Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236158
Title: Regulation of the gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae
Author: Ross, Joseph
ISNI:       0000 0001 2434 7955
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1989
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Abstract:
Lipoamide dehydrogenase is a component of the multienzyme complexes pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase in Saccharomyces cerevisiae, A mutant, Ipdl, lacking pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and lipoamide dehydrogenase activity had previously been isolated in yeast. In addition, this mutant had been used to screen a YEplS based yeast gene bank leading to the isolation of a 5,5 kb region of yeast DIA containing a gene, designated LFD, which complements the mutation, In this thesis the DIIA sequence of 2.7 kb of the 5,5 kb region is presented. The sequenced region contains a 1.5 kb open reading frame representing the LFD gene. From homology between the deduced amino acid sequence of this open reading frame and the primary sequence of E. coli and pig heart lipoamide dehydrogenase, the LFD gene has been shown to represent the structural gene for lipoamide dehydrogenase. The primary sequence encoded by the LPD gene also shows very strong homology to several other related amino acid sequences including glutathione reductase, mercuric reductase and the lipoamide dehydrogenase sequence from A, vinlandii and human liver cells. Analysis of the upstream region of the LFD gene led to the identification of several sites of homology between sequences within this region and known yeast regulatory motifs. These included three sites for the binding of the GCI4 protein, a sequence very similar to the UAS2 of CYC1, and three regions similar to the sequence TCACGTGA identified as an important element within the promoter of the TFF1 and GAL2 genes in S, cerevisiae, in the adenovirus major late promoter and as the binding site of the centromere-binding protein, CPI. Transcript analysis of LFD expression during conditions of amino acid, starvation was carried out. In addition, gel retardation and BUasel protection experiments, to investigate interactions between in vitro synthesised GCI4 and the upstream region of the LFD gene, were performed. The results from these experiments suggest but do not conclusively prove that the LFD gene is subject to general amino acid control. In an attempt to identify other DIA-binding proteins which interact specifically with the LPD gene, protein fractions from an heparinSepharose column were assayed by gel retardation for binding to DIA fragments from the 5' end of the gene. Several DIA-binding activities were identified including a DIA-binding protein which binds regions upstream and downstream of the translation start site. This DIA-binding activity could be competed by the addition of increasing amounts of a ligated oligonucleotide containing two copies of the TCACGTGA sequence. The specificity and the regulatory role of the proteins identified as binding to the 5' end of the LFD gene remain to be determined. In this thesis a preliminary analysis of the elements involved the regulation of this gene has been carried out and future directions, in the study of transcriptional control of the LFD gene, discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.236158  DOI: Not available
Keywords: Genetics
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