| Thiamine pyrophosphate (TPP) is a cofactor for several important enzymes like pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase. TPP is synthesized from two essential moieties, the pyrimidine moiety and the thiazole moiety, which are themselves synthesized by two separate branches. There have been many studies on the regulation of thiamine biosynthesis in bacteria and yeast. Recently, it has been found that many genes involved in the biosynthesis of cofactors such as TPP are regulated by riboswitches, a sequence in the mRNA to which the cofactor binds, altering the secondary structure of the transcript and thereby influencing expression of the gene. A study in the model green alga Chlamydomonas reinhardtii suggested that thiamine biosynthesis in C. reinhardtii can be effectively regulated at physiological concentrations of the vitamin. It is found that there are two genes in C. reinhardtii, THI4 and THIC, which contain riboswitches. The transcripts of these genes are found to be alternatively spliced when thiamine is added to the cultures, apparently by the presence of the riboswitches in the introns. In the pyr1 mutant of C. reinhardtii, there is a mutation in the THI4 riboswitch so that the alternative splicing does not occur. The effect of pyrithiamine addition on the expression of the THI genes in wild type and pyr1 mutant cells has been investigated. In addition, site directed mutagenesis will be carried out to determine if alteration of the riboswitch sequence interferes with alternative splicing activity.
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