Mutations Causing Constitutive Invertase Synthesis in Yeast: Genetic Interactions with snf Mutations

We have selected 210 mutants able to grow on sucrose in the presence of 2-deoxyglucose. We identified recessive mutations in three major complementation groups that cause constitutive (glucose-insensitive) secreted invertase synthesis. Two groups comprise alleles of the previously identified HXK2 and REG1 genes, and the third group was designated cid1 (constitutive invertase derepression). The effect of cid1 on SUC2 expression is mediated by the SUC2 upstream regulatory region, as judged by the constitutive expression of a SUC2-LEU2-lacZ fusion in which the LEU2 promoter is under control of SUC2 upstream sequences. A cid1 mutation also causes glucose-insensitive expression of maltase. The previously isolated constitutive mutation ssn6 is epistatic to cid1, reg1 and hxk2 for very high level constitutive invertase expression. Mutations in SNF genes that prevent derepression of invertase are epistatic to cid1, reg1 and hxk2; we have previously shown that ssn6 has different epistasis relationships with snf mutations. The constitutive mutation tup1 was found to resemble ssn6 in its genetic interactions with snf mutations. These findings suggest that CID1, REG1 and HXK2 are functionally distinct from SSN6 and TUP1.     

The DAL7 promoter consists of multiple elements that cooperatively mediate regulation of the gene''s expression.

Expression of the allantoin system genes in Saccharomyces cerevisiae is induced by allophanate or its analog, oxalurate. This work provides evidence for the involvement of distinct types of cis-acting elements in the induction process. The first element was found to have the properties of an upstream activation sequence (UAS). This element was localized to a 16-base-pair (bp) DNA fragment containing a short 5-bp sequence that occurred repeatedly in the upstream region of DAL7. When present in two or more copies, the 16-bp fragment supported high-level beta-galactosidase production in a CYC1-lacZ expression vector; there was, however, no response to the allantoin pathway inducer. The second element had the properties of a negatively acting element or upstream repression sequence (URS). This element was localized to a 16-bp DNA fragment containing an 8-bp sequence that was repeated four times in the upstream region of DAL7. A fragment containing the 8-bp repeated sequence placed adjacent to the UAS-containing fragment mediated inhibition of the ability of the UAS to support lacZ expression regardless of whether inducer was present. A third element, designated an upstream induction sequence (UIS), was required for response to inducer. The UIS was localized to a small DNA fragment containing an approximately 10-bp sequence that was repeated twice in the upstream region of DAL7. When a fragment containing the 10-bp repeated sequence was placed adjacent to these UAS and URS elements, the construction (UIS-UAS-URS) supported normal oxalurate-mediated induction of beta-galactosidase synthesis. These data are consistent with the suggestion that multiple, cis-acting elements participate in the induction process.     

Identification of a sequence containing the positive regulatory region of Saccharomyces cerevisiae gene ENO1

A DNA fragment which contains the 5'-flanking region of the Saccharomyces cerevisiae enolase 1 gene (ENO1) and a portion of the coding sequence was cloned in a plasmid pMC1587. This fragment was fused in frame to the lacZ gene of Escherichia coli. Many mutants which deleted a portion of the 5'-flanking region of ENO1 were isolated from this ENO1-lacZ fusion plasmid by in vitro recombination. Analysis of beta-galactosidase activity of these mutants indicated that the regulatory region responsible for an efficient expression of the ENO1-lacZ fused gene resides within an 86-bp sequence located at -487 to -402 upstream from the start codon of ENO1. We found that the segment encompassing the 86-bp region worked equally well in an inverted orientation, but the tandem duplication of the sequence did not enhance the expression of the fused gene.     

The secreted form of invertase in Saccharomyces cerevisiae is synthesized from mRNA encoding a signal sequence.

The SUC2 gene of Saccharomyces cerevisiae encodes two differently regulated mRNAs (1.8 and 1.9 kilobases) that differ at their 5' ends. The larger RNA encodes a secreted, glycosylated form of invertase and the smaller RNA encodes an intracellular, nonglycosylated form. We have determined the nucleotide sequence of the amino-terminal coding region of the SUC2 gene and its upstream flanking region and have mapped the 5' ends of the SUC2 mRNAs relative to the DNA sequence. The 1.9-kilobase RNA contains a signal peptide coding sequence and presumably encodes a precursor to secreted invertase. The 1.8-kilobase RNA does not include the complete coding sequence for the signal peptide. The nucleotide sequence data prove that SUC2 is a structural gene for invertase, and translation of the coding information provides the complete amino acid sequence of an S. cerevisiae signal peptide.     

Comparison of two yeast invertase genes: conservation of the upstream regulatory region.

The yeast genome contains a dispersed family of invertase structural genes (SUC1-SUC5, SUC7). Five of these genes are located very close to telomeres and are flanked by large regions of homologous sequence; recombination between telomeres could account for the dispersal of these SUC genes to different chromosomes. The SUC2 locus, in contrast, is not near a telomere and does not share large regions of flanking homology with the other loci. We examine here the relationship between SUC2 and one of the telomeric genes, SUC7. Sequence comparison revealed homology extending from about position -624 to +1791, which is close to the end of the mRNA. The 5' noncoding sequence includes two highly conserved regions: the region between -140 and +1, which contains the TATA box and presumably other promoter elements, and a second region extending from -508 to -400, which corresponds to the upstream regulatory region.     

The Rox1 repressor of the Saccharomyces cerevisiae hypoxic genes is a specific DNA-binding protein with a high-mobility-group motif.

The ROX1 gene encodes a repressor of the hypoxic functions of the yeast Saccharomyces cerevisiae. The DNA sequence of the gene was determined and found to encode a protein of 368 amino acids. The amino-terminal third of the protein contains a high-mobility-group motif characteristic of DNA-binding proteins. To determine whether the Rox1 repressor bound DNA, the gene was expressed in Escherichia coli cells as a fusion to the maltose-binding protein and this fusion was partially purified by amylose affinity chromatography. By using a gel retardation assay, both the fusion protein and Rox1 itself were found to bind specifically to a synthetic 32-bp DNA containing the hypoxic consensus sequence. We assessed the role of the general repressor Ssn6 in ANB1 repression. An ANB1-lacZ fusion was expressed constitutively in an ssn6 deletion strain, and deletion of the Rox1 binding sites in the ANB1 upstream region did not increase the level of derepression, suggesting that Ssn6 exerts its effect through Rox1. Finally, ROX1 was mapped to yeast chromosome XVI, near the ARO7-OSM2 locus.     

Nucleotide sequence of the yeast SUC2 gene for invertase.

The yeast SUC2 gene is a structural gene for both the secreted and intracellular forms of invertase. We have determined the nucleotide sequence of the coding region and the 5' and 3' flanking regions. The coding regions for the signal peptide-containing precursor to secreted invertase and for the intracellular invertase begin at different initiation codons within the SUC2 gene but share the same reading frame. The amino acid sequences predicted for the two forms of invertase from the nucleotide sequence are consistent with the properties of the purified enzymes. Potential sites for glycosylation of the secreted invertase are identified.