Biomedical subjects

F Winston

Publications and source records attributed to F Winston.

At least 73 records · Page 4Linked to original sources

PubMed1991

The products of the SPT10 and SPT21 genes of Saccharomyces cerevisiae increase the amplitude of transcriptional regulation at a large number of unlinked loci.

The 3' long terminal repeat (LTR) of yeast transposon Ty1 is not normally used as a promoter, although it contains sequences identical to those found in the 5' LTR, which does act as a promoter. We have isolated mutations that fall into two genes, SPT10 and SPT21, that allow the 3' LTRs of Ty1 elements inserted at various positions in the genome of Saccharomyces cerevisiae to act as promoters. We find that mutations in these two genes alter transcriptional regulation of Ty1 LTRs and also of certain non-Ty1-related promoters in two ways: (i) they allow the low-level expression of several genes under repressing conditions, and (ii) they allow transcription from the 5' LTR of Ty1 elements in the absence of a normally required activator, SPT3. Furthermore, the fully induced levels of transcription of several genes are reduced in these spt mutants. Hence, the products of these two genes increase the amplitude of transcriptional regulation of a wide variety of unlinked loci.

Acid Phosphatase↗

PubMed1990

Striking conservation of TFIID in Schizosaccharomyces pombe and Saccharomyces cerevisiae.

Eukaryotic promoters contain binding sites for basic transcription factors and gene-specific activator proteins. The transcription factors interact at the TATA box, which lies close to the position of transcription initiation. Activators typically bind to distant sites that can lie kilobases away from the initiation site. The factor TFIID binds specifically to the TATA box to initiate an ordered pathway of assembly of the basic transcription factors. Biochemical analyses have shown that human and Saccharomyces cerevisiae TFIID are functionally interchangeable in vitro. To study further the functional conservation of this critical factor, we are surveying proteins from divergent organisms that can substitute in vivo for the S. cerevisiae TFIID. We report here the isolation of a unique gene from Schizosaccharomyces pombe that fully complements a null mutation in SPT15, the gene that encodes TFIID in S. cerevisiae. The Schiz. pombe gene encodes a protein 93% identical (166/178) to S. cerevisiae TFIID in a region consisting of a direct repeat.

Amino Acid Sequence↗

PubMed1990

Affinity chromatography of mammalian and yeast nucleosomes. Two modes of binding of transcriptionally active mammalian nucleosomes to organomercurial-agarose columns, and contrasting behavior of the active nucleosomes of yeast.

The reasons for the selective binding of nucleosomes from transcriptionally active genes to the organomercurial-agarose columns have been investigated. At least two modes of binding are identified by a new two-stage elution procedure that discriminates between nucleosomes which are retained by the Hg-column because of their salt-labile associations with SH-reactive non-histone proteins, and nucleosomes in which a conformational change has made the thiol groups of histone H3 accessible to SH-reagents. The first class is released from the column in 0.5 M NaCl; the second class is eluted in 10 mM dithiothreitol which displaces the bound H3-thiols. In mammalian cells, both classes of Hg-bound nucleosomes are enriched in the DNA sequences being transcribed at the time, and their histones H3 and H4 are hyperacetylated. In yeast cells, in which histone H3 lacks cysteinyl residues, only a small fraction of nucleosomes binds to the mercury column, and it has no enrichment of DNA sequences derived from the actively transcribed GAL, HIS4, and ACT1 genes. Since few nucleosomes remain on the column after elution in 0.5 M NaCl, the bound nucleosomes of yeast are retained primarily because of salt-labile associations with thiol-reactive nonhistone proteins. Thus, the presence of histone H3-thiol groups appears to be essential for the mercury binding of the second class of nucleosomes which, in mammalian cells, is derived from the transcriptionally active genes. The results support models of reversible nucleosome unfolding during transcription in mammalian cells to reveal previously inaccessible H3-SH groups, and they also indicate that other thiol-containing proteins, including high mobility group 1 and 2, become closely but transiently associated with the chromatin subunits during their transcription.

Acetylation↗

PubMed1990

Isolation and characterization of mutants constitutive for expression of the fbp1 gene of Schizosaccharomyces pombe.

