Selection and characterization of nuclear genes coding mitochondrial proteins: Genetic complementation of yeast pet mutants

This chapter focuses on the selection and characterization of nuclear genes coding mitochondrial proteins. The ability to analyze yeast genes at the molecular level has increased markedly with the development of recombinant DNA technology and yeast transformation methods. The chapter describes the procedures used for the preparation of a yeast library encompassing the entire yeast genome and a two-stage screening procedure for the selection of a defined PET gene from this library of genomic DNA. A procedure is described to define and characterize rapidly the mitochondrial protein encoded by the selected PET gene. The yeast Escherichia coli shuttle vector YEp13 is used for the construction of a yeast genomic library. This vector consists of the entire bacterial plasmid pBR322, the LEU2 gene of yeast, and the origin of replication from the yeast 2μ circle DNA. There are several methods to confirm the location of the functional PET gene on the transforming vehicle. The most reliable of these is the demonstration of high-efficiency cotransformation of the Leu⁺ and Pet⁺ alleles. Plasmid DNA is first prepared from selected transformants using a modification of a small-scale isolation procedure for bacteriophage DNA.