| Summary: | This study has examined the regulation of thiamine biosynthesis in the bakers' yeast Saccharomyces cerevisiae. To facilitate this, promoters have been recovered from TH15 and TH112 and fused in-frame with lacZ reporter gene on a low copy plasmid vector. Along with a previously constructed TH14-lacZ translational fusion, these constructs enabled TH14, TH15 and TH112 expression to be monitored by assaying for -galactosidase activity under defined concentrations of thiamine. It has been shown that TH14, TH15 and TH112 expression is controlled by the intracellular concentration of the end product of thiamine biosynthesis. thiamine pyrophosphate (TPP). Transcriptional activation of TH14, TH15 and TH112 has been shown to be dependent upon the activator genes TH12 and TH13; additional work in this laboratory has shown that expression of these genes is also dependent upon PDC2. The analysis of TH112-lacZ expression also provided the first direct evidence that a second member of the TH15-like gene family was transcriptionally active. A successful screen was developed for the isolation of a new class of mutant strain that displayed derepressed expression of a TH14-lacZ reporter gene in medium containing thiamine. Eight single gene det mutants were isolated, of which five were due to recessive mutations and three to partially dominant mutations. Phenotypic analysis was carried out on the three partially dominant det mutants and one recessive det mutant, as these strains displayed derepressed TH14-lacZ expression levels comparable to a wild-type DET+ strain grown in the absence of thiamine (inducing conditions). This showed that the det phenotype was not caused by an inability to synthesise or transport thiamine, and that the mutations affected the expression of all thiamine-regulated genes tested. This latter result supports the theory that thiamine genes are controlled by a common set of regulatory factors. Attempts to clone the corresponding DET alleles were unsuccessful, although it was shown that they were not allelic to TH180 or PDC2. Finally, the gene RPII was identified as a multicopy activator of thiamine genes. This activation was dependent upon TH12, TH13 and PDC2. Analysis of an rpi1::TnHIS3 disruption strain found that it had no thiamine-associated phenotypes, from which it was concluded that RPII has no role in thiamine gene expression when single copy. However it was shown to be homologous to PDC2, due to the presence of asparagine-serine rich regions. Through the production of models of thiamine gene regulation, the method of RPII action has been proposed, and the possible identities of the det genes have been discussed.
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