As part of our effort to sequence the 100-megabase (Mb) genome of the nematode Caenorhabditis elegans, we have completed the nucleotide sequence of a contiguous 2,181,032 base pairs in the central gene cluster of chromosome III. Analysis of the finished sequence has indicated an average density of about one gene per five kilobases; comparison with the public sequence databases reveals similarities to previously known genes for about one gene in three. In addition, the genomic sequence contains several intriguing features, including putative gene duplications and a variety of other repeats with potential evolutionary implications.

Deletion of the bacterial two‐component response regulator homologue Skn7 results in sensitivity of yeast to oxidizing agents indicating that Skn7 is involved in the response to this type of stress. Here we demonstrate that following oxidative stress, Skn7 regulates the induction of two genes: TRX2, encoding thioredoxin, and a gene encoding thioredoxin reductase. TRX2 is already known to be induced by oxidative stress dependent on the Yap1 protein, an AP1‐like transcription factor responsible for the induction of gene expression in response to various stresses. The thioredoxin reductase gene has not previously been shown to be activated by oxidative stress and, significantly, we find that it too is regulated by Yap1. The control of at least TRX2 by Skn7 is a direct mechanism as Skn7 binds to the TRX2 gene promoter in vitro. This shows Skn7 to be a transcription factor, at present the only such eukaryotic two‐component signalling protein. Our data further suggest that Skn7 and Yap1 co‐operate on the TRX2 promoter, to induce transcription in response to oxidative stress.

Mutation of predicted 3′‐‐>5′ exonuclease active site residues of Saccharomyces cerevisiae POL3 DNA polymerase (delta) or deletion of the PMS1 mismatch repair gene lead to relative (to wild type) spontaneous mutation rates of approximately 130 and 41, respectively, measured at a URA3 reporter gene inserted near to a defined replication origin. The POL3 exonuclease‐deficient mutant pol3‐01 generated most classes of single base mutation in URA3, indicating a broad specificity that generally corresponds to that of the PMS1 system. pol3‐01 pms1 haploid cells ceased growth after a few divisions with no unique terminal cell morphology. A pol3‐01/pol3‐01 pms1/pms1 diploid was viable and displayed an estimated URA3 relative mutation rate of 2 × 10(4), which we calculate to be catastrophically high in a haploid. The relationship between the relative mutation rates of pol3‐01 and pms1 was multiplicative, indicating action in series. The PMS1 transcript showed the same cell cycle periodicity as those of a set of DNA replication genes that includes POL3, suggesting PMS1 is co‐regulated with these genes. We propose that the POL3 3′‐‐>5′ exonuclease and the PMS1 mismatch repair system act on a common pathway analogous to the dnaQ‐‐>mutHLS pathway of DNA replication error correction in Escherichia coli.

Specific transcription in late G1, mediated by the transcription factors SBF (Swi4p-Swi6p) and MBF (Mbplp-Swi6p), is crucial for cell cycle progression in budding yeast. In order to better understand the G1/S transition, we initiated a search for conditional mutations synthetic lethal with swi4A. One of the isolated mutants, rsf8swi4A, showed a growth defect due to cell lysis. rsf8 is allelic to PKCJ, encoding a protein kinase C homologue which controls cell integrity. In the presence of the rsf8/(pkcl-8) mutation, a functional SBF but not MBF is required for viability. Importantly, swi4A and swi6A strains are hypersensitive to calcofluor white and SDS, indicating that they possess a weakened cell wall. Overexpression or ectopic expression of CLN did not suppress the pkc1-8swi4A mutant phenotype, thus SBF must control cell integrity independently, rather than acting through CLN expression. We found that at least six genes involved in cell wall biosynthesis are periodically expressed at the G1/S phase boundary. In all six cases, cell cycleregulated expression is due mainly to Swi4p. Finally, we found that the PKCI MAP kinase pathway is a positive regulator of five of these cell wall genes, these genes being novel targets of regulation by this pathway. We suggest that SBF and the PKCJ MAP kinase pathway act in concert to maintain cell integrity during bud formation.

We have isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying mutations of DBF4, a gene that is required for the initiation of chromosomal DNA replication in Saccharomyces cerevisiae and that interacts with the CDC7 protein kinase. periodically during the cell cycle, peaking at the G2/M boundary. CDCS on a multicopy plasmid also suppresses temperature-sensitive cdc15, cdc2O, and dbf2 mutations which affect mitosis during the cell cycle.Protein phosphorylation by protein kinases plays an important role in regulating both the mitotic cell cycle and meiosis in eukaryotes (28, 49). In the yeast Saccharomyces cerevisiae, genes encoding more than 30 protein kinases have been identified (16). Among these, at least four protein kinase genes, CDC28, CDC7, DBF2, and HRR25, are associated with DNA metabolism in the mitotic cell cycle. CDC28 is essential for cell growth and is required both for entry into the S phase and for the G2/M transition (35). Although the cellular abundance of the Cdc28 protein remains constant throughout the cell cycle (30), its protein kinase is periodically active and is regulated by a physical interaction with G, and G2 cyclins (8, 37, 45). CDC7 is also essential for cell growth and is required for the GJIS transition. It has recently been shown that the Cdc7 polypeptide has an associated protein kinase activity and is phosphorylated in vivo (18). Its execution point is just before the initiation of DNA replication (12). After the CDC7 execution point, no protein synthesis is required for the initiation of DNA replication (15). The abundance of the CDC7 transcript remains constant throughout the cell cycle (42), and this is likely to be true for the Cdc7 polypeptide also. However, no direct relationship between the Cdc28 and Cdc7 protein kinases has been elucidated to date.Deletion of DBF2 is not lethal because a homolog ofDBF2 (DBF20) is able to substitute for DBF2 function (48). However, in dbf2 mutants DNA synthesis is transiently delayed and the cell cycle is blocked in late nuclear division at the restrictive temperature, suggesting that the Dbf2 protein is required for completion of the S and M phases (20). The DBF2 transcript accumulates periodically during the cell * Corresponding author.cycle (20), and it is likely that this is also true of the Dbf2 protein. HRR25 is not essential for cell growth, but deletion of HRR25 results in a delay at the G2/M boundary (17), suggesting an important role in the cell cycle. Mutant cells carrying hrr25 exhibit sensitivity to methyl methanesulfonate and X rays (17), suggesting that the gene is also required for DNA repair.In an attempt to identify the various proteins that might interact either with the Cdc7 protein kinase or with both the Cdc28 and Cdc7 protein kinases, we isolated a multicopy suppressor of the temperature-sensitive growth phenotype of organisms carrying cdc7 mutations (24). The suppressor was identified as the DBF4 gene, whose execution point is just before initiation of DNA replication ...