Transcription of the fbp1 gene of Schizosaccharomyces pombe, encoding fructose-1,6-bisphosphatase, is glucose repressible. We have constructed two hybrid genes, containing the fbp1 promoter, that allow selection for mutations that alter transcriptional regulation of fbp1. Strains carrying fbp1-ura4 and fbp1-lacZ fusions are phenotypically Ura-, resistant to 5-fluoro-orotic acid, and express a low level of beta-galactosidase activity when grown under repressing conditions (8% glucose). By selecting for Ura+ strains grown under repressing conditions, we have isolated 187 independent mutants that constitutively express the fbp1-ura4 fusion. These mutants identify ten complementation groups that represent ten unlinked git (glucose insensitive transcription) genes. The git gene products are required in trans for glucose repression of expression from the fbp1 promoter since these mutations also alter expression of the fbp1-lacZ fusion. We have shown that transcription of the wild type fbp1 gene in most git mutants is elevated to a level consistent with the increased expression of the fbp1-lacZ hybrid gene. Mutations in some git genes confer additional phenotypes such as slow growth, temperature-sensitive lethality and reduced spore viability. Therefore, some of these genes are likely to encode factors that are of general importance for S. pombe transcription.

Alleles↗

PubMed1990

SPT6, an essential gene that affects transcription in Saccharomyces cerevisiae, encodes a nuclear protein with an extremely acidic amino terminus.

SPT6 is an essential gene of Saccharomyces cerevisiae that appears to play a role in transcription. Mutations in the SPT6 (SSN20, CRE2) gene suppress delta insertion mutations in the 5' regions of HIS4 and LYS2 and mutations in cis- and/or trans-acting elements that are required for expression of SUC2 and ADH2. We report here that SPT6 encodes a 170-kilodalton highly charged protein with an extremely acidic amino terminus. By use of an epitope-tagged SPT6 protein, we have determined by indirect immunofluorescence that the SPT6 protein is located in the nucleus.

Amino Acid Sequence↗

PubMed1989

A transcriptionally regulated expression vector for the fission yeast Schizosaccharomyces pombe.

An expression vector for the fission yeast Schizosaccharomyces pombe is described. The vector is designed to facilitate the construction of transcriptional fusions to the promoter of the S. pombe fructose bisphosphatase gene. Transcription from this promoter is regulated by glucose repression over a range of greater than 100-fold. The tight regulation by this promoter should allow for the maintenance of genes whose products are lethal to S. pombe and for the high level production of their protein or RNA products. Intermediate levels of expression can also be achieved by growth on different carbon sources.

Base Sequence↗

PubMed1989

SPT15, the gene encoding the yeast TATA binding factor TFIID, is required for normal transcription initiation in vivo.

Mutations in the S. cerevisiae SPT15 gene were isolated as suppressors of insertion mutations that alter the transcription of adjacent genes. Molecular and genetic analysis of the cloned SPT15 gene has shown that it is the same as the gene that encodes the TATA binding factor TFIID. Analysis of spt15 mutants has demonstrated that alterations in TFIID can change transcription initiation in vivo. In addition, we demonstrate that TFIID is essential for growth and that spt15 mutations are pleiotropic, as spt15 mutants grow slowly and have defects in both mating and sporulation. Therefore, TFIID is an essential transcription factor in vivo, likely to be required for normal expression of a large number of genes.

Chromosome Mapping↗

PubMed1989

Ty1 transposition in Saccharomyces cerevisiae is nonrandom.

A large collection of Ty1 insertions in the URA3 and LYS2 loci was generated using a GAL1-Ty1 fusion to augment the transposition frequency. The sites of insertion of most of these Ty elements were sequenced. There appears to be a gradient of frequency of insertion from the 5' end (highest frequency) to the 3' end (lowest frequency) of both loci. In addition we observed hotspots for transposition. Twelve of the 82 Ty1 insertions in the URA3 locus were inserted in exactly the same site. Hotspots were also observed in the LYS2 locus. All hotspots were in the transcribed part of the genes. Alignment of the sites of insertion and of the neighboring sequences only reveals very weak sequence similarities.

Base Composition↗

PubMed1989

The Saccharomyces cerevisiae SPT13/GAL11 gene has both positive and negative regulatory roles in transcription.

To understand the function of SPT13, a gene encoding a trans-acting factor involved in regulation of Ty-mediated gene expression, we have cloned and sequenced the gene. Our analysis revealed that SPT13 is the same gene as GAL11, a gene in which mutations cause reduced expression of some GAL4-regulated genes. Further analysis of spt13/gal11 mutants suggested that the SPT13 gene also affects transcription of genes involved in mating-type specialization. We show here that SPT13 has both positive and negative regulatory roles in transcription.

Blotting, Northern↗

PubMed1988

Isolation and analysis of a novel class of suppressor of Ty insertion mutations in Saccharomyces cerevisiae.

