Recombinational analysis of oxi2 mutants and preliminary analysis of their translation products in S. cerevisiae

Abstract

Genetic and biochemical studies were performed with mutants allocated to the mitochondrial oxi2 gene.Recombinational analysis of 19 oxi2 mutants was performed using α and a mutant strains derived from the same genetic background. The frequencies of wild-type recombinants in oxi2 (-) × oxi2 (-) crosses varied from 0.002 to 17%. The map of oxi2 mutations constructed on the basis of these frequencies shows many internal inconsistencies. In the course of rho (-) deletion mapping five classes of oxi2 mutations were distinguished. The results of deletion analysis are in agreement with those of recombinational mapping.The analysis of mitochondrial translation products by SDS-polyacrylamide electrophoresis of 20 oxi2 mutants shows that 17 of them are connected with conspicuous changes of 22 kd polypeptide band corresponding to subunit III of cytochrome oxidase. At least four of them carried instead of subunit III clearly visible significantly shorter polypeptides (12.8 to 20.1 kd). These were, most likely, shorter fragments of subunit III resulting from chain termination mutations. Colinearity was observed between the lenght of new polypeptides and the positions of the respective mutations on the recombinational map. These data confirm hat oxi2 encodes subunit III of cytochrome oxidase and suggest that translation of the oxi2 gene is in the direction from V303 to V273.