Identification and cloning of a yeast nuclear gene (CBP1) involved in expression of mitochondrial cytochrome b.

Abstract

Nuclear pet mutants of Saccharomyces cerevisiae deficient in mitochondrial respiration were studied genetically and biochemically. Seven noncomplementing mutations leading to a deficiency of mitochondrial cytochrome b were assigned to a single complementation group (group 60). Examination of mitochondrial RNA by blot hybridization on diazobenzyloxymethyl-paper has revealed that group 60 mutants produce a large number of novel apocytochrome b transcripts not detected in wild-type yeast. The product of the gene affected in the mutants, therefore, appears to be required either for correct transcription or for processing of apocytochrome b premessenger RNA. The gene was designated CBP1. A representative mutant from complementation group 60 (N5-26) was transformed to respiratory competency with a recombinant plasmid pool consisting of random fragments of wild-type yeast nuclear DNA inserted into a vector capable of replicating in yeast and Escherichia coli. The complementation of the N5-26 mutation was shown for a number of independent transformants to be due to the presence of plasmid DNA. The plasmid pG60/T10 was further characterized to have a nuclear DNA insert of 6.7 kilobase pairs. This plasmid complements the mutations of all group 60 mutants, thus confirming that it contains the CBP1 gene.