nmt1 of fission yeast. A highly transcribed gene completely repressed by thiamine

Abstract

The first fully repressible gene in fission yeast is described. In minimal medium it is highly transcribed producing a mRNA which is 50-100 times more abundant than the cyc1 mRNA. By contrast, in minimal medium supplemented with thiamine at a concentration of 0.5 microM or greater the transcript is undetectable. The gene has been called nmt1 (for no message in thiamine). The 5' and 3' ends of the transcript have been mapped and indicate an unspliced mRNA of 1.3 kilobases with no evidence of heterogeneity at either end. The single major open reading frame encodes a protein of 39 kDa. The gene product is most likely involved in thiamine biosynthesis and consistent with this is the observation that the nmt1::ura4 disruption strain is a thiamine auxotroph. The kinetics of transcriptional repression and induction have been studied. Addition of thiamine to log phase cells growing minimal medium results in complete disappearance of the nmt1 message within 3 h. Removal of thiamine from the medium produces the first detectable message after 10 h and maximal steady-state levels after 16 h. Nuclear "run on" experiments demonstrate that control is exerted at the level of transcription initiation. The nmt1 promotor has been subcloned, and thiamine-mediated transcriptional control has been transferred to the bacterial reporter gene chloramphenicol acetyltransferase.