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F Winston

Publications and source records attributed to F Winston.

At least 55 records · Page 3Linked to original sources
PubMed1994

The SPT10 and SPT21 genes of Saccharomyces cerevisiae.

Mutations in the SPT10 and SPT21 genes were originally isolated as suppressors of Ty and LTR (delta) insertion mutations in Saccharomyces cerevisiae, and the genes were shown to be required for normal transcription at a number of loci in yeast. Now we have cloned, sequenced, mapped and mutagenized SPT10 and SPT21. Since the spt10 mutation used to clone SPT10 resulted in very poor transformation efficiency, a novel method making use of the kar1-1 mutation was used. Neither SPT gene is essential for growth, and constructed null alleles cause phenotypes similar to those caused by spontaneous mutations in the genes. spt10 null alleles are strong suppressor mutations and cause extremely slow growth. Certain spt10 spontaneous alleles are good suppressors but have a normal growth rate, suggesting that the SPT10 protein may have two distinct functions. An amino acid sequence motif that is similar to the Zn-finger motif was found in SPT10. Mutation of the second Cys residue in this motif resulted in loss of complementation of the suppression phenotype but a normal growth rate. Thus, this motif may reside in a part of the SPT10 protein that is important for transcriptional regulation but not for normal growth. Both the SPT10 and SPT21 proteins are relatively tolerant of large deletions; in both cases deletions of the C-terminus resulted in at least partially functional proteins; also, a large internal deletion in SPT21 was phenotypically wild type.

Amino Acid Sequence↗
PubMed1994

The Saccharomyces cerevisiae SPT8 gene encodes a very acidic protein that is functionally related to SPT3 and TATA-binding protein.

Mutations in the Saccharomyces cerevisiae SPT8 gene were previously isolated as suppressors of retrotransposon insertion mutations in the 5' regions of the HIS4 and LYS2 genes. Mutations in SPT8 confer phenotypes similar to those caused by particular mutations in SPT15, which encodes the TATA-binding protein (TBP). These phenotypes are also similar to those caused by mutations in the SPT3 gene, which encodes a protein that directly interacts with TBP. We have now cloned and sequenced the SPT8 gene and have shown that it encodes a predicted protein of 602 amino acids with an extremely acidic amino terminus. In addition, the predicted SPT8 amino acid sequence contains one copy of a sequence motif found in multiple copies in a number of other eukaryotic proteins, including the beta subunit of heterotrimeric G proteins. To investigate further the relationship between SPT8, SPT3 and TBP, we have analyzed the effect of an spt8 null mutation in combination with different spt3 and spt15 mutations. This genetic analysis has shown that an spt8 deletion mutation is suppressed by particular spt3 alleles. Taken together with previous results, these data suggest that the SPT8 protein is required, directly or indirectly, for TBP function at particular promoters and that the role of SPT8 may be to promote a functional interaction between SPT3 and TBP.

Amino Acid Sequence↗
PubMed1994

Equivalent mutations in the two repeats of yeast TATA-binding protein confer distinct TATA recognition specificities.

To investigate the process of TATA box recognition by the TATA box-binding protein (TBP), we have performed a detailed genetic and biochemical analysis of two Saccharomyces cerevisiae TBP mutants with altered DNA-binding specificity. The mutant proteins have amino acid substitutions (Leu-205 to Phe and Leu-114 to Phe) at equivalent positions within the two repeats of TBP that are involved in TATA element binding. In an in vivo assay that employs a nearly complete set of single point mutations of the consensus TATAAA sequence, one of the TBP mutants (TBP-L114F) recognizes the sequence TATAAG, while the other TBP mutant (TBP-L205F) recognizes one substitution at the first position of the TATA element, CATAAA, and three substitutions at the 3' end of the TATA box. Specificity patterns determined from in vitro transcription experiments with purified recombinant wild-type TBP and TBP-L205F agree closely with those observed in vivo, indicating that altered TATA utilization in the mutant strains is a direct consequence of altered TATA recognition by the mutant TBPs. The distinct TATA recognition patterns exhibited by TBP-L114F and TBP-L205F strongly suggest that in vivo, TBP binds to the TATA element in a specific orientation. The orientation predicted from these studies is further supported by the identification of intragenic suppressors that correct the defect of TBP-L205F. This orientation is consistent with that observed in vitro by crystallographic analyses of TBP-TATA box complexes. Finally, the importance of altered DNA-binding specificity in transcriptional regulation at the S. cerevisiae his4-912 delta promoter was addressed for TBP-L205F. A mutational analysis of this promoter region demonstrates that the nonconsensus TATA element CATAAA is required for a transcriptional effect of TBP-L205F in vivo. This finding suggests that the interaction of TBP with nonconsensus TATA elements may play an important regulatory role in transcription initiation.

