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J R Broach

Publications and source records attributed to J R Broach.

At least 55 records · Page 3Linked to original sources
PubMed1991

RAS genes in Saccharomyces cerevisiae: signal transduction in search of a pathway.

Ras proteins in budding yeasts initially appeared to regulate initiation of the cell cycle in response to nutrient availability. More recent work, while clarifying the mechanism of Ras-mediated signal transduction, has undermined our notion of the signal Ras transmits. We now suspect that Ras helps to coordinate cellular metabolism and mass accumulation, but what Ras responds to is not clear.

Cell Cycle↗
PubMed1991

Ras-regulated signaling processes in Saccharomyces cerevisiae.

Molecular genetics has proved quite successful in identifying the components of RAS-mediated signal transduction in the yeast Saccharomyces cerevisiae and in defining the nature of their interactions. Recently, the emphasis has shifted to a biochemical approach as the processes of guanine nucleotide exchange, GTPase stimulation activity and posttranslational modification of Ras proteins have all been reproduced in vitro.

Amino Acid Sequence↗
PubMed1991

Mutations in the HML E silencer of Saccharomyces cerevisiae yield metastable inheritance of transcriptional repression.

Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, mediated independently through two cis-acting sites, termed the E and I silencers. We have found that in the absence of the I silencer, deletion of any one of three distinct elements within E yields partial derepression of the mating-type genes resident at HML, whereas deletion of any two yields full derepression. These elements correspond to a binding site for the abundant DNA-binding protein RAP1, an autonomous replicating sequence (ARS), and an as yet undistinguished region. From detailed deletion analysis of the E site we conclude that the ARS element contributes to silencer function in a capacity distinct from its role as an initiator of DNA replication. In addition, we find that strains deleted for any one of these elements comprise two genetically identical but phenotypically distinct types of cells: Those with HML apparently fully derepressed, and those with HML apparently completely repressed. These results reinforce the notion that epigenetic inheritance is an intrinsic characteristic of silencer action.

Chromosomes, Fungal↗
PubMed1991

Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins.

The silent mating-type loci of Saccharomyces cerevisiae, HML and HMR, are flanked by transcriptional silencers that have ARS activity (i.e., they function as replication origins when in plasmids). To test whether these ARS elements are chromosomal origins, we mapped origins near HML (close to the left telomere of chromosome III). Our results indicate that the HML-associated ARS elements either do not function as chromosomal replication origins or do so at a frequency below our detection level, suggesting that replication from a silencer-associated origin in each S phase is not essential for the maintenance of transcriptional repression at HML. Our results also imply that the ability of a DNA fragment to function as an ARS element in a plasmid does not ensure its ability to function as an efficient chromosomal replication origin. Telomere proximity is not responsible for inactivating these ARS elements, because they are not detectably functional as chromosomal origins even in genetically modified strains in which they are far from the telomere.

Chromosome Mapping↗
PubMed1991

The CDC25 protein of Saccharomyces cerevisiae promotes exchange of guanine nucleotides bound to ras.

The product of the CDC25 gene of Saccharomyces cerevisiae, in its capacity as an activator of the RAS/cyclic AMP pathway, is required for initiation of the cell cycle. In this report, we provide an identification of Cdc25p, the product of the CDC25 gene, and evidence that it promotes exchange of guanine nucleotides bound to Ras in vitro. Extracts of strains containing high levels of Cdc25p catalyze both removal of GDP from and the concurrent binding of GTP to Ras. This same activity is also obtained with an immunopurified Cdc25p-beta-galactosidase fusion protein, suggesting that Cdc25p participates directly in the exchange reaction. This biochemical activity is consistent with previous genetic analysis of CDC25 function.

Cell Cycle Proteins↗
PubMed1991

The Saccharomyces cerevisiae YAK1 gene encodes a protein kinase that is induced by arrest early in the cell cycle.

Null mutations in the gene YAK1, which encodes a protein with sequence homology to known protein kinases, suppress the cell cycle arrest phenotype of mutants lacking the cyclic AMP-dependent protein kinase (A kinase). That is, loss of the YAK1 protein specifically compensates for loss of the A kinase. Here, we show that the protein encoded by YAK1 has protein kinase activity. Yak1 kinase activity is low during exponential growth but is induced at least 50-fold by arrest of cells prior to the completion of S phase. Induction is not observed by arrest at stages later in the cell cycle. Depending on the arrest regimen, induction can occur either by an increase in Yak1 protein levels or by an increase in Yak1 specific activity. Finally, an increase in Yak1 protein levels causes growth arrest of cells with attenuated A kinase activity. These results suggest that Yak1 acts in a pathway parallel to that of the A kinase to negatively regulate cell proliferation.

