Stasyk O. Identification of the GCR1 gene that controls catabolite repression in the methylotrophic yeast Hansenula polymorpha

3. The object: phenomenon and molecular mechanisms of catabolite repression in the methylotrophic yeasts. The aim: to investigate mutants of yeast H. polymorpha deficient in catabolite repression and to determine molecular nature of the corresponding mutations. The methods: methods of genetic analysis, biochemical: determination of enzymatic activities, of glycolitic intermediates concentrations, concentrations of nutrient sources; molecular-genetic: PCR, methods of recombinant DNAs; Western blotting; methods of ultrastructural studies: electron microscopy, immunocytochemistry, fluorescent microscopy; methods of computer analysis. It was demonstrated that identified GCR1 gene encodes for a protein homologous to glucose transporters. It participates in the mechanism of glucose catabolite repression. Functional role of the conserved serine-85 residue in Gcr1 sequence was demonstrated. Its substitution leads to a modification of Gcr1 function. Strain deleted in the GCR1 gene has been constructed. For thefirst time, mutants of H. polymorpha capable to constitutive peroxisome biogenesis in glucose medium were described. It was demonstruted that disaccharides have differnet from glucose-triggered signalling mechanism for repression in H. polymorpha. The phenomenon of "reverse diauxy" was established. The system for fluorescent labeling of peroxisomes in H. polymorpha was developed. Field of application: cell biology, biotechnology.