Base Sequence↗
PubMed1994

SPT10 and SPT21 are required for transcription of particular histone genes in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae genome contains four loci that encode histone proteins. Two of these loci, HTA1-HTB1 and HTA2-HTB2, each encode histones H2A and H2B. The other two loci, HHT1-HHF1 and HHT2-HHF2, each encode histones H3 and H4. Because of their redundancy, deletion of any one histone locus does not cause lethality. Previous experiments demonstrated that mutations at one histone locus, HTA1-HTB1, do cause lethality when in conjunction with mutations in the SPT10 gene. SPT10 has been shown to be required for normal levels of transcription of several genes in S. cerevisiae. Motivated by this double-mutant lethality, we have now investigated the interactions of mutations in SPT10 and in a functionally related gene, SPT21, with mutations at each of the four histone loci. These experiments have demonstrated that both SPT10 and SPT21 are required for transcription at two particular histone loci, HTA2-HTB2 and HHF2-HHT2, but not at the other two histone loci. These results suggest that under some conditions, S. cerevisiae may control the level of histone proteins by differential expression of its histone genes.

Fungal Proteins↗
PubMed1993

Molecular and genetic characterization of SPT4, a gene important for transcription initiation in Saccharomyces cerevisiae.

Mutations in the SPT4 gene of Saccharomyces cerevisiae were isolated as suppressors of delta insertion mutations that interfere with adjacent gene transcription. Recent genetic evidence indicates that the SPT4 protein functions with two other proteins, SPT5 and SPT6, in some aspect of transcription initiation. In this work we have characterized the SPT4 gene and we demonstrate that spt4 mutations, like spt5 and spt6 mutations, cause changes in transcription. Using the cloned SPT4 gene, spt4 null mutations were constructed; in contrast to spt5 and spt6 null mutants, which are inviable, spt4 null mutants are viable and have an Spt- phenotype. The DNA sequence of the SPT4 gene predicts a protein product of 102 amino acids that contains four cysteine residues positioned similarly to those of zinc binding proteins. Mutational analysis suggests that at least some of these cysteines are essential for SPT4 function. Genetic mapping showed that SPT4 is a previously unidentified gene that maps to chromosome VII, between ADE6 and CLY8.

Base Sequence↗
PubMed1993

Mutations that suppress the deletion of an upstream activating sequence in yeast: involvement of a protein kinase and histone H3 in repressing transcription in vivo.

Regulated transcription of most protein-encoding genes in Saccharomyces cerevisiae requires an upstream activating sequence (UAS); in the absence of UAS elements, little or no transcription occurs. In certain mutant strains, however, promoters that have been deleted for their UAS can direct significant levels of transcription, indicating that the remaining promoter elements (the basal promoter) are capable of directing higher levels of transcription, but they are normally represented in wild-type strains. To analyze this repression, we have selected for mutations that cause increased transcription of the SUC2 gene in the absence of its UAS. In addition to some previously studied genes, this selection has identified five genes that we have designated BUR1, BUR2, BUR3, BUR5 and BUR6 (for Bypass UAS Requirement). The bur mutations cause pleiotropic phenotypes, indicating that they affect transcription of many genes. Furthermore, some bur mutations suppress the requirement for the SNF5 trans-activator at both SUC2 and Ty. Additional analysis has demonstrated that BUR1 is identical to SGV1, which encodes a CDC28-related protein kinase. This result indicates that protein phosphorylation is important for repression of the SUC2 basal promoter as well as other aspects of transcription in vivo. Finally, BUR5 is identical to HHT1, encoding histone H3, further implicating chromatin structure as important for expression of SUC2.

Base Sequence↗
PubMed1992

Yeast SNF/SWI transcriptional activators and the SPT/SIN chromatin connection.

Genetic studies of many diversely regulated genes in the yeast Saccharomyces cerevisiae have identified two groups of genes with global functions in transcription. The first group comprises five SNF and SWI genes required for transcriptional activation. The other group, containing SPT and SIN genes, was identified by suppressor analysis and includes genes that encode histones. Recent evidence suggests that these SNF/SWI and SPT/SIN genes control transcription via effects on chromatin. SNF2/SWI2 sequence homologues have been identified in many organisms, suggesting that the SNF/SWI and SPT/SIN functions are conserved throughout eukaryotes.

Biological Evolution↗
PubMed1992

SPT4, SPT5 and SPT6 interactions: effects on transcription and viability in Saccharomyces cerevisiae.

