Nucleotide sequence of the yeast regulatory gene GAL80

The GAL80 gene in Saccharomyces cerevisiae encodes a negative regulatory protein for the set of inducible genes involving metabolism of galactose and melibiose. We have determined the nucleotide sequence of GAL80 and its flanking regions and assigned the 5' end of its mRNA to the sequence. The deduced coding sequence for GAL80 protein contains 1305 nucleotides and the calculated molecular weight of the peptide chain is 48309. The 5' end of the GAL80 mRNA maps about 67 nucleotides upstream from the translation initiating ATG. We have also determined the nucleotide sequence of uninducible alleles GAL80S-0, GAL80S-1 and GAL80S-2, and found single base substitution in each of these mutant genes which would lead to alteration of amino acid in GAL80 protein.     

The relationship of regulatory proteins and DNase I hypersensitive sites in the yeast GAL1-10 genes.

We have used yeast strains containing a disrupted positive (GAL4) and/or a disrupted negative (GAL80) regulatory gene to investigate the relationship of these regulatory proteins to the hypersensitive sites upstream of their target genes, GAL1-10. We find that neither of these regulatory proteins is required for the formation of the hypersensitive region. There is positive regulatory protein (dependent) binding to a portion of the hypersensitive region when GAL1 and 10 are expressed. However, similar binding can also occur under conditions in which the genes are not expressed. Thus, such binding is necessary but not sufficient for expression of GAL1 and 10 and control of GAL1-10 expression must also include processes which occur subsequent to GAL4/DNA binding. The negative regulatory protein GAL80 plays a significant role in these processes.     

GAL4 protein: purification, association with GAL80 protein, and conserved domain structure.

Expression of the yeast Saccharomyces cerevisiae GAL4 protein under its own (galactose-inducible) control gave 5 to 10 times the level of protein observed when the GAL4 gene was on a high-copy plasmid. Purification of GAL4 by a procedure including affinity chromatography on a GAL4-binding DNA column yielded not only GAL4 but also a second protein, shown to be GAL80 by its reaction with an antipeptide antibody. Sequence comparisons of GAL4 and other members of a family of proteins sharing homologous cysteine finger motifs identified an additional region of homology in the middle of these proteins shown by genetic analysis to be important for GAL4 function. GAL4 could be cleaved proteolytically at the boundary of the conserved region, defining internal and carboxy-terminal folded domains.     

The regulatory protein GAL80 is a determinant of the chromatin structure of the yeast GAL1-10 control region

Chromatin in the regions between the upstream activator sequence and the 5' ends of the yeast GAL1 and GAL10 genes has been analyzed by DNase I chromosomal footprinting and micrococcal nuclease digestion using the indirect end-labeling approach. Comparison of wild type chromatin digests to naked DNA digests shows that there are specific regions of these upstream sequences which are strongly protected in chromatin. Comparison to chromatin digests from cells disrupted for the positive regulatory gene, GAL4, or the negative regulatory gene, GAL80, and thus lacking GAL4 or GAL80 function, shows that these regions of protection in wild type chromatin are GAL80-dependent but not GAL4-dependent. The protected regions include DNA lying on (GAL10) or near (GAL1) the respective TATA boxes. These protections are present in both noninduced and induced cells. Both DNA strands are equally protected. Upstream of GAL1 there is a second protected region. This protection shows considerable expression and strand dependence. These observations provide the first evidence that the GAL80 function influences chromatin structure and suggest possible mechanisms by which GAL80 modulates the GAL1 and 10 promoters in induced cells. Micrococcal nuclease digests also suggest a role for GAL80 in a distinctive higher order organization of the intergenic region, perhaps involving multiprotein complexes.