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 ...

The transcription factor NF-kappaB is composed of homodimeric and heterodimeric complexes of Rel/NF-kappaB-family polypeptides, which include Rel-A, c-Rel, Rel-B, NF-kappaB/p50 and NF-kappaB2/p52 . The NF-kappaB1 gene encodes a larger precursor protein, p105, from which p50 is produced constitutively by proteasome-mediated removal of the p105 carboxy terminus. The p105 precursor also acts as an NFkappaB-inhibitory protein, retaining associated p50, c-Rel and Rel-A proteins in the cytoplasm through its carboxy terminus. Following cell stimulation by agonists, p105 is proteolysed more rapidly and released Rel subunits translocate into the nucleus. Here we show that TPL-2 , which is homologous to MAP-kinase-kinase kinases in its catalytic domain, forms a complex with the carboxy terminus of p105. TPL-2 was originally identified, in a carboxy-terminal-deleted form, as an oncoprotein in rats and is more than 90% identical to the human oncoprotein COT. Expression of TPL-2 results in phosphorylation and increased degradation of p105 while maintaining p50 production. This releases associated Rel subunits or p50-Rel heterodimers to generate active nuclear NF-kappaB. Furthermore, kinase-inactive TPL-2 blocks the degradation of p105 induced by tumour-necrosis factor-alpha. TPL-2 is therefore a component of a new signalling pathway that controls proteolysis of NF-kappaB1 p105.

It is widely believed that the cellular transcription factor DRTF1/E2F integrates cell cycle events with the transcription apparatus because during cell cycle progression in mammalian cells it interacts with molecules that are important regulators of cellular proliferation, such as the retinoblastoma tumour suppressor gene product (pRb), p107, cyclins and cyclin‐dependent kinases. Thus, pRb, which negatively regulates early cell cycle progression and is frequently mutated in tumour cells, and the Rb‐related protein p107, bind to and repress the transcriptional activity of DRTF1/E2F. Viral oncoproteins, such as adenovirus E1a and SV40 large T antigen, overcome such repression by sequestering pRb and p107 and in so doing are likely to activate genes regulated by DRTF1/E2F, such as cdc2, c‐myc and DHFR. Two sequence‐specific DNA binding proteins, E2F‐1 and DP‐1, which bind to the E2F site, contain a small region of similarity. The functional relationship between them has, however, been unclear. We report here that DP‐1 and E2F‐1 exist in a DNA binding complex in vivo and that they bind efficiently and preferentially as a heterodimer to the E2F site. Moreover, studies in yeast and Drosophila cells indicate that DP‐1 and E2F‐1 interact synergistically in E2F site‐dependent transcriptional activation.

The Saccharomyces cerevisiae Sln1 protein is a 'twocomponent' regulator involved in osmotolerance. Twocomponent regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.