Biomedical subjects

A E Ehrenhofer-Murray

Publications and source records attributed to A E Ehrenhofer-Murray.

8 recordsLinked to original sources

PubMed2001

The silencing complex SAS-I links histone acetylation to the assembly of repressed chromatin by CAF-I and Asf1 in Saccharomyces cerevisiae.

The acetylation state of histones plays a central role in determining gene expression in chromatin. The reestablishment of the acetylation state of nucleosomes after DNA replication and chromatin assembly requires both deacetylation and acetylation of specific lysine residues on newly incorporated histones. In this study, the MYST family acetyltransferase Sas2 was found to interact with Cac1, the largest subunit of Saccharomyces cerevisiae chromatin assembly factor-I (CAF-I), and with the nucleosome assembly factor Asf1. The deletions of CAC1 (cac1Delta), ASF1 (asf1Delta), and SAS2 (sas2Delta) had similar effects on gene silencing and were partially overlapping. Furthermore, Sas2 was found in a nuclear protein complex that included Sas4 and Sas5, a homolog of TAF(II)30. This complex, termed SAS-I, was also found to contribute to rDNA silencing. Furthermore, the observation that a mutation of H4 lysine 16 to arginine displayed the identical silencing phenotypes as sas2Delta suggested that it was the in vivo target of Sas2 acetylation. In summary, our data present a novel model for the reestablishment of acetylation patterns after DNA replication, by which SAS-I is recruited to freshly replicated DNA by its association with chromatin assembly complexes to acetylate lysine 16 of H4.

Acetyl Coenzyme A↗

PubMed1999

A role for the replication proteins PCNA, RF-C, polymerase epsilon and Cdc45 in transcriptional silencing in Saccharomyces cerevisiae.

Transcriptional silencing in the budding yeast Saccharomyces cerevisiae may be linked to DNA replication and cell cycle progression. In this study, we have surveyed the effect of 41 mutations in genes with a role in replication, the cell cycle, and DNA repair on silencing at HMR. Mutations in PCNA (POL30), RF-C (CDC44), polymerase epsilon (POL2, DPB2, DPB11), and CDC45 were found to restore silencing at a mutant HMR silencer allele that was still a chromosomal origin of replication. Replication timing experiments indicated that the mutant HMR locus was replicated late in S-phase, at the same time as wild-type HMR. Restoration of silencing by PCNA and CDC45 mutations required the origin recognition complex binding site of the HMR-E silencer. Several models for the precise role of these replication proteins in silencing are discussed.

Carrier Proteins↗

PubMed1998

The Sge1 protein of Saccharomyces cerevisiae is a membrane-associated multidrug transporter.

In this study, we report the further characterization of the Saccharomyces cerevisiae crystal violet-resistance protein Sge1. Sge1 is a highly hydrophobic 59 kDa protein with 14 predicted membrane-spanning domains. It shares homologies with several drug-resistance proteins and sugar transporters of the major facilitator superfamily. Here, we have demonstrated that Sge1 is not only a crystal violet-resistance protein, but that it also confers resistance to ethidium bromide and methylmethane sulfonate. Disruption of SGE1 leads to increased sensitivity towards all three compounds, thus designating Sge1 as a multiple drug-resistance protein. Subcellular fractionation as well as immunolocalization on whole yeast cells demonstrated that Sge1 was tightly associated with the yeast plasma membrane. Furthermore, Sge1 was highly enriched in preparations of yeast plasma membranes. In analogy to other multidrug-resistance proteins, we suggest that Sge1 functions as a drug export permease.

Amino Acid Sequence↗

PubMed1997

The origin recognition complex, SIR1, and the S phase requirement for silencing.

Silencing of transcription in Saccharomyces cerevisiae has several links to DNA replication, including a role for the origin recognition complex (ORC), the DNA replication initiator, in both processes. In addition, the establishment of silencing at the HML and HMR loci requires cells to pass through the S phase of the cell cycle. Passage through S phase was required for silencing of HMR even under conditions in which ORC itself was no longer required. The requirement for ORC in silencing of HMR could be bypassed by tethering the Sir1 protein to the HMR-E silencer. However, ORC had a Sir1-independent role in transcriptional silencing at telomeres. Thus, the role of ORC in silencing was separable from its role in initiation, and the role of S phase in silencing was independent of replication initiation at the silencers.

