The <i>LPD1</i> gene of <i>Saccharomyces cerevisiae</i> encoding lipoamide dehydrogenase (LPDH) has been shown to be subject to the general control of amino acid biosynthesis mediated via the <i>CGN4</i> gene product. It is subject to catabolite repression and was shown to require the <i>HAP2, HAP3</i> andf <i>HAP4</i> 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 <i>LPD1-lacZ</i> gene fusion integrated at the <i>ura3</i> locus. These <i>LPD1-lacZ</i> fusions differed in the amount of <i>LPD1</i> gene fused to the <i>lacZ</i> reporter. Comparison of the β-galactosidase activities of each strain during growth on glucose or ethanol revealed that part of the <i>LPD1</i> 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 <i>LPD1</i> 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 <i>LPD1</i> 3' enhancer function. The <i>LPD1</i> gene promoter contains three motifs which show strong homology to the core HAP2/3/4 binding motif. LPDH activities in wild-type and <i>hap2</i> 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 <i>hap2</i> mutant grown in similar conditions. Transcript analysis in wild-type and <i>hap2</i> mutants confirmed that the HAP2 protein regulates <i>LPD1</i> expression at the level of transcription in the same way as the <i>CYC1</i> gene. Similar studies (performed by others) comparing LPDH activities and <i>LPD1</i> gene transcription (assessed by constructing <i>hap</i> mutant strains carrying a single copy of the <i>LPD1</i> promoter fused in frame to the <i>lacZ</i> reporter gene integrated at the <i>ura3</i> locus) indicated that transcription of <i>LPD1</i> required HASP2, HAP3 and HPA4 for derepression on non-fermentative substrates. The <i>LPD1</i> gene promoter contains three matches to the consensus for control mediated by the GCN4 protein. Gel retardation analysis (performed by others) using <i>in-vitro</i> 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 <i>gcn4</i> mutant strain. Transcript analysis indicated that amino acid starvation affected levels of the <i>LPD1</i> 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 <i>HIS3</i> gene transcripts which reached peak levels betwen 30 min and 1 h, and then declined gradually.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:664209 |
| Date | January 1991 |
| Creators | Zaman, Zafar |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/11671 |
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