The SPT4, SPT5 and SPT6 genes of Saccharomyces cerevisiae were identified originally by mutations that suppress delta insertion mutations at HIS4 and LYS2. Subsequent analysis has demonstrated that spt4, spt5 and spt6 mutations confer similar pleiotropic phenotypes. They suppress delta insertion mutations by altering transcription and are believed to be required for normal transcription of several other loci. We have now analyzed interactions between SPT4, SPT5 and SPT6. First, the combination of mutations in any two of these three genes causes lethality in haploids. Second, some recessive mutations in different members of this set fail to complement each other. Third, mutations in all three genes alter transcription in similar ways. Finally, the results of coimmunoprecipitation experiments demonstrate that at least the SPT5 and SPT6 proteins interact physically. Taken together, these genetic and biochemical results indicate that SPT4, SPT5 and SPT6 function together in a transcriptional process that is essential for viability in yeast.

Chromosomal Proteins, Non-Histone↗
PubMed1992

A mutant tRNA affects delta-mediated transcription in Saccharomyces cerevisiae.

Mutations in the SPT3, SPT7, SPT8 and SPT15 genes define one class of trans-acting mutations that are strong suppressors of insertion mutations caused by Ty elements or by the Ty long terminal repeat sequence, delta. These SPT genes are required for normal transcription of Ty elements, and their gene products are believed to be involved in initiation of Ty transcription from delta sequences. We have isolated and analyzed extragenic suppressors of spt3 mutations. These new mutations, named rsp, partially suppress the requirement for SPT3, SPT7, SPT8 and SPT15 functions. In addition, rsp mutations cause changes in transcription of some delta insertions in an SPT+ genetic background. Interactions between mutations in the four identified RSP genes show a number of interesting genetic properties, including the failure of unlinked rsp mutations to complement for recessive phenotypes. Cloning and sequencing of one rsp mutant gene, rsp4-27, showed that it encodes a frameshift suppressor glycine tRNA. Our results indicate that the other three RSP genes also encode frameshift suppressor glycine tRNAs. In addition, other types of frameshift suppressor glycine tRNAs can confer some Rsp- phenotypes.

Alleles↗
PubMed1992

Evidence that SNF2/SWI2 and SNF5 activate transcription in yeast by altering chromatin structure.

Changes in chromatin structure have frequently been correlated with changes in transcription. However, the cause-and-effect relationship between chromatin structure and transcription has been hard to determine. In addition, identifying the proteins that regulate chromatin structure has been difficult. Recent evidence suggests that a functionally related set of yeast transcriptional activators (SNF2/SWI2, SNF5, SNF6, SWI1, and SWI3), required for transcription of a diverse set of genes, may affect chromatin structure. We now present genetic and molecular evidence that at least two of these transcriptional activators, SNF2/SWI2 and SNF5, function by antagonizing repression mediated by nucleosomes. First, the transcriptional defects in strains lacking these SNF genes are suppressed by a deletion of one of the two sets of genes encoding histones H2A and H2B, (hta1-htb1) delta. Second, at one affected promoter (SUC2), chromatin structure is altered in snf2/swi2 and snf5 mutants, and this chromatin defect is suppressed by (hta1-htb1) delta. Finally, analysis of chromatin structure at a mutant SUC2 promoter, in which the TATA box has been destroyed, demonstrates that the differences in SUC2 chromatin structure between SNF5+ and snf5 mutant strains are not simply an effect of different levels of SUC2 transcription. Thus, these results strongly suggest that SNF2/SWI2 and SNF5 cause changes in chromatin structure and that these changes allow transcriptional activation.

Adenosine Triphosphatases↗
PubMed1992

SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae.

Mutations in the Saccharomyces cerevisiae gene SPT15, which encodes the TATA-binding protein TFIID, have been shown to cause pleiotropic phenotypes and to lead to changes in transcription in vivo. Here, we report the cloning and analysis of one such mutation, spt15-21, which causes a single-amino-acid substitution in a conserved residue of TFIID. Surprisingly, the spt15-21 mutation does not affect the stability of TFIID, its ability to bind to DNA or to support basal transcription in vitro, or the ability of an upstream activator to function in vivo. To study further the spt15-21 defect, extragenic suppressors of this mutation were isolated and analyzed. All of the extragenic suppressors of spt15-21 are mutations in the previously identified SPT3 gene. Suppression of spt15-21 by these spt3 mutations is allele-specific, suggesting that TFIID and SPT3 interact and that spt15-21 impairs this interaction in some way. Consistent with these genetic data, coimmunoprecipitation experiments demonstrate that the TFIID and SPT3 proteins are physically associated in yeast extracts. Taken together, these results suggest that SPT3 is a TFIID-associated protein, required for TFIID to function at particular promoters in vivo.

Amino Acid Sequence↗
PubMed1992

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro.