Using a new scheme for the isolation of suppressor of Ty insertion mutations (spt mutations) in yeast, we have identified six new SPT genes. Mutations in two of these genes, SPT13 and SPT14, exhibit a novel suppression pattern: suppression of complete Ty insertion mutations, but not of solo delta insertion mutations. Transcriptional analysis shows that spt13- and spt14-mediated suppression of Ty insertion mutations is the result of an elevation in the levels of adjacent gene transcription. In spite of the failure of these mutations to suppress solo delta insertion mutations, they do cause changes in transcription of at least one solo delta insertion mutation. In addition, spt13 and spt14 mutations are epistatic to mutations in certain other SPT genes that do suppress solo delta insertion mutations. These results suggest that the SPT13 and SPT14 gene products may act via sequences in both the delta and epsilon regions of Ty elements. Finally, mutations in SPT13 cause sporulation and mating defects and SPT14 is essential for growth, suggesting that these two genes have important roles in general cellular functions.

Alleles↗

PubMed1988

Changes in histone gene dosage alter transcription in yeast.

Chromatin structure is believed to be important for a number of cellular processes, including transcription. However, the role of nucleosomes in transcription is not well understood. We have identified the yeast histone locus HTB1-HTB1, encoding histones H2A and H2B, as a suppressor of solo delta insertion mutations that inhibit adjacent gene expression. The HTA1-HTB1 locus causes suppression either when present on a high-copy-number plasmid or when mutant. These changes in HTA1-HTB1 after transcription of the genes adjacent to the delta insertions. On the basis of this result, we have examined the effects of increased and decreased histone gene dosage for all four yeast histone loci. From the types of histone gene dosage changes that cause suppression of insertion mutations, we conclude that altered stoichiometry of histone dimer sets can alter transcription in yeast.

DNA Transposable Elements↗

PubMed1988

Genetic evidence for promoter competition in Saccharomyces cerevisiae.

The his4-912 delta mutation is an insertion of the long terminal repeat (delta) of the yeast retrotransposon Ty into the HIS4 promoter region, such that the delta is 97 base pairs upstream of the HIS4 transcription initiation site. Strains carrying the his4-912 delta allele are His- at 23 degrees C; this phenotype can be reversed either by growth at 37 degrees C or by mutations in trans-acting SPT genes. Under conditions in which his4-912 delta confers a His- phenotype. HIS4 transcription initiates at the delta initiation site, rather than at the HIS4 initiation site, producing a longer, nonfunctional transcript. Under conditions in which the strain is His+, transcription initiates at the wild-type HIS4 initiation site. To understand how transcription is balanced between the delta and HIS4 promoters, we have selected for cis-acting suppressors of his4-912 delta. Two classes defined by six independent mutations restore synthesis of a functional HIS4 transcript. The first class is an A-to-G base change 1 base upstream of the proposed delta TATA sequence. These mutants do not synthesize the delta-initiated transcript; instead, they synthesize only the wild-type HIS4 transcript. The second class of mutations alters base pairs surrounding the functional HIS4 TATA sequence. The two strongest His+ mutants of this class synthesize the wild-type HIS4 transcript at levels consistent with their His+ phenotype. Surprisingly, these two mutants also have a reduced level of the delta-initiated transcript relative to the his4-912 delta parent. Analysis of these mutants indicates that the level of transcription from one promoter can affect the level of transcription from the other promoter and suggests that delta and HIS4 transcription signals compete for initiation of transcription from each site.

Base Sequence↗

PubMed1988

SPT3 is required for normal levels of a-factor and alpha-factor expression in Saccharomyces cerevisiae.

Mutations in the Saccharomyces cerevisiae SPT3 gene were previously found to cause suppression of Ty and delta insertion mutations in 5'-noncoding regions of genes. This suppression likely results from the fact that SPT3 is required for transcription initiation in delta sequences. Other additional phenotypes of spt3 mutants, including a mating defect, suggest that SPT3 is required for normal levels of expression of other genes. We analyzed the mating defect in spt3 mutants and showed that the levels of transcripts of the three major mating pheromone genes, MF alpha 1, MFa1, MFa2, were all reduced. The reduction in expression of these genes in spt3 mutants was not due to expression of a silent mating type cassette. Furthermore, we showed that the spt3 mating defect was manifest at the levels of both cellular fusion and nuclear fusion.

Genes↗

PubMed1987

A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli.

A procedure for the rapid isolation of DNA from the yeast Saccharomyces cerevisiae is described. To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform. Centrifugation of this mixture separates the DNA from cellular debris. E. coli can be efficiently transformed with plasmid present in the aqueous layer without further purification of the plasmid DNA. This procedure also releases chromosomal DNA. Following two ethanol precipitations, the chromosomal DNA can be digested by restriction endonucleases and analysed by Southern blot analysis.

