Cdc2 Gene Research Articles

Intracellular location of the Saccharomyces cerevisiae CDC6 gene product.

The CDC6 gene product from Saccharomyces cerevisiae is required for transition from late G1 to S phase of the cell cycle. We have investigated the subcellular localization of the CDC6 protein in yeast to explore where Cdc6p exerts its gene function (s). Using affinity-purified sera we localized Cdc6p to the cytoplasm and the nuclear matrix by both subcellular fractionation and indirect immunofluorescence microscopy. The nuclear localization was confirmed to be in the nuclear scaffold by the low-salt extraction method. The Cdc6p cannot be detected in the mitochondrial or plasma membrane fractions. Using indirect immunofluorescence, we found that a subpopulation of Cdc6p migrated into the nucleus after G1/S transition and diminished after M phase, suggesting its temporal role in nuclear DNA replication. The predicted Cdc6p polypeptide contains a conserved nuclear localization, 27PLKRKKL33, similar to that of the SV40 large T antigen and other nuclear proteins. To test whether this peptide segment plays a role in mediating nuclear transport, we have carried out site-directed mutagenesis to alter the conserved 29Lys to Thr and Arg. The wild-type nuclear localization signal of Cdc6p was found to mediate the LacZ reporter gene fused to CDC6 efficiently to the nucleus, but not the mutated versions of the nuclear localization motif. The results suggested that 29Lys is important in mediating nuclear localization, the 29Thr and 29Arg mutant versions of the CDC6 gene were also unable to complement the cdc6 temperature-sensitive mutant. However, when these mutants were expressed from a multicopy plasmid, the mutated genes could complement the mutation. Similar results were obtained in the cdc6-disrupted cells. Taken together, we suggest that (i) Cdc6p is predominantly located in the cytoplasm, (ii) the nuclear entry of Cdc6p is cell cycle dependent, and (iii) nuclear entry of Cdc6p is mediated by its nuclear localization signal. The presence of Cdc6p in both the nucleus and the cytoplasm suggests a model that Cdc6p exerts its gene function in DNA replication and mitotic restraint in the cell cycle.

The Cdc25 genes map to mouse chromosomes 2, 9, and 18.

The Cdc25 genes map to mouse chromosomes 2, 9, and 18.

Distinct classes of cdc2-related genes are differentially expressed during the cell division cycle in plants.

cdc2 and several related genes encode the catalytic subunits of cyclin-dependent kinases, which have been implicated in a number of cellular processes, including control of cell division. As a first step in exploring their function in plants, we isolated four cdc2-related genes from Antirrhinum. Two genes, cdc2a and cdc2b, encode proteins that contain a perfectly conserved PSTAIRE motif characteristic of cdc2 homologs, whereas the products of the two remaining genes, cdc2c and cdc2d, appear to represent a new subclass of proteins that have so far only been identified in plants. Transcripts of these novel genes were localized in isolated cells dispersed throughout actively dividing regions of the inflorescence. This localization is consistent with accumulation that is specific to particular phases of the cell cycle. Correlating cell labeling with nuclear condensation and double-labeling experiments using cdc2 and histone H4 as probes indicated that cdc2c transcripts accumulate during S phase as well as during the G2 and M transition, whereas cdc2d expression was specific to the G2 and M phases. All cells labeled with cdc2d also contained cdc2c label, Indicating that expression of cdc2d completely overlapped with that of cdc2c. Transcripts of cdc2a and cdc2b were detected in all cells within actively dividing regions, but at levels that were only slightly higher than those observed in nondividing areas. These transcripts did not appear to accumulate in a cell cycle-specific fashion. The genes cdc2a and cdc2b were able to partially complement a yeast cdc2 mutation, although all four genes appeared to interfere with the sizing mechanism of yeast cells. We propose that plants contain at least two classes of cdc2-related genes that differ in structure, expression, and perhaps function.

Paul Nurse: cycling up the hill

Paul Nurse: cycling up the hill

Regulation of the activity of M-phase promoting factor through protein kinase A-mediated pathway in LP1-1 cells.

