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Title: Regulation of expression of the LPD1 gene in Saccharomyces cerevisiae
Author: Zaman, Zafar
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1991
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The LPD1 gene of Saccharomyces cerevisiae encoding lipoamide dehydrogenase (LPDH) has been shown to be subject to the general control of amino acid biosynthesis mediated via the CGN4 gene product. It is subject to catabolite repression and was shown to require the HAP2, HAP3 andf HAP4 gene products for release from glucose repression. The gene also appears to contain a carbon source-regulated transcriptional enhancer that lies 3' to the translational start site. A defined set of isogenic yeast strains was constructed in which each strain contained a different LPD1-lacZ gene fusion integrated at the ura3 locus. These LPD1-lacZ fusions differed in the amount of LPD1 gene fused to the lacZ reporter. Comparison of the β-galactosidase activities of each strain during growth on glucose or ethanol revealed that part of the LPD1 coding region activates gene expression in a carbon source dependent manner. This activation occurred at the mRNA level and was not mediated by changes in mRNA stability. The 3' sequence of the LPD1 gene contains motifs homologous to the DNA binding elements of the ABF1 and RAP1 proteins and a sequence homologous to the CDE1 element. These motifs may represent potential candidates for the LPD1 3' enhancer function. The LPD1 gene promoter contains three motifs which show strong homology to the core HAP2/3/4 binding motif. LPDH activities in wild-type and hap2 mutant strains were expressed similarly at basal levels when grown on glucose. However, LPDH activity in the wild-type was derepressed 4-fold in raffinose medium but remained at near basal levels (as seen on glucose) in the hap2 mutant grown in similar conditions. Transcript analysis in wild-type and hap2 mutants confirmed that the HAP2 protein regulates LPD1 expression at the level of transcription in the same way as the CYC1 gene. Similar studies (performed by others) comparing LPDH activities and LPD1 gene transcription (assessed by constructing hap mutant strains carrying a single copy of the LPD1 promoter fused in frame to the lacZ reporter gene integrated at the ura3 locus) indicated that transcription of LPD1 required HASP2, HAP3 and HPA4 for derepression on non-fermentative substrates. The LPD1 gene promoter contains three matches to the consensus for control mediated by the GCN4 protein. Gel retardation analysis (performed by others) using in-vitro synthesized GCN4 protein revealed DNA:GCN4 complexes at two of the consensus motifs. When cells were grown on raffinose as a carbon substrate to partially relieve catabolite repression of the gene, levels of LPDH were derepressed about 2-fold in wild-type cells limited for histidine synthesis by the presence of 3-amino-1,2,4- triazole; this derepression did not occur in a gcn4 mutant strain. Transcript analysis indicated that amino acid starvation affected levels of the LPD1 transcript. Kinetic analysis indicated that subjecting cells to a sudden decrease in the availability of amino acids led to a marked increase in transcript levels within 30min, and that these continued to increase at a slower rate up to 6 hours after imposition of amino acid starvation. This differed from the response of HIS3 gene transcripts which reached peak levels betwen 30 min and 1 h, and then declined gradually.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available