A set of GAL2+ yeast strains that are isogenic to strain S288C have been constructed. They contain non-reverting mutations in genes commonly used for selection for recombinant plasmids. Strains from this collection are being used for the European Union Yeast Genome Sequencing Programme. Representative strains from this collection have been deposited with the ATCC.

Nucleosomes have been shown to repress transcription both in vitro and in vivo. However, the mechanisms by which this repression is overcome are only beginning to be understood. Recent evidence suggests that in the yeast Saccharomyces cerevisiae, many transcriptional activators require the SNF/SWI complex to overcome chromatin-mediated repression. We have identified a new class of mutations in the histone H2A-encoding gene HTA1 that causes transcriptional defects at the SNF/SWI-dependent gene SUC2. Some of the mutations are semidominant, and most of the predicted amino acid changes are in or near the N-and C-terminal regions of histone H2A. A deletion that removes the N-terminal tail of histone H2A also caused a decrease in SUC2 transcription. Strains carrying these histone mutations also exhibited defects in activation by LexA-GAL4, a SNF/SWI-dependent activator. However, these H2A mutants are phenotypically distinct from snf/swi mutants. First, not all SNF/SWI-dependent genes showed transcriptional defects in these histone mutants. Second, a suppressor of snf/swi mutations, spt6, did not suppress these histone mutations. Finally, unlike in snf/swi mutants, chromatin structure at the SUC2 promoter in these H2A mutants was in an active conformation. Thus, these H2A mutations seem to interfere with a transcription activation function downstream or independent of the SNF/SWI activity. Therefore, they may identify an additional step that is required to overcome repression by chromatin.In eukaryotic cells, DNA is complexed with histones and other proteins into chromatin (see reference 75 for a review). The primary component of chromatin is the nucleosome, which consists of approximately 146 bp of DNA wrapped around an octamer of histones (two histone H2A-H2B dimers and one [H3-H4] 2 tetramer; see reference 75 for a review). A growing body of evidence from both in vivo and in vitro studies has shown that the structure of chromatin influences gene expression (see references 25 and 53 for reviews). Biochemical experiments have shown that histones can repress transcription in vitro (see reference 53 for a review) and that transcriptional activators can overcome this repression (21,41,45,79,81,82). In vivo experiments in Saccharomyces cerevisiae have shown that the loss of transcriptional activators or of activator binding sites can be suppressed by mutations in histone genes (27,30,37,57). These results suggest that one function of transcriptional activators in vivo is to antagonize chromatin-mediated repression.In vivo studies of histone mutants have also suggested that histones play a variety of roles in transcriptional regulation. Small deletions and point mutations that alter the flexible N-terminal tails of different yeast histones have been shown to cause specific changes in transcription (see reference 25 for a review). Analysis of deletions and single amino acid changes in the N-terminal region of histone H4 has demonstrated that certain changes in the H4 N terminus abolish repression of the yeast silent mating-...

The TATA box binding protein (TBP) plays a central and essential role in transcription initiation. At TATA box‐containing genes transcribed by RNA polymerase II, TBP binds to the promoter and initiates the assembly of a multiprotein preinitiation complex. Several studies have suggested that binding of TBP to the TATA box is an important regulatory step in transcription initiation in vitro. To determine whether TBP is a target of regulatory factors in vivo, we performed a genetic screen in yeast for TBP mutants defective in activated transcription. One class of TBP mutants identified in this screen comprises inositol auxotrophs that are also defective in using galactose as a carbon source. These phenotypes are due to promoter‐specific defects in transcription initiation that are governed by the upstream activating sequence (UAS) and apparently not by the sequence of the TATA element. The finding that these TBP mutants are severely impaired in DNA binding in vitro suggests that transcription initiation at certain genes is regulated at the level of TATA box binding by TBP in vivo.

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.

Spt4p is a nonhistone protein of Saccharomyces cerevisiae that is believed to be required for normal chromatin structure and transcription. In this work we describe the isolation and analysis of a human gene, SUPT4H, that encodes a predicted protein 42% identical to Spt4p. When expressed in S. cerevisiae, SUPT4H complemented all spt4 mutant phenotypes. In human cells SUPT4H encodes a nuclear protein that is expressed in all tissues tested. In addition, hybridization analyses suggest that an SUPT4H-related gene is also present in mice. SUPT4H was localized to human chromosome 17 by PCR analysis of a human-rodent somatic cell hybrid panel. Thus, like other proteins that are components of or control the structure of chromatin, Spt4p appears to be conserved from S. cerevisiae to mammals.The importance of chromatin structure in the control of transcription has been demonstrated by a combination of genetic and biochemical studies (for a review, see reference 37). In particular, these studies show that nucleosomes can function to repress transcription. However, the mechanisms by which this control is exerted, in terms of establishing, maintaining, and overcoming the repression of transcription by nucleosomes, are not clear. Some clues have come from genetic studies with the yeast Saccharomyces cerevisiae. These studies have provided strong candidates for proteins that might regulate the assembly, organization, modification, and disassembly of chromatin structure (52). For example, substantial evidence suggests that the Snf/Swi complex of S. cerevisiae serves to overcome nucleosomal repression of transcription by altering nucleosomal structure, thus aiding the binding of transcription factors and the activation of transcription (38).Another set of S. cerevisiae genes, SPT4, SPT5, and SPT6, are candidates to encode functions that help nucleosomes to repress transcription (46, 52). These three genes are members of a large set of genes originally identified as mutations that suppress the transcriptional defects caused by insertions of the retrotransposon Ty or of its long terminal repeat, ␦, upstream of the HIS4 or LYS2 gene (Spt Ϫ phenotype) (50). On the basis of shared mutant phenotypes, SPT4, SPT5, and SPT6 were grouped within a set of genes believed to control transcription by modulating chromatin structure (46,50). This group also includes HTA1-HTB1, one of the two gene pairs encoding histones H2A and H2B (20). Mutations in spt4, spt5, and spt6 as well as a deletion of this histone gene pair, (hta1-htb1)⌬, cause an Spt Ϫ phenotype (13,26,44,45). In addition, mutations in this group of genes suppress snf/swi mutations (21,33,46). Suppression of snf/swi mutations by (hta1-htb1)⌬ occurs at the level of chromatin structure, presumably by reducing the number of nucleosomes via a reduction in the levels of histones H2A and H2B (21). By analogy, spt4, spt5, and spt6 mutations may also cause a reduced number of functional nucleosomes.Although spt4, spt5, and spt6 mutants share many phenotypes with (hta1-htb1)⌬ mutants, the pr...