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J R Broach

Publications and source records attributed to J R Broach.

At least 37 records · Page 2Linked to original sources
PubMed1997

Role of alpha2 protein in donor locus selection during mating type interconversion.

The homeodomain protein alpha2p plays a role both in transcriptional repression in the process of cell type determination and in donor selection during mating interconversion. We have explored the mechanism of alpha2p-directed donor selection by examining the effects on donor preference of mutants deficient in alpha2p-mediated transcriptional repression. As a transcriptional regulator, alpha2p interacts with Mcm1p, Tup1p, and Ssn6p to repress a-specific genes and with a1p, Tup1p, and Ssn6p to repress haploid-specific genes. We have found that mutant alleles of MATalpha2 that specifically diminish the interaction of alpha2p with Mcm1p or Tup1p behave as null alleles with regard to donor preference, while mutations of MATalpha2 that specifically diminish interaction of alpha2p with a1p behave as wild-type MATalpha2 in this capacity. Tup1p plays an essential role in alpha2p-mediated transcriptional repression, while Ssn6p has only a modest effect in repression. In a similar vein, we find that TUP1, but not SSN6, is required for proper donor selection. These results suggest that, in addition to regulating a-specific gene expression to establish the mating type of the cell, alpha2p-Mcm1p-Tup1p complex may indirectly regulate donor preference through transcriptional control of an a-specific gene. Alternatively, this complex may play a direct role in establishing donor preference via its DNA binding and chromatin organization capacity.

Alleles↗
PubMed1996

High-throughput screening for drug discovery.

Recent progress in elucidating the mechanisms underlying human disease has dramatically increased the number of protein targets available for potential drug treatment. Concurrently, new approaches have increased the number of compounds that can be tested for activity against these targets. Together, these trends have stimulated the adoption of high-throughput screening as a primary tool for early-stage drug discovery.

Drug Evaluation, Preclinical↗
PubMed1996

Silencers are required for inheritance of the repressed state in yeast.

Transcriptional silencers in the yeast Saccharomyces induce position-specific, sequence-independent repression by promoting formation of a heterochromatin-like structure across sequences adjacent to them. We have examined the role of silencers in maintenance and inheritance of repression at the silent mating-type cassettes in yeast by monitoring the expression state of one of these cassettes following in vivo deletion of the adjacent silencer. Our experiments indicate that although silencer sequences are dispensable for the maintenance of repression in the absence of cell-cycle progression, silencers are required for the stable inheritance of a repressed state. That is, silenced loci from which the silencer is deleted most often become derepressed within one generation of losing the silencer. Thus, the heritability of a repressed state is not intrinsic to a silenced locus or to the chromatin encompassing it; rather, heritability of repression appears to be a property of the silencer itself.

DNA Nucleotidyltransferases↗
PubMed1996

A ceramide-activated protein phosphatase mediates ceramide-induced G1 arrest of Saccharomyces cerevisiae.

Certain mammalian growth modulators, such as tumor necrosis factor alpha, interleukin-1beta, and gamma-interferon, induce an antiproliferative response-terminal differentiation, apoptosis, or cell cycle arrest-through a novel signal transduction pathway mediated by the lipid ceramide as a second messenger. Both a ceramide-activated protein phosphatase and a ceramide-activated protein kinase have been implicated in transmitting the signals elicited by ceramide. We have determined that ceramide addition to the yeast Saccharomyces causes a similar antiproliferative response, resulting in arrest of cells in the G1 phase of the cell cycle. We have also determined that yeast cells contain a ceramide-activated protein phosphatase composed of regulatory subunits encoded by TPD3 and CDC55 and a catalytic subunit encoded by SIT4. Because mutation of any one of these three genes renders strains resistant to ceramide inhibition, we conclude that the G1 effects of ceramide are mediated at least in part by the yeast ceramide-activated protein phosphatase. These results highlight the conservation of signaling systems in yeast and mammalian cells and provide a novel approach to dissecting this ubiquitous signal transduction pathway.

Apoptosis↗
PubMed1996

Efficient transcriptional silencing in Saccharomyces cerevisiae requires a heterochromatin histone acetylation pattern.