Base Sequence↗
PubMed1990

SRV2, a gene required for RAS activation of adenylate cyclase in yeast.

We have identified a gene, SRV2, mutations of which alleviate stress sensitivity in strains carrying an activated RAS gene. Epistasis analysis suggests that the gene affects accumulation of cAMP in the cell. Direct assays of cAMP accumulation indicate that mutations of the gene diminish the rate of in vivo production of cAMP following stimulation by an activated RAS allele. Null mutations of srv2 result in lethality, which cannot be suppressed by mutational activation of the cAMP-dependent protein kinase. The sequence of the gene indicates that it encodes an adenylate cyclase-associated protein. These results demonstrate that SRV2 protein is required for RAS-activated adenylate cyclase activity, but that it participates in other essential cellular functions as well.

Adenylyl Cyclases↗
PubMed1989

RAS2 protein of Saccharomyces cerevisiae is methyl-esterified at its carboxyl terminus.

Yeast and mammalian RAS gene products are GTP-binding proteins that are posttranslationally localized to the inner surface of the plasma membrane. This localization is accomplished by the addition of a lipid moiety to a conserved cysteine residue close to the carboxyl terminus. In a previous report we showed that the mammalia Ha-ras protein is also modified posttranslationally by methyl esterification. Here we show that the yeast RAS2 protein is posttranslationally modified by methyl esterification at or near the carboxyl terminus. We also present evidence indicating that the methyl ester is linked to the conserved cysteine residue, implying that RAS2 protein is cleaved to expose this cysteine as the carboxyl-terminal residue. This maturation pathway may be shared by a family of proteins that are initially synthesized as soluble proteins and must become membrane-localized to function.

Chromatography, Liquid↗
PubMed1989

A general screen for mutant of Saccharomyces cerevisiae deficient in tRNA biosynthesis.

We have devised a general screen for isolating conditional lethal mutants defective in synthesis of mature tRNA in Saccharomyces cerevisiae. Using this screen, we have identified several new genes in yeast that are required for production of mature tRNA. These genes most likely encode essential functions, since the mutations we isolated are recessive and cause temperature-sensitive growth. One of the mutants, tpd3, is defective in de novo transcription of 4S RNA at the nonpermissive temperature. A second mutant, tpd1, is specifically defective in excision of intervening sequence from a variety of tRNA species. Finally, two other mutants are defective in production of tRNA from a suppressor tRNA locus, as measured by an in vitro suppression assay. The specific lesion in these strains, though, is not known. These data confirm that the screen does, in fact, yield a broad spectrum of mutants defective in tRNA maturation.

Blotting, Northern↗
PubMed1989

The HML mating-type cassette of Saccharomyces cerevisiae is regulated by two separate but functionally equivalent silencers.

Mating-type genes resident in the silent cassette HML at the left arm of chromosome III are repressed by the action of four SIR gene products, most likely mediated through two cis-acting sites located on opposite sides of the locus. We showed that deletion of either of these two cis-acting sites from the chromosome did not yield any detectable derepression of HML, while deletion of both sites yielded full expression of the locus. In addition, each of these sites was capable of exerting repression of heterologous genes inserted in their vicinity. Thus, HML expression is regulated by two independent silencers, each fully competent for maintaining repression. This situation was distinct from the organization of the other silent locus, HMR, at which a single silencer served as the predominant repressor of expression. Examination of identifiable domains and binding sites within the HML silencers suggested that silencing activity can be achieved by a variety of combinations of various functional domains.

Chromosomes, Fungal↗
PubMed1988

Autoregulation of 2 micron circle gene expression provides a model for maintenance of stable plasmid copy levels.

The yeast plasmid 2 micron circle actively maintains high but stable copy levels in the cell, even though the plasmid confers no selective advantage to its host. To address the mechanism by which stable copy control is achieved, we have examined the level of expression of the genes resident on the yeast plasmid 2 micron circle as a function of the presence of proteins encoded by the plasmid. We find that transcription of the site-specific recombinase gene, FLP, is repressed at least 100-fold by the concerted action of the products of two other plasmid genes, REP1 and REP2. In addition, these products repress transcription of the REP1 gene itself. These results can be formulated into a consistent model for plasmid copy control.

DNA Nucleotidyltransferases↗