DNA, Fungal↗

PubMed1987

Three genes are required for trans-activation of Ty transcription in yeast.

Mutations in the SPT3 gene were isolated as one class of suppressors of Ty and solo delta insertion mutations in Saccharomyces cerevisiae. Previous work has shown that null mutations in SPT3 abolish the normal Ty delta-delta transcript; instead, a transcript that initiates 800 bases farther downstream is made, suggesting that SPT3 is required for transcription initiation in delta sequences. We have selected for new spt mutations and have screened for those with the unique suppression pattern of spt3 mutations with respect to two insertion mutations. Our selection and screen has identified two additional genes, SPT7 and SPT8, that are also required for transcription initiation in delta sequences. We show that mutations in SPT7 or SPT8 result in the same alteration of Ty transcription as do mutations in SPT3. In addition, mutations in all three genes cause a sporulation defect. By assay of a Ty-lacZ fusion we have shown that spt3, spt7 and spt8 mutations reduce transcription from a delta sequence by 10-25-fold. Finally, we show that SPT3 mRNA levels are unaffected in either spt7 or spt8 mutants, suggesting that these two genes do not regulate transcription of SPT3.

DNA Transposable Elements↗

PubMed1987

The SPT6 gene is essential for growth and is required for delta-mediated transcription in Saccharomyces cerevisiae.

Mutations in the Saccharomyces cerevisiae SPT6 gene were originally identified as one class of extragenic suppressors of Ty and delta insertion mutations in the 5' noncoding regions of HIS4 and LYS2. We cloned SPT6 and constructed a null allele by gene disruption. Haploid spores carrying the spt6 null allele were inviable, indicating that the SPT6 gene is essential for mitotic growth. SPT6 was mapped to the right arm of chromosome VII, 44 centimorgans (cM) from ADE6 and 9 cM from CLY8. We showed that spt6 mutations suppress delta insertion mutations at the level of transcription but have no qualitative or quantitative effect on Ty transcription. In addition, we observed interesting SPT6 gene dosage effects. An SPT6 strain containing a high-copy-number plasmid clone of SPT6 showed suppression of delta insertion mutations, and a diploid strain with half its normal dose of SPT6 (SPT6/spt6 null) also exhibited suppression of delta insertion mutations. Therefore, having either too many or too few copies of SPT6 causes a mutant phenotype. Finally, this study and that in the accompanying paper (L. Neigeborn, J. L. Celenza, and M. Carlson, Mol. Cell. Biol. 7:679-686, 1986) showed that spt6 and ssn20 mutations (isolated as suppressors of snf2 and snf5 [sucrose nonfermenting] mutations) identify the same gene. SPT6 and SSN20 have the same genetic map position and share an identical restriction map. Furthermore, spt6 and ssn20 mutations fail to complement each other, and ssn20 mutations suppress solo delta insertion mutations at HIS4 and LYS2. These results, taken in conjunction with the SPT6 dosage effects and the fact that SPT6 is an essential gene, suggest that SPT6 plays a fundamental role in cellular transcription, perhaps by interaction with other transcription factors.

Alleles↗

PubMed1986

Analysis of the yeast SPT3 gene and identification of its product, a positive regulator of Ty transcription.

Previous work has demonstrated that the yeast SPT3 gene is required for transcription from delta sequences, the long terminal repeats that flank yeast Ty elements. In spt3 null mutants, transcription fails to initiate in delta sequences and instead initiates farther downstream. Null mutations in SPT3 cause other mutant phenotypes, including defects in sporulation and diploid formation. In this paper we report further genetic and physical characterization of the SPT3 gene and protein. By extensive linker insertion mutagenesis, we have delimited the region necessary for SPT3 function. From DNA sequence analysis, SPT3 encodes a protein of 337 amino acids. We have identified this protein with an anti-SPT3 antibody. Finally, we show that overproduction of the SPT3 gene product does not alter the level of Ty transcription.

Amino Acid Sequence↗

PubMed1984

Identification of a Ty insertion within the coding sequence of the S. cerevisiae URA3 gene.

Insertion mutations in yeast caused by the transposable element Ty have been identified at several genetic loci. In all cases so far, the site of Ty insertion has been in the 5' non-coding region of the affected gene. Experiments presented here demonstrate that the ura3-52 mutation, a non-reverting ura3 mutation, is caused by a Ty insertion mutation within the coding region of the URA3 gene. This Ty insertion within a gene has a similar structure to those in non-coding regions.

Base Sequence↗