Heterochromatin in metazoans induces transcriptional silencing, as exemplified by position effect variegation in Drosophila melanogaster and X-chromosome inactivation in mammals. Heterochromatic DNA is packaged in nucleosomes that are distinct in their acetylation pattern from those present in euchromatin, although the role these differences play in the structure of heterochromatin or in the effects of heterochromatin on transcriptional activity is unclear. Here we report that, as observed in the facultative heterochromatin of the inactive X chromosome in female mammalian cells, histones H3 and H4 in chromatin spanning the transcriptionally silenced mating-type cassettes of the yeast Saccharomyces cerevisiae are hypoacetylated relative to histones H3 and H4 of transcriptionally active regions of the genome. By immunoprecipitation of chromatin fragments with antibodies specific for H4 acetylated at particular lysine residues, we found that only three of the four lysine residues in the amino-terminal domain of histone H4 spanning the silent cassettes are hypoacetylated. Lysine 12 shows significant acetylation levels. This is identical to the pattern of histone H4 acetylation observed in centric heterochromatin of D. melanogaster. These two observations provide additional evidence that the silent cassettes are encompassed in the yeast equivalent of metazoan heterochromatin. Further, mutational analysis of the amino-terminal domain of histone H4 in S. cerevisiae demonstrated that this observed pattern of histone H4 acetylation is required for transcriptional silencing. This result, in conjunction with prior mutational analyses of yeast histones H3 and H4, indicates that the particular pattern of nucleosome acetylation found in heterochromatin is required for its effects on transcription and is not simply a side effect of heterochromatin formation.

Acetylation↗
PubMed1996

Yeast alpha mating factor structure-activity relationship derived from genetically selected peptide agonists and antagonists of Ste2p.

alpha-Factor, a 13-amino-acid pheromone secreted by haploid alpha cells of Saccharomyces cerevisiae, binds to Ste2p, a seven-transmembrane, G-protein-coupled receptor present on haploid alpha cells, to activate a signal transduction pathway required for conjugation and mating. To determine the structural requirements for alpha-factor activity, we developed a genetic screen to identify from random and semirandom libraries novel peptides that function as agonists or antagonists of Ste2p. The selection scheme was based on autocrine strains constructed to secrete random peptides and respond by growth to those that were either agonists or antagonists of Ste2p. Analysis of a number of peptides obtained by this selection procedure indicates that Trp1, Trp3, Pro8, and Gly9 are important for agonist activity specifically. His2, Leu4, Leu6, Pro10, a hydrophobic residue 12, and an aromatic residue 13 are important for both agonist and antagonist activity. Our results also show that activation of Ste2p can be achieved with novel, unanticipated combinations of amino acids. Finally, the results suggest the utility of this selection scheme for identifying novel ligands for mammalian G-protein-coupled receptors heterologously expressed in S. cerevisiae.

Amino Acid Sequence↗
PubMed1995

Ras membrane targeting is essential for glucose signaling but not for viability in yeast.

Ras proteins are small GTP binding proteins that serve as critical relays in a variety of signal transduction pathways in eukaryotic cells. Like most metazoan Ras proteins, yeast Ras is post-translationally modified by addition of a farnesyl and a palmitoyl moiety, and these modifications are required for targeting the protein to the cytoplasmic face of the plasma membrane and for biological activity of the protein. We have constructed mutants of the yeast (Saccharomyces cerevisiae) Ras that are farnesylated in vivo but are not palmitoylated. These mutant proteins are not localized to the plasma membrane but function in the cell as well as the wild-type protein. Such mutants are viable but fail to induce a transient increase in intracellular cAMP concentration in response to glucose addition, although this deficiency does not yield a marked growth phenotype. These results are consistent with the hypothesis that the essential role of the farnesyl moiety on yeast Ras is to enhance productive interaction between Ras and its essential downstream target, adenylyl cyclase, rather than to localize Ras to the plasma membrane.

Alleles↗
PubMed1995

Mutations affecting donor preference during mating type interconversion in Saccharomyces cerevisiae.