Treatment of cAMP analogs, dibutyryl cAMP and 8-bromo-cAMP, was found to inhibit the proliferation of mouse fibroblast LP1-1 cells and the p34cdc2 kinase activity of M-phase promoting factor (MPF). However, it showed relatively little effect on expression of the cyclin B1 and cdc2 genes. On the other hand, when the nuclear extracts obtained from the cells at early G2 phase were treated with cAMP analogs, the kinase activity was significantly decreased as compared to the untreated control. Furthermore, the inhibitory effect of cAMP analogs could be reversed upon treating with okadaic acid even in the presence of the cAMP analogs, implying that cdc25 remains in an active form. In addition, the treatment of okadaic acid stimulated the cell progression. These results suggest that down-regulation of MPF activity through protein kinase A-mediated pathway is under post-translational control and cdc25 activation pathway involving okadaic acid-sensitive phosphatase play a role in the regulation of MPF activity.

Novel alleles of cdc13 and cdc2 isolated as suppressors of mitotic catastrophe in Schizosaccharomyces pombe.

Cell cycle control in the fission yeast Schizosaccharomyces pombe involves interplay amongst a number of regulatory molecules, including the cdc2, cdc13, cdc25, wee1, and mik1 gene products. Cdc2, Cdc13, and Cdc25 act as positive regulators of cell cycle progression at the G2/M boundary, while Wee1 and Miky1 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 both cdc2 and cdc13, including a novel wee allele of cdc2, cdc2-5w. Here, we characterize cdc2-5w and two alleles of cdc13, which have implications for the understanding of details of the interactions amongst Cdc2, Cdc13, and Wee1.

The Cdc25 protein of Saccharomyces cerevisiae is required for normal glucose transport

The essential CDC25 gene product of Saccharomyces cerevisiae is the most upstream known component of the RAS/adenylate cyclase pathway. Cdc25 is a GTP-exchange protein involved in activating RAS in response to fermentable carbon sources. In this paper it is reported that the Cdc25 protein, in addition to its stimulatory role in the RAS/adenylate cyclase pathway, regulates glucose transport. Continuous culture studies and glucose uptake experiments showed that the cdc25-1 and the cdc25-5 temperature-sensitive mutants exhibit decreased glucose uptake activity at the restrictive temperature under both repressed and derepressed conditions as compared to the wild-type strain. Because the cdc25-1 mutant is not impaired in its cAMP metabolism, it is concluded that this effect on glucose transport is independent of cAMP levels. Furthermore, it is shown that the decrease in glucose uptake activity is not due to a decrease in protein synthesis or to an arrest in the G1 phase of the cell cycle. In addition to a defect in glucose uptake, the cdc25-5 mutant strain exhibited differences in glucose metabolism, probably due to the decreased cAMP level and hence decreased protein kinase A activity. Because the Cdc25 protein is localized at the membrane, these results indicate that Cdc25 is directly involved in glucose transport and may be in direct contact with the glucose transporters.

A connection between pre-mRNA splicing and the cell cycle in fission yeast: cdc28+ is allelic with prp8+ and encodes an RNA-dependent ATPase/helicase.

The fission-yeast gene cdc28+ was originally identified in a screen for temperature-sensitive mutants that exhibit a cell-division cycle arrest and was found to be required for mitosis. We undertook a study of this gene to understand more fully the general requirements for entry into mitosis. Cells carrying the conditional lethal cdc28-P8 mutation divide once and arrest in G2 after being shifted to the restrictive temperature. We cloned the cdc28+ gene by complementation of the temperature-sensitive growth arrest in cdc28-P8. DNA sequence analysis indicated that cdc28+ encodes a member of the DEAH-box family of putative RNA-dependent ATPases or helicases. The Cdc28 protein is most similar to the Prp2, Prp16, and Prp22 proteins from budding yeast, which are required for the splicing of mRNA precursors. Consistent with this similarity, the cdc28-P8 mutant accumulates unspliced precursors at the restrictive temperature. Independently, we isolated a temperature-sensitive pre-mRNA splicing mutant prp8-1 that exhibits a cell-cycle phenotype identical to that of cdc28-P8. We have shown that cdc28 and prp8 are allelic. These results suggest a connection between pre-mRNA splicing and progression through the cell cycle.

Yeast ubiquitin-like genes are involved in duplication of the microtubule organizing center.

