ABSTRACT
Mutations in the yeast SNF3 gene affect glucose sensing and snf3 mutants show defective growth on glucose. DNA sequence dependent suppressing elements (DDSEs) are regions located in the promoters of yeast glucose transporter (HXT) genes that when present in high copy suppress the snf3 growth defect. Here we provide evidence that the multicopy DDSE suppression is due to the titration of the Rgt1p transcriptional repressor. The DDSE region from HXT4 was found to function as a UAS sequence rendering a UAS(gal)-less LacZ gene fused to the GAL1 promoter responsive to glucose induction. Expression mediated by the UAS(DDSE) was dependent upon the presence of Snf3p. Expression was elevated to a high level in an rgt1 mutant in the absence of Snf3p suggesting that this DDSE region contains binding sites for the Rgt1p transcriptional repressor/activator. The UAS(DDSE) led to expression in a grr1 mutant background, which confers a defect in inactivation of Rgt1p, as predicted from the model. The presence of tandem repeats of the putative Rgt1p binding site gave results similar to those of the DDSE, suggesting that loss of repression is due to the presence of Rgt1p footprint in the multicopy DDSE.
Subject(s)
Membrane Proteins/genetics , Monosaccharide Transport Proteins/genetics , Promoter Regions, Genetic/genetics , Saccharomyces cerevisiae Proteins , Saccharomyces/genetics , Saccharomyces/metabolism , Signal Transduction , Base Sequence , DNA-Binding Proteins , Gene Deletion , Gene Expression Regulation, Fungal , Glucose/metabolism , Glucose Transport Proteins, Facilitative , Molecular Sequence Data , Monosaccharide Transport Proteins/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Saccharomyces/growth & development , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription FactorsABSTRACT
HXT4, a new member of the hexose transporter (HXT) family in Saccharomyces cerevisiae was identified by its ability to suppress the snf3 mutation in multicopy. Multicopy HXT4 increases both high and low affinity glucose transport in snf3 strains and increases low and high transport in wild-type strains. Characterization of HXT4 led to the discovery of a new class of multicopy suppressors of glucose transport defects: regulatory elements in the promoters of the HXT genes. We have designated these sequences DDSEs (DNA sequence dependent suppressing element). Multicopy HXT4 and DDSEs in the HXT1, HXT2, HXT3 and HXT4 promoters were found to restore growth to snf3 and grr1 strains on low glucose media. The DDSE in the HXT4 promoter was refined to a 340-bp sequence 450 bp upstream of the HXT4 translational start. This region was found to contain an 183-amino acid open reading frame. Extensive analysis indicates that the DNA sequence itself and not the encoded protein is responsible for suppression. The promoters of SNF3 and of other glycolytic genes examined did not suppress snf3 in multicopy. Suppression of snf3 by DDSE is dependent on the presence of either HXT2 or HXT3.
Subject(s)
Carrier Proteins , Genes, Fungal , Glucose/metabolism , Monosaccharide Transport Proteins/metabolism , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/genetics , Suppression, Genetic , Ubiquitin-Protein Ligases , Amino Acid Sequence , Base Sequence , F-Box Proteins , Fungal Proteins/genetics , Fungal Proteins/metabolism , Glucose Transport Proteins, Facilitative , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Monosaccharide Transport Proteins/genetics , Open Reading Frames , Plasmids , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/metabolismABSTRACT
Alcohol dehydrogenase (ADH) and the genes encoding this enzyme have been studied intensively in a broad range of organisms. Little, however, has been reported on ADH in the free-living nematode Caenorhabditis elegans. Extracts of wild-type C. elegans contain ADH activity and display a single band of activity on a native polyacrylamide gel. Reaction rate for alcohol oxidation is more rapid with higher molecular weight alcohols as substrate than with ethanol. Primary alcohols are preferred to secondary alcohols. C. elegans is sensitive to allyl alcohol, a compound that has been used to select for ADH-null mutants of several organisms. Allyl alcohol-resistant mutant strains were selected from ethylmethanesulfonate (EMS)-mutagenized nematode populations. ADH activity was measured in extracts from eight of these strains and was found to be low or nondetectable. These results form a basis for molecular and genetic characterization of ADH expression in C. elegans.
