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Query: UNIPROT:P06889 (Mol)
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The Saccharomyces cerevisiae DNA-binding protein RAP1 is capable of binding in vitro to sequences from a wide variety of genomic loci, including upstream activating sequence elements, the HML and HMR silencer regions, and the poly(G1-3T) tracts of telomeres. Recent biochemical and genetic studies have suggested that RAP1 physically and functionally interacts with the yeast telomere. To further investigate the role of RAP1 at the telomere, we have identified and characterized three intragenic suppressors of a temperature-sensitive allele of RAP1, rap1-5. These telomere deficiency (rap1t) alleles confer several novel phenotypes. First, telomere tract size elongates to up to 4 kb greater than sizes of wild-type or rap1-5 telomeres. Second, telomeres are highly unstable and are subject to rapid, but reversible, deletion of part or all of the increase in telomeric tract length. Telomeric deletion does not require the RAD52 or RAD1 gene product. Third, chromosome loss and nondisjunction rates are elevated 15- to 30-fold above wild-type levels. Sequencing analysis has shown that each rap1t allele contains a nonsense mutation within a discrete region between amino acids 663 and 684. Mobility shift and Western immunoblot analyses indicate that each allele produces a truncated RAP1 protein, lacking the C-terminal 144 to 165 amino acids but capable of efficient DNA binding. These data suggest that RAP1 is a central regulator of both telomere and chromosome stability and define a C-terminal domain that, while dispensable for viability, is required for these telomeric functions.
Mol Cell Biol 1992 Nov
PMID:C-terminal truncation of RAP1 results in the deregulation of telomere size, stability, and function in Saccharomyces cerevisiae. 140 88

RAP1 is an essential sequence-specific DNA-binding protein in Saccharomyces cerevisiae whose binding sites are found in a large number of promoters, where they function as upstream activation sites, and at the silencer elements of the HMR and HML mating-type loci, where they are important for repression. We have examined the involvement of specific regions of the RAP1 protein in both repression and activation of transcription by studying the properties of a series of hybrid proteins containing RAP1 sequences fused to the DNA-binding domain of the yeast protein GAL4 (amino acids 1 to 147). GAL4 DNA-binding domain/RAP1 hybrids containing only the carboxy-terminal third of the RAP1 protein (which lacks the RAP1 DNA-binding domain) function as transcriptional activators of a reporter gene containing upstream GAL4 binding sites. Expression of some hybrids from the strong ADH1 promoter on multicopy plasmids has a dominant negative effect on silencers, leading to either partial or complete derepression of normally silenced genes. The GAL4/RAP1 hybrids have different effects on wild-type and several mutated but functional silencers. Silencers lacking either an autonomously replicating sequence consensus element or the RAP1 binding site are strongly derepressed, whereas the wild-type silencer or a silencer containing a deletion of the binding site for another silencer-binding protein, ABF1, are only weakly affected by hybrid expression. By examining a series of GAL4 DNA-binding domain/RAP1 hybrids, we have mapped the transcriptional activation and derepression functions to specific parts of the RAP1 carboxy terminus.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Cell Biol 1992 Mar
PMID:Dissection of a carboxy-terminal region of the yeast regulatory protein RAP1 with effects on both transcriptional activation and silencing. 154 2

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.
Mol Cell Biol 1991 Oct
PMID:Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins. 192 50

Copies of the mating-type genes are present at three loci on chromosome III of the yeast Saccharomyces cerevisiae. The genes at the MAT locus are transcribed, whereas the identical genes at the silent loci, HML and HMR, are not transcribed. Several genes, including the four SIR genes, and two sites, HMR-E and HMR-I, are required for repression of transcription at the HMR locus. Three elements have been implicated in the function of the HMR-E silencer: a binding site for the RAP1 protein, a binding site for the ABF1 protein, and an 11-bp consensus sequence common to nearly all autonomously replicating sequence (ARS) elements (putative origins of DNA replication). RAP1 and ABF1 binding sites of different sequence than those found at HMR-E were joined with an 11-bp ARS consensus sequence to form a synthetic silencer. The synthetic silencer was able to repress transcription of the HMRa1 gene, confirming that binding sites for RAP1 and ABF1 and the 11-bp ARS consensus sequence were the functional components of the silencer in vivo. Mutations in the ABF1 binding site or in the ARS consensus sequence of the synthetic silencer caused nearly complete derepression of transcription at HMR. The ARS consensus sequence mutation also eliminated the ARS activity of the synthetic silencer. These data suggested that replication initiation at the HMR-E silencer was required for establishment of the repressed state at the HMR locus.
Mol Cell Biol 1991 Nov
PMID:A synthetic silencer mediates SIR-dependent functions in Saccharomyces cerevisiae. 192 68

We have isolated a class of mutants, aar2, showing the alpha mating type due to a defect in a1-alpha 2 repression but with alpha 2 repression activity from a nonmater strain of Saccharomyces cerevisiae expressing both a and alpha mating-type information in duplicate. Cells of the aar2 mutant and the aar2 disruptant also show a growth defect. A DNA fragment complementing the aar2 mutation contains an open reading frame consisting of 355 amino acid codons. Northern hybridization showed that cells of the aar2 mutant and disruptant contained alpha 1 and alpha 2 transcripts of the MAT alpha gene (or HML alpha in sir3 cells), but their a1 transcript of MATa (or HMRa in sir3 cells) migrated more slowly than that of the wild-type cells on gel electrophoresis and gave a diffused band. Primer extension analysis showed that the aar2 mutant and disruptant have a defect in splicing two short introns of the a1 pre-mRNA but not in splicing pre-mRNA of ACT1. The alpha mating type, but not the slow-growing phenotype, of the aar2 mutant was suppressed by introduction of an intronless MATa1 DNA. Thus, the AAR2 gene is involved in splicing pre-mRNA of the a1 cistron and other genes that are important for cell growth. The AAR2 locus was mapped on chromosome II beside the SSA3 locus, with a 276-bp space, but was not allelic to either PRP5 or PRP6, which are both located on chromosome II and function in splicing pre-mRNA of ACT1.
Mol Cell Biol 1991 Nov
PMID:AAR2, a gene for splicing pre-mRNA of the MATa1 cistron in cell type control of Saccharomyces cerevisiae. 192 71

