Cloning of yeast mitochondrial DNA in the Escherichia coli plasmid pBR322. Identification of tRNA genes.

Fragments of mitochondrial DNA from Saccharomyces cerevisiae were joined to the Escherichia coli plasmid pBR322 by the poly(dA):poly(dT) tailing method. A total of 347 transformants containing DNA inserts were isolated. The size of the inserted DNA ranges from 0.5 to 5 kilobases. To screen for mitochondrial tRNA genes, we isolated mitochondrial tRNAs, end-labeled them with 32P, and hybridized them to the transformed E. coli clones affixed to nitrocellulose filters. Mitochondrial tRNA hybridized to the DNA of 62 of the transformants (16% of the total). As a first step toward correlating the cloned mitochondrial tRNA genes with their gene products, we took advantage of previous work that mapped tRNA genes with respect to antibiotic resistance markers through the use of the petite deletion mapping technique. Petite DNAs known to carry chloramphenicol (C), erythromycin (E), paramomycin (P), or oligomycin (01) markers, as well as a limited subset of mitochondrial tRNA genes, were nick-translated and used as hybridization probes. In this way, we were able to map the cloned sequences with respect to C, E, P, or 01 markers and predict which tRNA genes they were most likely to carry. Recombinants corresponding to the 01 region of the mtDNA were screened with mitochondrial seryl-tRNA. The tRNAS”’ gene is contained in a Hpa II fragment of 320 base pairs.