Homothallic strains of Saccharomyces cerevisiae can convert mating type from a to alpha or alpha to a as often as every generation, by replacing genetic information specifying one mating type at the expressor locus, MAT, with information specifying the opposite mating type. The cryptic mating type information that is copied and inserted at MAT is contained in either of two loci, HML or HMR. The particular locus selected as donor during mating type interconversion is regulated by the allele expressed at MAT. MATa cells usually select HML, and MAT alpha cells usually select HMR, a process referred to as donor preference. To identify factors required for donor preference, we isolated and characterized a number of mutants that frequently selected the nonpreferred donor locus during mating type interconversion. Many of these mutants were found to harbor chromosome rearrangements or mutations at MAT or HML that interfered with the switching process. However, one mutant carried a recessive allele of CHL1, a gene previously shown to be required for efficient chromosome segregation during mitosis. Homothallic strains of yeast containing a null allele of CHL1 exhibited almost random selection of the donor locus in a MATa background but were normal in their ability to select HMR in a MAT alpha background. Our results indicate that Chl1p participates in the process of donor selection and are consistent with a model in which Chl1p helps establish an intrinsic bias in donor preference.

Alleles↗
PubMed1995

Nutrient availability and the RAS/cyclic AMP pathway both induce expression of ribosomal protein genes in Saccharomyces cerevisiae but by different mechanisms.

By differential hybridization, we identified a number of genes in Saccharomyces cerevisiae that are activated by addition of cyclic AMP (cAMP) to cAMP-depleted cells. A majority, but not all, of these genes encode ribosomal proteins. While expression of these genes is also induced by addition of the appropriate nutrient to cells starved for a nitrogen source or for a sulfur source, the pathway for nutrient activation of ribosomal protein gene transcription is distinct from that of cAMP activation: (i) cAMP-mediated transcriptional activation was blocked by prior addition of an inhibitor of protein synthesis whereas nutrient-mediated activation was not, and (ii) cAMP-mediated induction of expression occurred through transcriptional activation whereas nutrient-mediated induction was predominantly a posttranscriptional response. Transcriptional activation of the ribosomal protein gene RPL16A by cAMP is mediated through a upstream activation sequence element consisting of a pair of RAP1 binding sites and sequences between them, suggesting that RAP1 participates in the cAMP activation process. Since RAP1 protein decays during starvation for cAMP, regulation of ribosomal protein genes under these conditions may directly relate to RAP1 protein availability. These results define additional critical targets of the cAMP-dependent protein kinase, suggest a mechanism to couple ribosome production to the metabolic activity of the cell, and emphasize that nutrient regulation is independent of the RAS/cAMP pathway.

Cyclic AMP↗
PubMed1995

Yeast silencers can act as orientation-dependent gene inactivation centers that respond to environmental signals.

The mating-type loci located at the ends of chromosome III in Saccharomyces cerevisiae are transcriptionally repressed by a region-specific but sequence-nonspecific silencing apparatus, mediated by cis-acting silencer sequences. Previous deletion analyses have defined the locations and organizations of the silencers in their normal context and have shown that they are composed of various combinations of replication origins and binding sites for specific DNA-binding proteins. We have evaluated what organization of silencer sequences is sufficient for establishing silencing at a novel location, by inserting individual silencers next to the MAT locus and then assessing expression of MAT. The results of this analysis indicate that efficient silencing can be achieved by inserting either a single copy of the E silencer from HMR or multiple, tandem copies of either the E or I silencer from HML. These results indicate that while all silencers are functionally equivalent, they have different efficiencies; HMR E is more active than HML E, which itself is more active than HML I. Both HMR E and HML E exhibit orientation-dependent silencing, and the particular organization of binding elements within the silencer domain is critical for function. In some situations, silencing of MAT is conditional: complete silencing is obtained when cells are grown on glucose, and complete derepression occurs when cells are shifted to a nonfermentable carbon source, a process mediated in part by the RAS/cyclic AMP signaling pathway. Finally, the E silencer from HMR is able to reestablish repression immediately upon a shift back to glucose, while the silencers from HML exhibit a long lag in reestablishing repression, thus indicating distinctions between the two silencers in their reestablishment capacities. These results demonstrate that silencers can serve as nonspecific gene inactivation centers and that the attendant silencing can be rendered responsive to potential developmental cues.

Base Sequence↗
PubMed1994

Cdc25p, the guanine nucleotide exchange factor for the Ras proteins of Saccharomyces cerevisiae, promotes exchange by stabilizing Ras in a nucleotide-free state.