KAR1 is required for duplication of the Saccharomyces cerevisiae microtubule organizing center, the spindle pole body (SPB) (Rose, M.D., and G.R. Fink, 1987. Cell. 48:1047-1060). Suppressors of a kar1 allele defective for SPB duplication were isolated in two genes, CDC31 and DSK2 (Vallen, E.A., W.H., M. Winey, and M.D. Rose. 1994. Genetics. 137:407-422). To elucidate the role of DSK2 in SPB duplication, we cloned the gene and found it encodes a novel ubiquitin-like protein containing an NH2 terminus 36% identical to ubiquitin. The only other known yeast ubiquitin-like protein is encoded by the nucleotide excision repair gene RAD23 (Watkins, J.F.,P. Sung, L. Prakash, and S. Prakash. 1993. Mol. Cell. Bio. 13:7757-7765). Unlike ubiquitin, the NH2-terminal domain of Dsk2p is not cleaved from the protein, indicating that Dsk2p is not conjugated to other proteins. Although the DSK2-1 mutation alters a conserved residue in the Dsk2p ubiquitin-like domain, we detect no differences in Dsk2p or Cdc31p stability. Therefore, DSK2 does not act by interfering with ubiquitin-dependent protein degradation of these proteins. Although DSK2 is not essential, a strain deleted for both DSK2 and RAD23 is temperature sensitive for growth due to a block in SPB duplication. In addition, overexpression of DSK2 is toxic, and the DSK2-1 allele causes a block in SPB duplication. Therefore, DSK2 dosage is critical for SPB duplication. We determined that CDC31 gene function is downstream of DSK2 and KAR1. Dsk2p is a nuclear-enriched protein, and we propose that Dsk2p assists in Cdc31 assembly into the new SPB.

Control of root growth and development by cyclin expression.

Root development is plastic, with post-embryonic organogenesis being mediated by meristems. Although cell division is intrinsic to meristem initiation, maintenance and proliferative growth, the role of the cell cycle in regulating growth and development is unclear. To address this question, we examined the expression of cdc2 and cye genes, which encode the catalytic and regulatory subunits, respectively, of cyclin-dependent protein kinases that control progression through the cell cycle. Unlike cdc2, which is expressed not only in apical meristems but also before lateral root initiation in quiescent, pericycle cells arrested in the G2 phase of the cell cycle, cyc1At transcripts accumulate specifically in dividing cells immediately before cytokinesis. Ectopic expression of cyc1At under the control of the cdc2aAt promoter in Arabidopsis plants markedly accelerates growth without altering the pattern of lateral root development or inducing neoplasia. Thus cyclin expression is a limiting factor for growth, which in turn drives indeterminate development of the root system.

Characterization of a novel CDC gene (ORC1) partly homologous to CDC6 of Saccharomyces cerevisiae.

A novel cell cycle gene was identified by a computer search for genes partly homologous to known CDC genes, CDC6 of Saccharomyces cerevisiae and CDC18 of Schizosaccharomyces pombe, using the nucleotide sequence data base for S. cerevisiae produced by the Yeast Sequencing Project. The protein sequence coded by the cloned gene was found to be identical to that of purified ORC1 protein. Disruption of the gene and subsequent tetrad analysis revealed that the gene was essential for growth. The function of the gene product was analyzed by depleting the protein from the cell using a mutant haploid strain containing the disrupted ORC1 gene on the chromosome and a galactose-inducible gene coding for HA-tagged ORC1 protein on a single copy plasmid. The HA-tagged protein was expressed during growth in the presence of galactose but began to decrease rapidly upon depletion of galactose. Analysis of the cell cycle progression of the mutant cells by FACS after the removal of galactose from the medium, and microscope observations of cells and their nuclei revealed that the normal progression of 2N cells was immediately impeded as the ORC1 protein started to decrease. This was blocked completely in the cells that had progressed to the S phase under conditions deficient in ORC1 protein followed by cell death. Two-dimensional gel analysis of the replication intermediates after the galactose removal revealed that the depletion of ORC1 protein caused a decrease in the frequency of initiation of chromosomal replication, eventually resulting in the inhibition of replication as a whole. The function of the ORC1 protein in the cell cycle progression of S. cerevisiae is discussed in light of current information on ORC.

