A DNA fragment conferring resistance to zinc and cadmium ions in the yeast Saccharomyces cerevisiae was isolated from a library of yeast genomic DNA. Its nucleotide sequence revealed the presence of a single open reading frame (ORF; 1326 bp) having the potential to encode a protein of 442 amino acid residues (molecular mass of 48.3 kDa). A frameshift mutation introduced within the ORF abolished resistance to heavy metal ions, indicating the ORF is required for resistance. Therefore, we termed it the ZRC1 (zinc resistance conferring) gene. The deduced amino acid sequence of the gene product predicts a rather hydrophobic protein with six possible membrane-spanning regions. While multiple copies of the ZRC1 gene enable yeast cells to grow in the presence of 40 mM Zn2+, a level at which wild-type cells cannot survive, the disruption of the chromosomal ZRC1 locus, though not a lethal event, makes cells more sensitive to zinc ions than are wild-type cells.
The yeast genome has DNA replication fork blocking sites, that we have named sog sites, in the ribosomal RNA gene (rDNA) cluster. These are located at the 3' end of the 35S rRNA transcription unit and they block replication fork movement in a direction opposite to that of RNA polymerase I. We cloned this replication blocking site into a YEp-type plasmid and analyzed DNA replication intermediates, using two-dimensional (2D) agarose gel electrophoresis. The blocking activity remained even on a plasmid not involved in 35S rRNA transcription and inhibited fork movement in the same polar fashion as on the yeast chromosome. To define the site further, smaller fragments were subcloned into the YEp-type plasmid. A small 109 bp region exhibited sog activity and was located near the enhancer region for 35S rRNA transcription. It overlaps an essential element of the recombinational hot spot HOT1.
In the yeast Saccharomyces cerevisiae, Sic1, an inhibitor of Clb-Cdc28 kinases, must be phosphorylated and degraded in G1 for cells to initiate DNA replication, and Cln-Cdc28 kinase appears to be primarily responsible for phosphorylation of Sic1. The Pho85 kinase is a yeast cyclin-dependent kinase (Cdk), which is not essential for cell growth unless both CLN1 and CLN2 are absent. We demonstrate that Pho85, when complexed with Pcl1, a G1 cyclin homologue, can phosphorylate Sic1 in vitro, and that Sic1 appears to be more stable in pho85Delta cells. Three consensus Cdk phosphorylation sites present in Sic1 are phosphorylated in vivo, and two of them are required for prompt degradation of the inhibitor. Pho85 and other G1 Cdks appear to phosphorylate Sic1 at different sites in vivo. Thus at least two distinct Cdks can participate in phosphorylation of Sic1 and may therefore regulate progression through G1.
GAL)) was first identified as a gene required for full expression of some of the galactose-inducible genes in the yeast Saccharomyces cerevisiae. A null mutation within the GAL)) locus causes defects in mating, growth on nonfermentable carbon sources, and sporulation of gal)) homozygotes. The mating defect was observed only in MATh gall) strains. Northern hybridization analysis revealed that a gal) mutation impaired transcription of a-specific genes (MFal and STE3) but not of an a-specific gene (STE2). Furthermore, this mutation reduced expression of the MATa locus, suggesting that a deficiency in MATal protein is responsible for the reduced expression of a-specific genes. Since general regulatory factor I (GRFI)/repressor/activator site binding protein 1 (RAP1)/ translation upstream factor (TUF) is believed to be an activator of MATa expression, we examined whether PYKI, which is known to be regulated by GRFI/RAP1/TUF, is also affected by the gall) mutation. It was determined that the level ofPYKI message was significantly lowered by the mutation. The requirement for functional GAL)) in transcriptional activation was bypassed when either the upstream activating sequence of galactose-inducible genes or of PYKI was placed very close to the TATA box, suggesting that one of the Gaull protein functions is to mediate the activation signal of Gal4 and GRFI/RAP1/TUF, when the respective binding site is situated at the naturally occurring distance from the TATA box.Investigations over the last few years have generated a large body of information concerning transcriptional regulation of eukaryotic genes in terms of DNA-protein interactions. It appears that for even the simplest promoters, an activator protein binding to a site far upstream of a gene called an upstream activating sequence (UAS) can cause stimulation of transcription ofthe gene. One of the most intriguing problems in the field is the basis of this activating mechanism, which is able to tolerate manipulation of the distance separating the UAS and TATA box regions over a wide range of distances without affecting the efficiency of activation. The activation is now known to be mediated by binding of a common factor, transcription factor IID (TFIID), to its target site, the TATA box. There are a number of indications that interaction between TFIID and the UAS-binding activator protein requires at least one other protein (for review, see ref. 1 and references therein). This paper deals with a candidate for this hypothetical protein in the yeast Saccharomyces cerevisiae.The regulatory circuit for galactose-inducible genes in yeast may be the best studied of all eukaryotic genes of this type. Two regulatory proteins, Gal4 and Gal80, have been identified, while a UAS has been found upstream of all galactose-inducible genes (for review, see ref.2). Gal4 binds to the UAS of galactose-inducible genes (UASG) to stimulate transcription ofthe gene, and Gal80 is proposed to bind Gal4, resulting in a block of its action (3). Previously we suggested involvement of a...
Rho1p is an essential small GTPase that plays a key role in the morphogenesis of Saccharomyces cerevisiae. We show here that the activation of Rho1p is regulated by a cyclin-dependent kinase (CDK). Rho1p is activated at the G1/S transition at the incipient-bud sites by the Cln2p (G1 cyclin) and Cdc28p (CDK) complex, in a process mediated by Tus1p, a guanine nucleotide exchange factor for Rho1p. Tus1p interacts physically with Cln2p/Cdc28p and is phosphorylated in a Cln2p/Cdc28p-dependent manner. CDK phosphorylation consensus sites in Tus1p are required for both Cln2p-dependent activation of Rho1p and polarized organization of the actin cytoskeleton. We propose that Cln2p/Cdc28p-dependent phosphorylation of Tus1p is required for appropriate temporal and spatial activation of Rho1p at the G1/S transition.
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