In Saccharomyces cerevisiae, adenylate cyclase activity is controlled by Ras1p and Ras2p. Activation of the Ras proteins is in turn controlled by the GTPase-activating proteins (GAPs), Ira1p and Ira2p, and the guanine nucleotide exchange factor (GNEF), Cdc25p. We have characterized Cdc25p enzymologically in order to gain information about the mechanism of Cdc25p-mediated guanine nucleotide exchange and to appreciate how the activity of a GNEF is integrated as a part of a basic molecular switch module consisting of Ras, GNEF, and GAP. Using Ras2p and a catalytic fragment of Cdc25p, both expressed in and purified from Escherichia coli, we determined that Cdc25p has a Km for Ras2p-GDP of 160 nM and a maximal rate of 0.20 s-1. The Km of Cdc25p for Ras2p complexed to GTP is 3-fold greater than that for Ras2p complexed to GDP. The Km of free GDP is about 2-fold higher than the Km of free GTP. This suggests that Cdc25p activates Ras2p primarily by equilibrating Ras2p with the pool of free guanine nucleotides in the cell rather than by driving Ras2p inexorably into the activated state. This renders Ras activation potentially subject to energy charge fluctuations in the cell. The free guanine nucleotide affects kcat, indicating that the rate-limiting step is nucleotide association. Finally, we demonstrated that dominant negative alleles of Ras2p are potent competitive inhibitors of Cdc25p. These data, in conjunction with the kinetic data, are consistent with the hypothesis that Cdc25p catalyzes guanine nucleotide exchange by stabilizing a nucleotide-free intermediate of Ras.

Cell Cycle Proteins↗
PubMed1994

TPD1 of Saccharomyces cerevisiae encodes a protein phosphatase 2C-like activity implicated in tRNA splicing and cell separation.

The Saccharomyces cerevisiae TPD1 gene has been implicated in tRNA splicing because a tpd1-1 mutant strain accumulates unspliced precursor tRNAs at high temperatures (W. H. van Zyl, N. Wills, and J. R. Broach, Genetics 123:55-68, 1989). The wild-type TPD1 gene was cloned by complementation of the tpd1-1 mutation and shown to encode a protein with substantial homology to protein phosphatase 2C (PP2C) of higher eukaryotes. Expression of Tpd1p in Escherichia coli results in PP2C-like activity. Strains deleted for the TPD1 gene exhibit multiple phenotypes: temperature-sensitive growth, accumulation of unspliced precursor tRNAs, sporulation defects, and failure of cell separation during mitotic growth. On the basis of the presence of these observable phenotypes and the fact that Tpd1p accounts for a small percentage of the observed PP2C activity, we argue that Tpd1p is a unique member of the PP2C family.

Amino Acid Sequence↗
PubMed1993

Transcriptional silencing in yeast is associated with reduced nucleosome acetylation.

Two classes of sequences in the yeast Saccharomyces cerevisiae are subject to transcriptional silencing: the silent mating-type cassettes and telomeres. In this report we demonstrate that the silencing of these regions is strictly associated with acetylation of the epsilon-amino groups of lysines in the amino-terminal domains of three of the four core histones. Both the silent mating-type cassettes and the Y domains of telomeres are packaged in nucleosomes in vivo that are hypoacetylated relative to those packaging active genes. This difference in acetylation is eliminated by genetic inactivation of silencing: The silent cassettes from sir2, sir3, or sir4 cells show the same level of acetylation as other active genes. The correspondence of silencing and hypoacetylation of the mating-type cassettes is observed even for an allele lacking a promoter, indicating that silencing per se, rather than the absence of transcription, is correlated with hypoacetylation. Finally, overexpression of Sir2p, a protein required for transcriptional silencing in yeast, yields substantial histone deacetylation in vivo. These studies fortify the hypothesis that silencing in yeast results from heterochromatin formation and argue that the silencing proteins participate in this formation.

Acetylation↗
PubMed1992

The 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae are regulated differentially by phosphorylation via cAMP-dependent protein kinase.

