Preliminary characterization of the transcriptional and translational products of the Saccharomyces cerevisiae cell division cycle gene CDC28.

The CDC28 gene was subcloned from a plasmid containing a 6.5-kilobase-pair segment of Saccharomyces cerevisiae DNA YRp7(CDC28-3) by partial digestion with Sau3A and insertion of the resulting fragments into the BamHI sites of YRp7 and pRC1. Recombinant plasmids were obtained containing inserts of 4.4 and 3.1 kilobase pairs which were capable of complementing a cdc28(ts) mutation. R-loop analysis indicated that each yeast insert contained two RNA coding regions of about 0.8 and 1.0 kilobase pairs, respectively. In vitro mutagenesis experiments suggested that the smaller coding region corresponded to the CDC28 gene. When cellular polyadenylic acid-containing RNA, separated by agarose gel electrophoresis after denaturation with glyoxal and transferred to nitrocellulose membrane, was reacted with labeled DNA from the smaller coding region, and RNA species of about 1 kilobase in length was detected. Presumably, the discrepancy in size between the R-loop and electrophoretic determinations is due to a segment of polyadenylic acid which is excluded from the R-loops. By using hybridization of the histone H2B mRNAs to an appropriate probe as a previously determined standards, it was possible to estimate the number of CDC28 mRNA copies per haploid cell as between 6 and 12 molecules. Hybrid release translation performed on the CDC29 mRNA directed the synthesis of a polypeptide of 27,000 daltons, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. This polypeptide was not synthesized when mRNA prepared from a cdc28 nonsense mutant was translated in a parallel fashion. However, if the RNA from a cell containing the CDC28 gene on a plasmid maintained at a high copy number was translated, the amount of in vitro product was amplified fivefold.     

Molecular cloning of Saccharomyces cerevisiae CDC6 gene. Isolation, identification, and sequence analysis

The CDC6 gene product is required for entering the S phase of the cell cycle in Saccharomyces cerevisiae. It has been isolated on recombinant plasmids by selection for complementation of temperature-sensitive alleles with a yeast genomic library. The entire complementing activity is carried on a 1.8-kilobase chromosomal DNA fragment, as revealed by deletion mapping. Northern blotting shows that the size of the CDC6 mRNA is about 1.7 kilobases. A Southern blot of yeast chromosomes which were separated by the field inversion gel electrophoresis method indicates that the isolated DNA fragment is derived from chromosome X. The locus from which the clone was derived was marked by integration with a nutritional marker and found by meiotic mapping to cosegregate with CDC6. Thus, we conclude that we have isolated the authentic CDC6 gene. Nucleotide sequence analysis of the CDC6 gene has revealed an open reading frame that encodes a protein with Mr = 57,969. There are five potential Asn-X-(Ser/Thr) glycosylation sites and a highly conserved nucleotide-binding site in the CDC6 sequence. Although computer surveys indicate overall sequence homology between S. cerevisiae CDC6 protein and Saccharomyces pombe CDC10 START protein, they may not be functionally equivalent as evaluated by the complementation assay.     

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.     

DNA polymerase III, a second essential DNA polymerase, is encoded by the S. cerevisiae CDC2 gene

Three nuclear DNA polymerases have been described in yeast: DNA polymerases I, II, and III. DNA polymerase I is encoded by the POL1 gene and is essential for DNA replication. Since the S. cerevisiae CDC2 gene has recently been shown to have DNA sequence similarity to the active site regions of other known DNA polymerases, but to nevertheless be different from DNA polymerase I, we examined cdc2 mutants for the presence of DNA polymerases II and III. DNA polymerase II was not affected by the cdc2 mutation. DNA polymerase III activity was significantly reduced in the cdc2-1 cell extracts. We conclude that the CDC2 gene encodes yeast DNA polymerase III and that DNA polymerase III, therefore, represents a second essential DNA polymerase in yeast.     

Mutations in cell division cycle genes CDC36 and CDC39 activate the Saccharomyces cerevisiae mating pheromone response pathway.

Conditional mutations in the genes CDC36 and CDC39 cause arrest in the G1 phase of the Saccharomyces cerevisiae cell cycle at the restrictive temperature. We present evidence that this arrest is a consequence of a mutational activation of the mating pheromone response. cdc36 and cdc39 mutants expressed pheromone-inducible genes in the absence of pheromone and conjugated in the absence of a mating pheromone receptor. On the other hand, cells lacking the G beta subunit or overproducing the G alpha subunit of the transducing G protein that couples the receptor to the pheromone response pathway prevented constitutive activation of the pathway in cdc36 and cdc39 mutants. These epistasis relationships imply that the CDC36 and CDC39 gene products act at the level of the transducing G protein. The CDC36 and CDC39 gene products have a role in cellular processes other than the mating pheromone response. A mating-type heterozygous diploid cell, homozygous for either the cdc36 or cdc39 mutation, does not exhibit the G1 arrest phenotype but arrests asynchronously with respect to the cell cycle. A similar asynchronous arrest was observed in cdc36 and cdc39 cells where the pheromone response pathway had been inactivated by mutations in the transducing G protein. Furthermore, cdc36 and cdc39 mutants, when grown on carbon catabolite-derepressing medium, did not arrest in G1 and did not induce pheromone-specific genes at the restrictive temperature.     