The mating-type genes at MAT in Saccharomyces cerevisiae are expressed, whereas the same genes located at HML and HMR are transcriptionally repressed. The DNA element responsible for repression at HMR has been termed a silencer and contains an autonomous replication sequence, a binding site for GRFI/RAPI, and a binding site for ABFI. A double-mutant HMR-E silencer that contains single nucleotide substitutions in both the GRFI/RAPI- and ABFI-binding sites no longer binds either factor in vitro, nor represses transcription at HMR in vivo. In MAT alpha cells, this derepression of a information results in a nonmating phenotype. Second-site suppressor mutations were isolated that restored the alpha mating phenotype to MAT alpha cells containing the double-mutant silencer. One of these suppressors, designated sas1-1, conferred a temperature-sensitive lethal phenotype to the cell. SAS1 was found to be identical to CDC7, a gene which encodes a protein kinase required for the initiation of DNA replication. This new allele of CDC7 was designated cdc7-90. cdc7-90 restored the alpha mating phenotype by restoring silencing. The original allele of CDC7, isolated on the basis of the cell cycle phenotype it confers, also restored silencing, and overexpression of CDC7 interfered with silencing. cdc7-90 did not restore detectable binding of GRFI/RAPI or ABFI to the double-mutant silencer in vitro. These results indicate that a reduced level of CDC7 function restores silencing to a locus defective in binding two factors normally required for silencing.
Mol Cell Biol 1991 Feb
PMID:A role for CDC7 in repression of transcription at the silent mating-type locus HMR in Saccharomyces cerevisiae. 199 Feb 68

The N-terminal serine and four conserved lysine residues near the N-terminus of yeast histone H4 are acetylated. We found that a mutation that changed the fourth lysine to alanine resulted in specific derepression of the silent mating type locus HML, while mutations that altered the N-terminal serine or the first three lysines had only minor phenotypic effects. Our results support an active role for histone H4 in the silencing of gene expression at this locus.
Mol Cell Biol 1990 Sep
PMID:Point mutations in the yeast histone H4 gene prevent silencing of the silent mating type locus HML. 211 3

We have identified a novel integrated form of the yeast retrotransposon Ty consisting of multiple elements joined into large arrays. These arrays were first identified among Ty-induced alpha-pheromone-resistant mutants of MATa cells of Saccharomyces cerevisiae which contain Ty insertions at HML alpha that result in the expression of that normally silent cassette. These insertions are multimeric arrays of both the induced genetically marked Ty element and unmarked Ty elements. Structural analysis of the mutations indicated that the arrays include tandem direct repeats of Ty elements separated by only a single long terminal repeat. The Ty-HML junction fragments of one mutant were cloned and shown to contain a 5-base-pair duplication of the target sequence that is characteristic of a Ty transpositional insertion. In addition, the arrays include rearranged Ty elements that do not have normal long terminal repeat junctions. We have also identified multimeric Ty insertions at other chromosomal sites and as insertions that allow expression of a promoterless his3 gene on a plasmid. The results suggest that Ty transposition includes an intermediate that can undergo recombination to produce multimers.
Mol Cell Biol 1990 Jun
PMID:Multimeric arrays of the yeast retrotransposon Ty. 216 May 87

After UV irradiation, the transcriptionally active MAT alpha locus in Saccharomyces cerevisiae is preferentially repaired compared with the inactive HML alpha locus. The effect of rad mutations from three different epistasis groups on differential repair was investigated. Three mutants, rad9, rad16, and rad24, were impaired in the removal of UV dimers from the inactive HML alpha locus, whereas they had generally normal repair of the active MAT alpha locus. Since RAD9 is necessary for G2 arrest after UV irradiation, we propose that the G2 stage plays a role in making the dimers accessible for repair, at least in the repressed HML alpha locus.
Mol Cell Biol 1990 Sep
PMID:Differential repair of UV damage in rad mutants of Saccharomyces cerevisiae: a possible function of G2 arrest upon UV irradiation. 220 99

The silent mating-type genes (HML and HMR) of Saccharomyces cerevisiae are kept under negative transcriptional control by the trans-acting products of the four MAR/SIR loci. MAR/SIR gene mutations result in the simultaneous derepression of HML and HMR gene expression. The sum1-1 mutation was previously identified as an extragenic suppressor of mutations in MAR1 (SIR2) and MAR2 (SIR3). As assayed genetically, sum1-1 is capable of restoring repression of silent mating-type information in cells containing mar1 or mar2 null mutations. We show here that the mating-type phenotype associated with sum1-1 results from a dramatic reduction in the steady-state level of HML and HMR gene transcripts. At the same time, the sum1-1 mutation has no significant effect on the level of each of the four MAR/SIR mRNAs.
Mol Cell Biol 1990 Jan
PMID:The sum1-1 mutation affects silent mating-type gene transcription in Saccharomyces cerevisiae. 240 45


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