Positive Regulation of cdc2 Gene Activity by Protein Phosphatase Type 2A

Several lines of evidence indicate that serine/threonine protein phosphatases may act as negative regulators of cellular growth. For example, treatment of cells with the tumor-promoter okadaic acid, an inhibitor of certain types of these phosphatases, resulted in the increased expression of several proto-oncogenes, indicating a negative role of the respective phosphatases in gene regulation. However, it was puzzling to find that okadaic acid-treated cells, even in the presence of highly expressed proto-oncogenes, did not proliferate, but were arrested at certain points of the cell cycle. To further analyze this discrepancy, we investigated the involvement of protein phosphatases in the control of other cell cycle regulatory genes, such as cdc2 which encodes an essential cell cycle regulatory kinase. We found that cdc2 gene expression was blocked by okadaic acid, but stimulated by protein phosphatase 2A. Protein phosphatase 2A is shown to be a positive regulator of cdc2 gene activity and to be required for cdc2 expression. Thus, our findings identify protein phosphatase 2A as a positive regulator of a major cell cycle regulatory gene and therefore suggest a stimulatory role of this enzyme in this aspect of cellular growth control.

THE ROLE OF CDC2, CDC25 AND CYCLIN A GENES IN THE MAINTENANCE OF IMMORTALIZATION AND GROWTH ARREST IN A RAT EMBRYONIC FIBROBLAST CONDITIONAL CELL LINE

Immortalization of rodent cells by oncogenes is a complex biological process which involves the abnormal regulation of genes who control cellular proliferation. The role of the cell cycle control genes cdc2, cdc25 and cyclin A in the maintenance of immortalization and in growth arrest was examined in the tsa14, a SV40 T antigen rat embryonic fibroblast conditional for growth cell line. Analysis of RNA expression showed minimal levels of cdc2 mRNA in both proliferating and growth-arrested tsa14 cells. In contrast, cyclin A mRNA was found downregulated in growth-arrested tsa14 cells, as well as in senescent primary rat embryonic fibroblasts (REFS). The ability of cdc2, or cdc25, or cyclin A genes to maintain the tsa14 immortal phenotype was also examined by electroporations of these genes into the tsa14 cells. Clones over-expressing the electroporated cdc2, or cdc25, or cyclin A, or combinations of these genes growth arrested at the non-permissive conditions similar to controls, thereby suggesting that the expression of these genes alone is insufficient for tsa14 maintenance of immortalization.

A single fission yeast mitotic cyclin B p34cdc2 kinase promotes both S-phase and mitosis in the absence of G1 cyclins.

Deletion of the fission yeast mitotic B-type cyclin gene cdc13 causes cells to undergo successive rounds of DNA replication. We have used a strain which expresses cdc13 conditionally to investigate re-replication. Activity of Start genes cdc2 and cdc10 is necessary and p34cdc2 kinase is active in re-replicating cells. We tested to see whether other cyclins were required for re-replication using cdc13delta. Further deletion of cig1 and puc1 had no effect, but deletion of cig2/cyc17 caused a severe delay in re-replication. Deletion of cig1 and cig2/cyc17 together abolished re-replication completely and cells arrested in G1. This, and analysis of the temperature sensitive cdc13-117 mutant, suggests that cdc13 can effectively substitute for the G1 cyclin activity of cig2/cyc17. We have characterized p56cdc13 activity and find evidence that in the absence of G1 cyclins, S-phase is delayed until the mitotic p34cdc2-p56cdc13 kinase is sufficiently active. These data suggest that a single oscillation of p34cdc2 kinase activity provided by a single B-type cyclin can promote ordered progression into both DNA replication and mitosis, and that the level of cyclin-dependent kinase activity may act as a master regulator dictating whether cells undergo S-phase or mitosis.