Isolation and characterization of the HO gene from the yeast Saccharomyces paradoxus

A DNA fragment homologous to the homothallism (HO) gene of Saccharomyces cerevisiae was isolated from Saccharomyces paradoxus and was found to contain an open reading frame that was 90.9% identical to the coding sequence of the S. cerevisiae HO gene. The putative HO gene was shown to induce diploidization in a heterothallic haploid strain from S. cerevisiae. Phylogenetic analysis revealed that the coding and 5'-upstream regulatory regions from five Saccharomyces sensu stricto HO genes have coevolved, and that S. paradoxus is phylogenetically closer to S. cerevisiae than to S. bayanus. Finally, heterothallic haploid strains were isolated from the original homothallic type strain of S. paradoxus by disrupting the S. paradoxus HO gene with the S. cerevisiae URA3 gene.     

CDC45, a novel yeast gene that functions with the origin recognition complex and Mcm proteins in initiation of DNA replication.

The CDC45 gene of Saccharomyces cerevisiae was isolated by complementation of the cold-sensitive cdc45-1 mutant and shown to be essential for cell viability. Although CDC45 genetically interacts with a group of MCM genes (CDC46, CDC47, and CDC54), the predicted sequence of its protein product reveals no significant sequence similarity to any known Mcm family member. Further genetic characterization of the cdc45-1 mutant demonstrated that it is synthetically lethal with orc2-1, mcm2-1, and mcm3-1. These results not only reveal a functional connection between the origin recognition complex (ORC) and Cdc45p but also extend the CDC45-MCM genetic interaction to all known MCM family members that were shown to be involved in replication initiation. Initiation of DNA replication in cdc45-1 cells was defective, causing a delayed entry into S phase at the nonpermissive temperature, as well as a high plasmid loss rate which could be suppressed by tandem copies of replication origins. Furthermore, two-dimensional gels directly showed that chromosomal origins fired less frequently in cdc45-1 cells at the nonpermissive temperature. These findings suggest that Cdc45p, ORC, and Mcm proteins act in concert for replication initiation throughout the genome.     

Molecular characterization of CDC42, a Saccharomyces cerevisiae gene involved in the development of cell polarity

The Saccharomyces cerevisiae CDC42 gene product is involved in the morphogenetic events of the cell division cycle; temperature-sensitive cdc42 mutants are unable to form buds and display delocalized cell-surface deposition at the restrictive temperature (Adams, A. E. M., D. I. Johnson, R. M. Longnecker, B. F. Sloat, and J. R. Pringle. 1990. J. Cell Biol. 111:131-142). To begin a molecular analysis of CDC42 function, we have isolated the CDC42 gene from a yeast genomic DNA library. The use of the cloned DNA to create a deletion of CDC42 confirmed that the gene is essential. Overexpression of CDC42 under control of the GAL10 promoter was not grossly deleterious to cell growth but did perturb the normal pattern of selection of budding sites. Determination of the DNA and predicted amino acid sequences of CDC42 revealed a high degree of similarity in amino acid sequence to the ras and rho (Madaule, P., R. Axel, and A. M. Myers. 1987. Proc. Natl. Acad. Sci. 84:779-783) families of gene products. The similarities to ras proteins (approximately 40% identical or related amino acids overall) were most pronounced in the regions that have been implicated in GTP binding and hydrolysis and in the COOH-terminal modifications leading to membrane association, suggesting that CDC42 function also involves these biochemical properties. The similarities to the rho proteins (approximately 60% identical or related amino acids overall) were more widely distributed through the coding region, suggesting more extensive similarities in as yet undefined biochemical properties and functions.     

Isolation of a CDC28 homologue from Cryptococcus neoformans that is able to complement cdc28 temperature-sensitive mutants of Saccharomyces cerevisiae

A partial cDNA fragment of the Cryptococcus neoformans homologue of the main cell cycle control gene CDC28/cdc2 was isolated using degenerate primer RT-PCR. A subsequent search in the C. neoformans genome database identified several sequences similar to CDC28/cdc2. A part of the sequence which showed the highest similarity to CDC28/cdc2 turned out to be identical to the partial cyclin-dependent kinase (Cdk) cDNA fragment isolated by degenerate RT-PCR. The full-length coding region of this Cdk homologue was amplified by RT-PCR using primers designed to target regions around start and stop codons, and the gene was named CnCdk1. To determine its function, an analysis of deduced amino acid sequence of the CnCdk1 was performed and its ability to rescue Saccharomyces cerevisiae cdc28-temperature sensitive mutants was tested. S. cerevisiae cdc28-4 and cdc28-1N strains transformed with the pYES2- CnCdk1 construct exhibited growth at 36.5 degrees C in galactose-raffinose medium, but not in glucose medium. Results of the sequence analysis and the fact that CnCdk1 is able to complement the S. cerevisiae cdc28-ts mutation support its assumed role as the CDC28/cdc2 homologue in C. neoformans.     

