular functions including the control of the division cycle. Increasing numbers of sequence and biochemical data show the involvement of cyclin-dependent kinases (CDKs) and cyclins in regulation of the cell cycle progression in higher plants. The complexity represented by different types of CDKs and cyclins in a single species such as alfalfa, indicates that multicomponent regulatory pathways control G₂/M transition. A set of cdc2-related genes (cdc2Ms A, B, D and F) was expressed in G₂ and M cells. Phosphorylation assays also revealed that at least three kinase complexes (Cdc2Ms A/B, D and F) were successively active in G₂/M cells after synchronization. Interaction between alfalfa mitotic cyclin (Medsa;CycB2;1) and a kinase partner has been reported previously. The present yeast two-hybrid analyses showed differential interaction between defined D-type cyclins and Cdc2Ms kinases functioning in G₂/M phases. Localization of Cdc2Ms F kinase to the preprophase band (PPB), the perinuclear ring in early prophase, the mitotic spindle and the phragmoplast indicated a pivotal role for this kinase in mitotic plant cells. So far limited research efforts have been devoted to the functions of phosphatases in the control of plant cell division. A homologue of dual phosphatase, cdc25, has not been cloned yet from alfalfa; however tyrosine phosphorylation was indicated in the case of Cdc2Ms A kinase and the p^13suc1-bound kinase activity was increased by treatment of this complex with recombinant Drosophila Cdc25. The potential role of serine/threonine phosphatases can be concluded from inhibitor studies based on okadaic acid or endothall. Endothall elevated the kinase activity of p13^suc1-bound fractions in G₂-phase alfalfa cells. These biochemical data are in accordance with observed cytological abnormalities. The present overview with selected original data outlines a conclusion that emphasizes the complexity of G₂/M regulatory events in flowering plants.     

Identification and functional characterization of <Emphasis Type="Italic">Candida albicans CDC4</Emphasis>

Summary The CDC4 gene of Saccharomyces cerevisiae encodes an essential function that is required for G1-S and G2-M transitions during mitosis and at various stages during meiosis. We have isolated a functional homologue of CDC4 (CaCDC4) from the pathogenic yeast Candida albicans by complementing the S. cerevisiae cdc4-3 mutation with CaCDC4 expressed from its own promoter on a single-copy vector. The predicted product of CaCDC4 has 37% overall identity to the S. cerevisiae Cdc4 protein, although this identity is biased towards the C-terminal region of the two proteins which contains eight copies of the degenerate WD-40 motif, an element found in proteins that regulate diverse biological processes and an F-box domain proximal to the first iteration of the WD-40 motif. Both the F-box domain and WD-40 motifs appear necessary for the mitotic functions of Cdc4 in both yeasts. In contrast to its conserved role in mitosis, C. albicans CDC4 is unable to rescue the meiotic deficiency in a S. cerevisiae cdc4 homozygous diploid under restrictive conditions, even when expressed from an efficient S. cerevisiae promoter. In opposition to S. cerevisiae CDC4 being essential, C. albicans CDC4 appears to be nonessential and in its absence is critical for filamentous growth in C. albicans.     

Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants.

Summary Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 andcdc2Os-2) encoding structural homologues of thecdc2 ⁺/CDC28(cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62 % identical toCDC28 ofSaccharomyces cerevisiae and much more similar to the yeast and mammalian p34^cdc2 kinases than to riceR2, acdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1,cdc2Os-2 andR2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings.cdc2Os-1 could complement a temperature-sensitive yeast mutant ofcdc28. However, despite the similarity in structure, bothcdc2Os-2 andR2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of thecdc2 cell cycle kinase in rice.The nucleotide sequence data in this paper have been deposited in the EMBL database under accession number X60374 (cdc2Os-1) and X60375 (cdc2Os-2)