In Saccharomyces cerevisiae, the transcriptional expression of the galactose-melibiose catabolic pathway genes is under the control of at least three regulatory genes, GAL4, GAL80, and GAL3. We have isolated the GAL80 gene and have studied the effect of a null mutation on the carbon-controlled regulation of the MEL] and GAL cluster genes. The null mutation was achieved in vivo by replacing the chromosomal wild-type GAL80 allele with an in vitro-created GAL80 deletion-disruption mutation. Enzyme activities and RNA levels for the GAL cluster and MEL] genes were constitutively expressed in the null mutant strain grown on glycerol-lactate and were higher than in the isogenic wild-type yeast strain when compared after growth on galactose. Carbon catabolite repression of the GAL cluster and MEL] genes, which occurs at the level of transcription, is retained in the null mutant. Deletion of the GAL80 gene in a gal4 cell does not restore GAL cluster and MEL) gene expression. The data demonstrate that (i) the GAL80 protein is a purely negative regulator, (ii) the GAL80 protein does not mediate carbon catabolite repression, and (iii) the GAL4 protein is not simply an antagonizer of GAL80-mediated repression.At least three genetically defined regulatory genes (GAL4, GAL80, and GAL3) control the galactose-inducible expression of five structural genes which code for enzymes required for galactose or melibiose catabolism. The appearance of the enzyme activities specified by the MELI (axgalactosidase), GAL2 (galactose permease), and the tightly linked GAL cluster genes, GAL] (galactokinase), GAL7 (Gal-i-P uridyl transferase), and GALIO (UDPG-4-epimerase) is coordinately controlled (3,4,7,16). These structural genes comprise separate transcriptional units (13, 30). Considerable genetic evidence indicates that the GAL4 and GAL80 regulatory genes function via diffusable proteins (8,17,20,21) to activate or inactivate coordinately the transcription of the structural genes (12,27,30).The GAL4 protein has been assigned a positive regulatory function based on genetic data (6). The GAL3 function is necessary for normal rapid induction; gal3 mutants exhibit slower induction and a prolonged induction lag (33) which occurs at transcription (T. E. Torchia and J. E. Hopper, manuscript in preparation).The GAL80 protein is thought to be necessary to prevent structural gene expression in the absence of galactose, as suggested by the constitutive phenotype of cells with recessive gal80 mutations and the noninducible phenotype of cells with dominant GAL80S mutations (6,8,25 tein may be multifunctional with some required role in gene expression. Although cells bearing a recessive gal80 nonsense mutation are constitutive, it is not known whether this mutation occurs early or late in the protein sequence (8). Defined deletion mutations in GAL80, which could be used to resolve the role of the GAL80 protein, have not been studied.In addition to galactose control, this system is repressed by glucose (1). The role of the regulatory proteins in glucose ...

GAL3 gene expression is required for rapid GAL4-mediated galactose induction of the galactose-melibiose regulon genes in Saccharomyces cerevisiae. Here we show by Northern (RNA) blot analysis that GAL3 gene expression is itself galactose inducible. Like the GALl, GAL7, GALIO, and MEL] genes, the GAL3 gene is severely glucose repressed. Like the MEL) gene, but in contrast to the GALl, GAL7, and GALIO genes, GAL3 is expressed at readily detectable basal levels in cells grown in noninducing, nonrepressing media. We determined the sequence of the S. cerevisiae GAL3 gene and its 5'-noncoding region. Within the 5'-noncoding region of the GAL3 gene, we found two sequences similar to the UASGa,, elements of the other galactosemelibiose regulon genes. Deletion analysis indicated that only the most ATG proximal of these sequences is required for GAL3 expression. The coding region of GAL3 consists of a 1,275-base-pair open reading frame in the direction of transcription. A comparison of the deduced 425-amino-acid sequence with the protein data bank revealed three regions of striking similarity between the GAL3 protein and the GALl-specified galactokinase of Saceharomyces carlsbergensis. One of these regions also showed striking similarity to sequences within the galactokinase protein of Escherichia coli. On the basis of these protein sequence similarities, we propose that the GAL3 protein binds a molecule identical to or structurally related to one of the substrates or products of the galactokinase-catalyzed reaction.

We have used deoxyribonuclease I (DNase I) and methidium-propyl-EDTA.Fe(II) digestion to characterize the chromosomal structure of the single-copy autonomously replicating sequence ARS1. The major feature of this chromatin is a region of strong hypersensitivity to both cleavage agents. The hypersensitive region contains most of the DNA sequences which have been suggested by in vitro mutagenesis studies [Celniker, S., Sweder, K., Srienc, F., Bailey, J., & Campbell, J. (1984) Mol. Cell. Biol. 4, 2455-2466] to be important in ARS function. It lies at the downstream end of the TRP1 gene. A chromosomal DNase I footprinting analysis was carried out on the hypersensitive region. These data give direct evidence for several localized DNA/protein contacts within the hypersensitive region. The most prominent of these chromatin-dependent contacts is located on the functionally most important 11 base pairs of ARS DNA. On the TRP1 side of the hypersensitive region, there are positioned nucleosomes. On the other side of the hypersensitive region, there is a complex (and possibly heterogeneous) structure.

During the galactose adaptation period of a Saccharomyces cerevisiae strain bearing a naturally occurring gal3 allele, we found a longer induction lag and slower rate of accumulation of GAL10 and MEL1 RNAs compared to wild-type strains. A strain of genotype gal3 gal1 gal7 is noninducible for MEL1 gene expression, but this expression block is bypassed by overexpression of the GAL4 gene or by deletion of the GAL80 gene, either of which causes a constitutive phenotype. An otherwise wild-type strain that bears a chromosomal gal3 gene disruption mutation does not produce wild-type GAL3 RNA and exhibits induction comparable to a strain bearing the naturally occurring gal3. Based on this array of results, we conclude that the GAL3 gene product executes its function at a point before GAL4 mediated transcription of the GAL1-10-7 and MEL1 genes. Thus, the data are consistent with the previously advanced hypothesis that the GAL3 gene product functions to synthesize the inducer or coinducer molecule. In experiments in which the presence of either the plasmid-carried cloned GAL3 gene or the plasmid-carried cloned GAL1-10-7 genes allows MEL1 induction of a gal3 gal1 gal7 cell, we find that loss of the plasmid results in the shutoff of MEL1 expression even when galactose is continuously present. Either GAL3 function or GAL1-10-7 functions are therefore required for both the initiation and the maintenance of the induced state. Since the strains bearing either the naturally occurring gal3 allele or the gal3 disruption (null) allele do induce, the plasmid loss experiments indicate the existence of two completely independent induction initiation-maintenance pathways, one requiring GAL3 function, the other requiring GAL1-10-7 function. Finally, Northern blot analysis reveals two major GAL3 transcripts that differ in size by approximately 500 nucleotides.