Characterization of the five replication factor C genes of Saccharomyces cerevisiae.

Replication factor C (RFC) is a five-subunit DNA polymerase accessory protein that functions as a structure-specific, DNA-dependent ATPase. The ATPase function of RFC is activated by proliferating cell nuclear antigen. RFC was originally purified from human cells on the basis of its requirement for simian virus 40 DNA replication in vitro. A functionally homologous protein complex from Saccharomyces cerevisiae, called ScRFC, has been identified. Here we report the cloning, by either peptide sequencing or by sequence similarity to the human cDNAs, of the S. cerevisiae genes RFC1, RFC2, RFC3, RFC4, and RFC5. The amino acid sequences are highly similar to the sequences of the homologous human RFC 140-, 37-, 36-, 40-, and 38-kDa subunits, respectively, and also show amino acid sequence similarity to functionally homologous proteins from Escherichia coli and the phage T4 replication apparatus. All five subunits show conserved regions characteristic of ATP/GTP-binding proteins and also have a significant degree of similarity among each other. We have identified eight segments of conserved amino acid sequences that define a family of related proteins. Despite their high degree of sequence similarity, all five RFC genes are essential for cell proliferation in S. cerevisiae. RFC1 is identical to CDC44, a gene identified as a cell division cycle gene encoding a protein involved in DNA metabolism. CDC44/RFC1 is known to interact genetically with the gene encoding proliferating cell nuclear antigen, confirming previous biochemical evidence of their functional interaction in DNA replication.     

Characterization of the oncogene (erb) of avian erythroblastosis virus and its cellular progenitor.

Avian erythroblastosis virus (AEV) induces primarily erythroblastosis when injected intravenously into susceptible chickens. In vitro, the hematopoietic target cells for transformation are the erythroblasts. Occasional sarcomas are also induced by intramuscular injection, and chicken or quail fibroblasts can be transformed in vitro. The transforming capacity of AEV was shown to be associated with the presence of a unique nucleotide sequence denoted erb in its genomic RNA. Using a simplified procedure, we prepared radioactive complementary DNA (cDNAaev) representative of the erb sequence at a high yield. Using a cDNAaev excess liquid hybridization technique adapted to defective retroviruses, we determined the complexity of the erb sequence to be 3,700 +/- 370 nucleotides. AEV-transformed erythroblasts, as well as fibroblasts, contained two polyadenylated viral mRNA species of 30 and 23S in similar high abundance (50 to 500 copies per cell). Both species were efficiently packaged into the virions. AEV-transformed erythroblasts contained additional high-molecular-weight mRNA species hybridizing with cDNAaev and cDNA5' but not with cDNA made to the helper leukosis virus used (cDNArep). The nature and the role, if any, of these bands remain unclear. The erb sequence had its counterpart in normal cellular DNA of all higher vertebrate species tested, including humans and fish (1 to 2 copies per haploid genome in the nonrepetitive fraction of the DNA). These cellular sequences (c-erb) were transcribed at low levels (1 to 2 RNA copies per cell) in chicken and quail fibroblasts, in which the two alleged domains of AEV-specific sequences corresponding to the 75,000- and 40,000-molecular-weight proteins seemed to be conserved phylogenetically and transcribed at similar low rates.     

Sequence of the cell division gene CDC2 from Schizosaccharomyces pombe; patterns of splicing and homology to protein kinases

The complete nucleotide sequence of a 2.9-kb DNA fragment containing the CDC2 gene-complementing activity from Schizosaccharomyces pombe has been determined. Within this region lies a 1.69-kb DNA sequence whose predicted amino acid sequence shows extensive homology to that previously deduced for the CDC28 gene product from Saccharomyces cerevisiae [L?rincz and Reed, Nature 307 (1984) 183-185]. Taken with the earlier observation that mutants in CDC2 can be rescued by the presence of the CDC28 gene [Beach, Durkacz and Nurse, Nature 300 (1982) 706-709], these results strongly suggest that the two genes code for similar functions. In contrast to the CDC28 gene, however, which contains no introns, the CDC2 coding sequence is split by four introns and from a comparison of the two sequences a consensus sequence for intron splicing in S. pombe can be established. Both CDC2 and CDC28 contain the consensus sequences for the ATP binding and phosphorylation acceptor sites of protein kinases such as bovine cAMP-dependent protein kinase (bov PK) and the src family of viral oncogene products.