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

Amino Acid Sequence↗
PubMed1991

The SNF2, SNF5 and SNF6 genes are required for Ty transcription in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae SNF2, SNF5 and SNF6 genes were initially identified as genes required for expression of SUC2 and other glucose repressible genes. The Suc- defect in all three of these classes of mutants is suppressed by mutations in the SPT6 gene. Since mutations in SPT6 had also been identified as suppressors of Ty and solo delta insertion mutations at the HIS4 and LYS2 loci, we have examined Ty transcription in snf2, snf5 and snf6 mutants and have found that Ty transcription is abolished or greatly reduced. The snf2, snf5 and snf6 defect for Ty transcription, like the defect for SUC2 transcription, is suppressed by spt6 mutations. In contrast to other mutations that abolish or greatly reduce Ty transcription (in the SPT3, SPT7 and SPT8 genes), mutations in these SNF genes do not cause suppression of insertion mutations. This result suggests that the SNF2, SNF5 and SNF6 gene products act by a distinct mechanism from the SPT3, SPT7 and SPT8 gene products to promote transcription of Ty elements. This result also suggests that a reduction of Ty transcription is not always sufficient for activation of adjacent gene expression.

Blotting, Northern↗
PubMed1991

Glucose repression of transcription of the Schizosaccharomyces pombe fbp1 gene occurs by a cAMP signaling pathway.

Transcription of the fbp1 gene, encoding fructose-1,6-bisphosphatase, of Schizosaccharomyces pombe is subject to glucose repression. Previous work has demonstrated that several genes (git genes) are required for this repression. In this report we demonstrate that one of these genes, git2, is the same as the cyr1 gene, which encodes adenylate cyclase, and that loss-of-function mutations in git2 cause constitutive fbp1 transcription. Addition of cAMP to the growth medium suppresses the transcriptional defect in git2 mutants as well as in strains that carry mutations in any of six additional git genes. Similarly, exogenous cAMP represses fbp1 transcription in wild-type cells grown on a derepressing carbon source. Different levels of adenylate cyclase activity in different git2 mutants, coupled with the result that some git2 mutants display intragenic complementation, strongly suggest that adenylate cyclase acts as a multimer and that different git2 mutations alter distinct activities of adenylate cyclase, including catalytic activity and response to glucose. Additional experiments demonstrate that this cAMP signaling pathway is independent of the S. pombe ras1 gene and works by activation of cAMP-dependent protein kinase.

Cloning, Molecular↗
PubMed1991

Mutations in SPT16/CDC68 suppress cis- and trans-acting mutations that affect promoter function in Saccharomyces cerevisiae.

SPT16 was previously identified as a high-copy-number suppressor of delta insertion mutations in the 5' regions of the HIS4 and LYS2 genes of Saccharomyces cerevisiae. We have constructed null mutations in the SPT16 gene and have demonstrated that it is essential for growth. Temperature-sensitive-lethality spt16 alleles have been isolated and shown to be pleiotropic; at a temperature permissive for growth, spt16 mutations suppress delta insertion mutations, a deletion of the SUC2 upstream activating sequence, and mutations in trans-acting genes required for both SUC2 and Ty expression. In addition, SPT16 is identical to CDC68, a gene previously shown to be required for passage through the cell cycle control point START. However, at least some transcriptional effects caused by spt16 mutations are independent of arrest at START. These results and those in the accompanying paper (A. Rowley, R. A. Singer, and G. C. Johnston, Mol. Cell. Biol. 11:5718-5726, 1991) indicate that SPT16/CDC68 is required for normal transcription of many loci in S. cerevisiae.

DNA, Fungal↗
PubMed1991

SPT5, an essential gene important for normal transcription in Saccharomyces cerevisiae, encodes an acidic nuclear protein with a carboxy-terminal repeat.

Mutations in the SPT5 gene of Saccharomyces cerevisiae were isolated previously as suppressors of delta insertion mutations at HIS4 and LYS2. In this study we have shown that spt5 mutations suppress the his4-912 delta and lys2-128 delta alleles by altering transcription. We cloned the SPT5 gene and found that either an increase or a decrease in the copy number of the wild-type SPT5 gene caused an Spt- phenotype. Construction and analysis of an spt5 null mutation demonstrated that SPT5 is essential for growth, suggesting that SPT5 may be required for normal transcription of a large number of genes. The SPT5 DNA sequence was determined; it predicted a 116-kDa protein with an extremely acidic amino terminus and a novel six-amino-acid repeat at the carboxy terminus (consensus = S-T/A-W-G-G-A/Q). By indirect immunofluorescence microscopy we showed that a bifunctional SPT5-beta-galactosidase protein was located in the yeast nucleus. This molecular analysis of the SPT5 gene revealed a number of interesting similarities to the previously characterized SPT6 gene of S. cerevisiae. These results suggest that SPT5 and SPT6 act in a related fashion to influence essential transcriptional processes in S. cerevisiae.

Alleles↗