Transcriptional regulation of GCV2 expression in Saccharomyces cerevisiae [thesis]

Nadia Minarovic
2010
Cellular metabolism must be regulated in order for a cell to conserve energy, and also maintain an appropriate level of biosynthesis in response to nutrients available in the environment. GCV2 encodes a subunit of the Saccharomyces cerevisiae glycine decarboxylase complex, a multi-enzyme complex that is involved in one-carbon metabolism. Preliminary studies indicated that two sequences in the GCV2 promoter might be regulated according to nutritional cues. One of the sequences, GATAAG, is an
more » ... blished binding site for transcription factors that mediate regulation in response to nitrogen quality. The second sequence is a palindrome, AAGGACCTT, which has been implicated in GCV2 regulation according to rich medium. A mutation analysis of the S. cerevisiae GVC2 palindrome sequence utilising lacZ expression vectors was undertaken in order to determine how the palindrome might regulate GCV2 expression when cells are grown in a rich medium. Depending on the mutation, both positive and negative changes in GCV2 expression were observed. A combined mutation of the palindrome and GATA sequences resulted in a decrease in GCV2 expression in rich and poor nutritional conditions. These results demonstrated that the palindrome was involved in regulating GCV2 in an auxiliary manner, rather than acting as an independent regulatory element. Expression analysis of GCV2 under a range of nitrogen sources indicated that transcription of the gene was under the control of nitrogen catabolite repression, which regulates genes according to nitrogen quality. A decrease in GCV2 expression when each of the four transcription factors that are known to mediate nitrogen regulation were deleted indicated that all four were involved in GCV2 regulation. A bioinformatics analysis of the yeast genome to identify genes that contained a similar promoter structure to GCV2 identified eleven genes or unannotated open reading frames. The expression of a selection of these genes was assayed in rich medium using quantitative PCR to determine if they were al [...]
doi:10.26190/unsworks/23575 fatcat:ske4yd5pancznenzfnvzaks2k4