35] Transformation of nuclear respiratory deficient mutants of yeast

Abstract

This chapter focuses on the transformation of the nuclear respiratory deficient mutants of yeast. The chapter describes the methods for (1) transformation of pet mutants with a plasmid pool and the isolation of plasmid(s) carrying the wild-type genes complementing the mutations; (2) transfer of the selected plasmid into Escherichia coli to amplify and purify the wild-type DNA insert for restriction analyses and sequencing; and (3) transformation of the pet mutant with a plasmid preparation from E. coli to confirm the presence of the wild-type gene copy in the insert of the isolated plasmid. The vector CV13 plasmid (YEp 13) has three components: pBR322, Eco RI fragment, and a Pst I. The Pst I fragment of nuclear yeast DNA contains the LEU2 gene that confers a means of positive selection for leu2 cells acquiring plasmid during transformation. To complete the identification of the CV13 plasmid isolated in E. coli as the carrier of the wild-type gene complementing a yeast nuclear mutation, a small-scale E. coli DNA preparation is used to transform the original pet mutant.