REGULATION OF THI4 (MOL1), A THIAMINE-BIOSYNTHETIC GENE OF SACCHAROMYCES-CEREVISIAE

THI4, a Saccharomyces cerevisiae gene originally identified as a resul t of transient expression in molasses medium and named MOL1 is regulat ed by thiamine. Using a THI4 promoter-lacZ fusion on a centromeric yea st vector, we have shown that the THI4 is completely repressed through out batch culture by thiamine at a concentration around 1 mu M, but sh ows high level constitutive expression in thiamine-free medium. The tr ansient expression pattern observed in molasses medium can be mimicked by the addition of 0.15 mu M-thiamine to defined minimal medium. Cell s grown in thiamine-free medium have an intracellular thiamine concent ration of around 9 pmol/10(7) cells. A low level (1 mu M) Of exogenous thiamine is completely sequestered from the medium within 30 min; int racellular thiamine concentrations rise rapidly, followed by a gradual decrease as a result of dilution during growth. A saturating extracel lular level of thiamine leads to a maximal intracellular concentration of around 1600 pmol/10(7) cells, at which point the transport system is shut down. After transfer from repressing to non-repressing medium, THI4 becomes induced when the intracellular concentration of thiamine falls to 20 pmol/10(7) cells. A. thi4::URA3 disruption strain is auxo trophic for thiamine, but can grow in the presence of hydroxyethyl thi azole, indicating that the gene product is involved in the biosyntheti c pathway leading to the formation of the thiazole precursor of thiami ne.