Evidence is presented that demonstrated that the 45- and 104-kDa forms of phosphatidate phosphatase from Saccharomyces cerevisiae (Morlock, K. R., McLaughlin, J. J., Lin, Y.-P., and Carman, G. M. (1991) J. Biol. Chem. 266, 3586-3593) were regulated differentially by phosphorylation. Purified 45-kDa phosphatidate phosphatase was phosphorylated by cAMP-dependent protein kinase whereas purified 104-kDa phosphatidate phosphatase was not phosphorylated. cAMP-dependent protein kinase catalyzed the phosphorylation of pure 45-kDa phosphatidate phosphatase at a serine residue which resulted in a stimulation (2.4-fold) of phosphatidate phosphatase activity. Alkaline phosphatase catalyzed the dephosphorylation of pure 45-kDa phosphatidate phosphatase which resulted in an inhibition (1.3-fold) of phosphatidate phosphatase activity. Results of studies using mutants (bcy1 and cyr1) defective in cAMP-dependent protein kinase activity corroborated the results of the phosphorylation studies using pure preparations of phosphatidate phosphatase. The 45-kDa phosphatidate phosphatase phosphorylated in vitro and in vivo had phosphopeptides in common. The activation of the GAL10-RAS2val19 allele in mutant cells resulted in an increase in the synthesis of diacylglycerols and triacylglycerols. These results were consistent with the phosphorylation and activation of 45-kDa phosphatidate phosphatase by cAMP-dependent protein kinase in vivo.

Alleles↗
PubMed1992

Direct simulation of yeast 2-microns circle plasmid amplification.

The 2-microns circle is a plasmid found in most strains of the yeast Saccharomyces cerevisiae at approximately 60-100 copies per cell. The plasmid possesses the novel capacity for replicative amplification induced by site-specific recombination. To address the question of whether the recombination model is adequate to account for observed rates of 2-microns circle amplification, we developed a direct computational simulation of the amplification system. Results of this simulation show that theoretically at least six copies per plasmid can be produced in each generation, and that previously unanticipated replication intermediates contribute largely to this degree of amplification.

Computer Simulation↗
PubMed1992

Donor locus selection during Saccharomyces cerevisiae mating type interconversion responds to distant regulatory signals.

Mating type interconversion in homothallic strains of the yeast Saccharomyces cerevisiae results from directed transposition of a mating type allele from one of the two silent donor loci, HML and HMR, to the expressing locus, MAT. Cell type regulates the selection of the particular donor locus to be utilized during mating type interconversion: MATa cells preferentially select HML alpha and MAT alpha cells preferentially select HMRa. Such preferential selection indicates that the cell is able to distinguish between HML and HMR during mating type interconversion. Accordingly, we designed experiments to identify those features perceived by the cell to discriminate HML and HMR. We demonstrate that discrimination does not derive from the different structures of the HML and HMR loci, from the unique sequences flanking each donor locus nor from any of the DNA distal to the HM loci on chromosome III. Moreover, we find that the sequences flanking the MAT locus do not function in the preferential selection of one donor locus over the other. We propose that the positions of the donor loci on the left and right arms of chromosome III is the characteristic utilized by the cell to distinguish HML and HMR. This positional information is not generated by either CEN3 or the MAT locus, but probably derives from differences in the chromatin structure, chromosome folding or intranuclear localization of the two ends of chromosome III.

Base Sequence↗
PubMed1992

Inactivation of the protein phosphatase 2A regulatory subunit A results in morphological and transcriptional defects in Saccharomyces cerevisiae.

We have determined that TPD3, a gene previously identified in a screen for mutants defective in tRNA biosynthesis, most likely encodes the A regulatory subunit of the major protein phosphatase 2A species in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence of the product of TPD3 is highly homologous to the sequence of the mammalian A subunit of protein phosphatase 2A. In addition, antibodies raised against Tpd3p specifically precipitate a significant fraction of the protein phosphatase 2A activity in the cell, and extracts of tpd3 strains yield a different chromatographic profile of protein phosphatase 2A than do extracts of isogenic TPD3 strains. tpd3 deletion strains generally grow poorly and have at least two distinct phenotypes. At reduced temperatures, tpd3 strains appear to be defective in cytokinesis, since most cells become multibudded and multinucleate following a shift to 13 degrees C. This is similar to the phenotype obtained by overexpression of the protein phosphatase 2A catalytic subunit or by loss of CDC55, a gene that encodes a protein with homology to a second regulatory subunit of protein phosphatase 2A. At elevated temperatures, tpd3 strains are defective in transcription by RNA polymerase III. Consistent with this in vivo phenotype, extracts of tpd3 strains fail to support in vitro transcription of tRNA genes, a defect that can be reversed by addition of either purified RNA polymerase III or TFIIIB. These results reinforce the notion that protein phosphatase 2A affects a variety of biological processes in the cell and provide an initial identification of critical substrates for this phosphatase.

Amino Acid Sequence↗