In Saccharomyces cerevisiae, expression of arginine catabolic genes CAR1 and CAR2 in response to exogenous nitrogen availability is mediated by the Ume6 (CargRI)-Sin3 (CargRII)-Rpd3 (CargRIII) complex.

The products of three genes named CARGRI, CARGRII, and CARGRIII were shown to repress the expression of CAR1 and CAR2 genes, involved in arginine catabolism. CARGRI is identical to UME6 and encodes a regulator of early meiotic genes. In this work we identify CARGRII as SIN3 and CARGRIII as RPD3. The associated gene products are components of a high-molecular-weight complex with histone deacetylase activity and are recruited by Ume6 to promoters containing a URS1 sequence. Sap30, another component of this complex, is also required to repress CAR1 expression. This histone deacetylase complex prevents the synthesis of the two arginine catabolic enzymes, arginase (CAR1) and ornithine transaminase (CAR2), as long as exogenous nitrogen is available. Upon nitrogen depletion, repression at URS1 is released and Ume6 interacts with ArgRI and ArgRII, two proteins involved in arginine-dependent activation of CAR1 and CAR2, leading to high levels of the two catabolic enzymes despite a low cytosolic arginine pool. Our data also show that the deletion of the UME6 gene impairs cell growth more strongly than the deletion of the SIN3 or RPD3 gene, especially in the Sigma1278b background.

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

The yeast YBR235w gene encodes a homolog of the mammalian electroneutral Na(+)-(K+)-C1- cotransporter family.

The YBR235w gene of the yeast Saccharomyces cerevisiae was found during sequencing of chromosome II. Here, we show that the 1120 aa protein (Ybr235p) encoded by this gene shares strong sequence similarity with the highly related electroneutral Na(+)-C1- and Na(+)-K(+)-C1- cotransporters of animal cells. We hypothesize that this yeast protein also mediates active uptake of C1- into the cell.

Sequencing and functional analysis of a 32,560 bp segment on the left arm of yeast chromosome II. Identification of 26 open reading frames, including the KIP1 and SEC17 genes.

We report here the DNA sequence of a segment (alpha 1006.13: YBLO5) of chromosome II of Saccharomyces cerevisiae, extending over 32.5 kb. The segment contains 26 open reading frames (ORFs) from YBLO501 to YBLO526. YBL0505 corresponds to the SEC17 gene and YBL0521 to the KIP1 gene. YBL0516 contains an intron, YBL0513 shows homology with the RAT protein phosphatase and YBL0526 contains a zinc-finger motif. Disruption of 14 genes by insertion of a URA3 cassette has been performed and these mutants were analysed for their mating and sporulation ability, and for their growth on different carbon sources. YBL0515 and YBL0526 ORFs seem to be involved in the sporulation process.

The complete sequence of a 9,543 bp segment on the left arm of chromosome III reveals five open reading frames including glucokinase and the protein disulfide isomerase.

We report here the DNA sequence of a 9.5 kb segment of chromosome III. The sequence was determined by subcloning the segment into subfragments generated by appropriate restriction enzymes followed by oligonucleotide-directed sequencing. The segment contains at least five open reading frames, YCL311, YCL312, YCL313, YCL314, YCL315. YCL311 and YCL315 extend in the adjacent fragments, A4H and A6C respectively. YCL312 encodes glucokinase, and YCL313 the protein disulfide isomerase. Disruption of YCL311, 314 and 315 by insertion of a URA3 cassette does not lead to a detectable phenotype, whereas disruption of YCL313 provokes cell lethality.

Determination of the sequence of the yeast YCL313 gene localized on chromosome III. Homology with the protein disulfide isomerase (PDI gene product) of other organisms.

We have determined the nucleotide sequence of the YCL313 gene as part of the YIp5 A1G clone localized on the left arm of chromosome III. This YCL313 gene encodes a protein of 522 amino acids (MW 58.3 kDa) which has large homologies with the human, mouse, chicken, bovine and rat PDI gene products. In these organisms the PDI gene encodes the protein disulfide isomerase (EC 5.3.4.1) also called S-S rearrangase, an enzyme that catalyses the rearrangements of S-S bonds in proteins. This enzyme is probably involved in protein folding within the lumen of the endoplasmic reticulum. These sequence homologies suggest that YCL313 is the yeast equivalent of the PDI gene. Gene disruption of YCL313 leads to a lethal phenotype indicating that this gene is essential for cell survival.

Induction of "General Control" and thermotolerance in cdc mutants of Saccharomyces cerevisiae.

In Saccharomyces cerevisiae starvation for a single amino acid activates the transcription of a set of genes belonging to different amino acid biosynthetic pathways (General Control, GC). We show that mutants affected in GC regulation are also affected in their response to thermal stress. Moreover, growth conditions that are known to induce heat shock proteins induce the GC response. However, unlike heat shock proteins, the transcriptional activator of GC, GCN4, is not induced after a short exposure to heat, and in gcn mutant strains induction of heat resistance is normal.