The protein factor which binds to the upstream activating sequence of Saccharomyces cerevisiae ENO1 gene.

Using a gel retardation assay it was shown that the 87 bp DNA fragment (UAS87) containing the upstream activating sequence (UAS) of S. cerevisiae EN01 gene and a nuclear extract gave rise to three migration-retarded species specific to UAS87. Heat- or proteinase-treatment of the nuclear extract revealed that these species were protein-DNA complexes. The precise binding region of the protein identified by DNaseI protection analysis was found to include a CCAAACA sequence which forms a dyad-symmetrical structure. The amount of one of the three migration-retarded species significantly increased when cells were grown in medium containing a gluconeogenic carbon source. The introduction of pGCR8, a multicopy plasmid containing GCR1 gene, a regulatory gene controlling the expression of several glycolytic enzymes, showed no effect on the amount of three migration-retarded species.     

Characterisation of the DNA binding domain of the yeast RAP1 protein.

The 827 amino acid yeast RAP1 protein interacts with DNA to regulate gene expression at numerous unrelated loci in the yeast genome. By a combination of amino, carboxy and internal deletions, we have defined an internal 235 amino acid fragment of the yeast RAP1 protein that can bind efficiently to the RAP1 binding site of the PGK Upstream Activation Sequence (UAS). This domain spans residues 361 to 596 of the full length protein and lacks any homology to the DNA binding 'zinc finger' or 'helix-turn-helix' structural motifs. All the RAP1 binding sites we have tested bind domain 361-596, arguing that RAP1 binds all its chromosomal sites via this domain. The domain could not be further reduced in size suggesting that it represents the minimal functional DNA binding domain. The relevance of potential regions of secondary structure within the minimal binding domain is discussed.     

The yeast inositol-sensitive upstream activating sequence, UASINO, responds to nitrogen availability

The INO1 gene of yeast is expressed in logarithmically growing, wild-type cells when inositol is absent from the medium. However, the INO1 gene is repressed when inositol is present during logarithmic growth and it is also repressed as cells enter stationary phase whether inositol is present or not. In this report, we demonstrate that transient nitrogen limitation also causes INO1 repression. The repression of INO1 in response to nitrogen limitation shares many features in common with repression in response to the presence of inositol. Specifically, the response to nitrogen limitation is dependent upon the presence of a functional OPI1 gene product, it requires ongoing phosphatidylcholine biosynthesis and it is mediated by the repeated element, UASINO, found in the promoter of INO1 and other co-regulated genes of phospholipid biosynthesis. Thus, we propose that repression of INO1 in response to inositol and in response to nitrogen limitation occurs via a common mechanism that is sensitive to the status of ongoing phospholipid metabolism.     

Histone gene organization of fission yeast: a common upstream sequence.

Histone genes of the fission yeast Schizosaccharomyces pombe were cloned from Charon 4A and cosmid gene libraries by hybridization, and their nucleotide sequences were determined. The genome of S. pombe has a single, isolated H2A, a pair of H2A-H2B and three pairs of H3-H4 (one H2B, two H2A and three each of H3 and H4). This non-assorted histone gene organization is distinct from that of the budding yeast which has two pairs of H2A-H2B and H3-H4. The predicted amino acid sequences of S. pombe histone H2As, H3s and H4s were identical except for three residue changes in H2As. Compared with those os S. cerevisiae and human, variable residues were clustered near the NH2- and COOH-terminal regions of H2A and H2B. Sequence homologies to the two organisms were roughly the same in H2A (79-83%), H3 (92-93%) and H4 (91%), but differed in H2B (82% to S. cerevisiae and 68% to human). The coding sequences in pairs of S. pombe histone genes were divergently directed. A 17-bp long highly homologous sequence (AACCCT box) that had internal 6-bp direct repeats was present in the intergene spacer sequences or in the 5' upstream region of all the cloned histone genes. A possible regulatory role of the common upstream sequence for histone gene expression is discussed.