Aldose-Ketose Isomerases↗

PubMed1997

The role of Sas2, an acetyltransferase homologue of Saccharomyces cerevisiae, in silencing and ORC function.

Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers. Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMRa-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR. Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMRa-e**. Silencing conferred by the removal of SAS2 (sas2 delta) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC). Restoration of silencing by sas2 delta required ORC2 and ORC5, but not SIR1 or RAP1. Furthermore, sas2 delta suppressed the temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1. Moreover, sas2 delta had opposing effects on silencing of HML and HMR. The putative Sas2 protein bears similarities to known protein acetyltransferases. Several models for the role of Sas2 in silencing are discussed.

Alleles↗

PubMed1995

Separation of origin recognition complex functions by cross-species complementation.

Transcriptional silencing at the HMRa locus of Saccharomyces cerevisiae requires the function of the origin recognition complex (ORC), the replication initiator of yeast. Expression of a Drosophila melanogaster Orc2 complementary DNA in the yeast orc2-1 strain, which is defective for replication and silencing, complemented the silencing defect but not the replication defect; this result indicated that the replication and silencing functions of ORC were separable. The orc2-1 mutation mapped to the region of greatest homology between the Drosophila and yeast proteins. The silent state mediated by DmOrc2 was epigenetic; it was propagated during mitotic divisions in a relatively stable way, whereas the nonsilent state was metastable. In contrast, the silent state was erased during meiosis.

Animals↗

PubMed1994

The Saccharomyces cerevisiae SGE1 gene product: a novel drug-resistance protein within the major facilitator superfamily.

Several pleiotropic drug sensitivities have been described in yeast. Some involve the loss of putative drug efflux pumps analogous to mammalian P-glycoproteins, others are caused by defects in sterol synthesis resulting in higher plasma membrane permeability. We have constructed a Saccharomyces cerevisiae strain that exhibits a strong crystal violet-sensitive phenotype. By selecting cells of the supersensitive strain for normal sensitivity after transformation with a wild-type yeast genomic library, a complementing 10-kb DNA fragment was isolated, a 3.4-kb subfragment of which was sufficient for complementation. DNA sequence analysis revealed that the complementing fragment comprised the recently sequenced SGE1 gene, a partial multicopy suppressor of gal11 mutations. The supersensitive strain was found to be a sge1 null mutant. Overexpression of SGE1 on a high-copy-number plasmid increased the resistance of the supersensitive strain. Disruption of SGE1 in a wild-type strain increased the sensitivity of the strain. These features of the SGE1 phenotype, as well as sequence homologies of SGE1 at the amino acid level, confirm that the Sge1 protein is a member of the drug-resistance protein family within the major facilitator superfamily (MFS).

Amino Acid Sequence↗

PubMed1994

Characterization of the trp5-27 allele used to monitor drug-induced mitotic gene conversion in the Saccharomyces cerevisiae tester strain D7.

Mitotic gene conversions, among other recombinagenic events, can play an important role in the multistep process of carcinogenesis. The ability of chemicals to induce such gene conversions can easily be monitored in the Saccharomyces cerevisiae tester strain YHE2, a derivative of strain D7. For the detection of drug-induced gene conversions, two mutations in the TRP5 locus are used, trp5-12 and trp5-27. Here we report on the characterization of the stable allele trp5-27. Our analysis revealed two relevant mutations in trp5-27: (a) a transition C to T at position 121 after ATG that results in an amber stop codon and abolishes gene expression and (b) a transversion A to T at position 1555 that creates an ochre stop codon. Simultaneous amber and ochre suppression with the suppressors SUP3 and SUP11, respectively, was capable of relieving the tryptophan-requiring phenotype of strains carrying the trp5-27 allele. These findings have implications on the length of gene conversion tracts in conversion events between trp5-12 and trp5-27: conversion tracts can cover several kilobases, if the site of the mutation in trp5-12 lies outside of the positions mutated in trp5-27. Conversely, the maximal length is limited to 1435 bp, if the mutation in trp5-12 is located between the positions mutated in trp5-27.

Alleles↗