Date of Award

8-7-2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Lambeth, David O.

Abstract

Succinyl-CoA synthetase (SCS) in eukaryotes is an alpha-beta heterodimer with the nucleotide specificity residing within the beta subunit. It was previously believed that animals have the GTP-specific isoform, while plants and bacteria possess the ATP-specific isoform. However, recent work demonstrated that both isoforms of SCS exist in mammals, and the cDNA sequences for the GTP and ATP-specific beta subunits have been determined for mouse and human. The complete genomic sequence for any of the subunits of SCS in a eukaryotic organism had not been elucidated. The promoter structures of the SCS subunits, as well as the relative expression of the genes in mouse and both human adult and fetal tissues, should provide insight into understanding tissue distribution and regulation of enzyme activity. The 5′ untranslated regions (UTRs) of the mouse A-beta and G-beta sequences were determined by 5′ RACE (rapid amplification of 5′ cDNA ends). Once the sequences for the 5′ UTRs were acquired, the upstream genomic sequences were determined using the Genome Walking kit by Clontech. This kit has five mouse genomic libraries digested by five different restriction endonucleases, with adaptor molecules ligated to the 5′ ends. Nested gene-specific primers located in the coding region and adaptor primers were used to amplify upstream genomic sequences for the mouse A-beta, G-beta and alpha sequences. Dual luciferase assays were used to determine promoter activities of the upstream sequences. In addition, northern blots were used to determine relative amounts of A-beta, G-beta and alpha SCS transcripts in eight different mouse tissues. Real-time RT-PCR was used to determine the relative amounts of A-beta, G-beta and alpha SCS transcripts in human adult and fetal tissues. The upstream sequences for the A-beta and alpha SCS genes were shown to act as promoters in the dual luciferase assay. However, the G-beta upstream sequence obtained had very weak promoter activity, and was later found to be the upstream sequence of a pseudogene. Northern blots showed that the A-beta SCS gene produced had two transcripts; the smaller transcript is a result of alternative polyadenylation. Real-time PCR showed that A-beta and G-beta are expressed in all fetal and adult tissues examined, but their relative levels vary.

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