Candida albicans CDK1 and CYB1: cDNA homologues of the cdc2/CDC28 and cdcl3/CLB1/CLB2 cell cycle control genes

Major transitions in the eukaryotic cell cycle are regulated by the cyclin-dependent protein kinases (CDK). In particular, the G2/M transition is initiated by the activity of a complex formed by a CDK of the Cdc2/Cdc28 family and B-type cyclins of the Cdc13/Clb family in the yeasts, Schizosaccharomyces pombe (Sp) and Saccharomyces cerevisiae (Sc). To study the molecular mechanisms that control the G2/M transition in the dimorphic pathogenic yeast, Candida albicans, we have cloned and characterized cDNAs corresponding to CDK1 and CYB1. The CDK1 cDNA encodes a 317-amino-acid (aa) protein that shares 76.8 and 62.3% identity with the Sc CDC28 and Sp cdc2 gene products, respectively. The CYB1 cDNA encodes a 493-aa protein that is 34.8, 34.4 and 35.5% identical to Sc Clbl and Clb2, and to Sp Cdc13, respectively. Cyb1 contains characteristic mitotic destruction and cyclin boxes. The CDK1 and CYB1 cDNAs are functional homologues, as they are able to complement Sp cdc2 and cdc13 temperature-sensitive (ts) mutations, respectively, and their gene products interact in vivo in Sc to form an active histone H1 kinase.

5441880 Human cdc25 genes, encoded products and uses thereof : Beach David H; Galaktionov Konstantin Huntington Bay, NY, United States assigned to Cold Spring Harbor Laboratory

5441880 Human cdc25 genes, encoded products and uses thereof : Beach David H; Galaktionov Konstantin Huntington Bay, NY, United States assigned to Cold Spring Harbor Laboratory

Membrane-anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor

The CDC25 gene product from Saccharomyces cerevisiae, the prototype of the family of ras guanine nucleotide exchange factors, is expressed as a 180-kDa polypeptide, tightly bound to a membrane fraction. The ability to complement a cdc25 defect is located in the 3' part of the gene (codons 877–1589). Sequence analysis reveals only a short hydrophobic domain (residues 1459–1471) and no consensus sequence for post-translational acylation. The SH3 domain present in the N-terminal part of Cdc25p is not involved nor required for membrane localization, since the N-terminal part of Cdc25p did not fractionate with a membrane pellet. In contrast, the C-terminal part was attached to a 18000 g pellet after subcellular fractionation and immunoblotting. This subcellular localization was conserved in a ras1ras2 double disruption mutant and in a ira2 disruption mutant. Immunofluorescence analysis showed a patchy staining, mainly at the periphery of the cells. These patches were quite distinct from actin patches by double immunolabeling. By analysing a set of truncated derivatives, the elements required for a particulate localization were restricted to residues 1441–1552.

5441880 Human cdc25 genes, encoded products and uses thereof : Beach David H; Galaktionov Konstantin Huntington Bay, NY, United States assigned to Cold Spring Harbor Laboratory

5441880 Human cdc25 genes, encoded products and uses thereof : Beach David H; Galaktionov Konstantin Huntington Bay, NY, United States assigned to Cold Spring Harbor Laboratory

Isolation and molecular characterisation of the POL3 gene from Candida albicans

In Saccharomyces cerevisiae, the CDC2 gene encodes the large subunit of DNA polymerase III, the analogue of mammalian DNA polymerase δ. We have isolated DNA fragments from a library of Candida albicans genomic DNA in the vector pRS316 that rescue temperature sensitive cdc2 mutations in S. cerevisiae. These fragments contain an ORF coding for a protein of 1038 aa with a predicted molecular mass of 118.8 kDa. The predicted protein shows homology to a number of eukaryotic DNA polymerases, with 62% identity over its length to the S. cerevisiae Cdc2 protein. It also contains a number of motifs which are characteristic of DNA polymerases in general and viral polymerases in particular, as well as the conserved motif which interacts with proliferating cell nuclear antigen. These results indicate that this gene is C. albicans POL3. Analysis of the expression of C. albicans POL3 revealed that the transcript is present throughout the mitotic cell cycle, which contrasts with the expression of S. cerevisiae CDC2

Guided myeloid leukemia cell differentiation by blocking a specific cell cycle stage.

Since malignant cell proliferation occurs at the expense of cell differentiation, we examined the possibility of differentiating malignant cells from mature cells by altering the regulation of the cell cycle. Using the human promyelocytic leukemia cell line HL-60, which expresses p34cdc2, the product of the cdc2 gene that controls the cell cycle, we showed that guided internalization of an anti-cdc2 monoclonal antibody could initiate differentiation not yet described for other cells of myeloid malignant origin. Experimental cellular manipulation may be employed as a method of inducing in vitro differentiation for transplantation purposes.