A protocol is introduced for probing the organization and regulation of expression of the yeast mitochondrial genome, termed "zygotic gene rescue." The procedure is based on the notion that genes retained on mitochondrial DNA of petites can be expressed in zygotes of a cross between petite and wild type. To test the validity of this notion, we have taken advantage of our ability to discriminate, by mobility differences on sodium dodecyl sulfate/polyacrylamide gels, different forms of the product of alleles of the mitochondrial gene, varl. In petite strains that have retained the varl gene, its characteristic product appears in zygotes 4-5 hr after mating; no product is observed in petite strains deleted in the varl locus. Our studies indicate that (i) expression in the zygote of the varl gene in the petite genome is not exclusively the result of recombination with mitochondrial DNA of the wild-type tester, and (ii) the var) gene is probably reiterated in the petite mitochondrial genome. The strength of the technique of zygotic gene rescue in the analysis of the mitochondrial genome is discussed.Recent advances in genetic and physical mapping of the yeast mitochondrial genome have provided considerable information on the identification and localization of a number of mitochondrial genes. Among these are included drug resistance loci (1-3), genes specifying rRNAs (4-6) and tRNAs (7-9), and a variety of loci termed mit (10, 11). The latter, when mutated, lead to an inability of the strain to grow on nonfermentable carbon sources as a result of specific alterations in cytochrome oxidase, the cytochrome be1 complex, or the oligomycin-sensitive ATPase complex (12). These complexes are composed of nuclear encoded, cytoplasmically synthesized subunits and subunits that are translated on mitochondrial ribosomes and generally believed to be encoded by mitochondrial DNA (mtDNA). It is of obvious interest to know the precise location of sequences on the yeast mitochondrial genome corresponding to the structural genes for these and other, less well characterized gene products of mtDNA.In this communication we introduce a procedure for probing the organization of the yeast mitochondrial genome. This procedure, termed "zygotic gene rescue," is based on the premise that the mtDNA of yeast cells carrying the vegetative petite mutation (p-) is senseful and could, in principle, express structural gene sequences in tlvo given a functional transcriptional and translational apparatus.It is now well documented that petites are the result of extensive deletions of wild-type mtDNA sequences (13-s15) and, moreover, that individual petite isolates can retain any part of the mitochondrial genome (16, 17), most probably in a geneamplified configuration (18)(19)(20) (22) and mit-loci (11). What is intrinsically uncertain in such analyses, however, is whether or not the particular allele localized by the phenotype measured represents the structural gene sequence for a specified polypeptide product. We reasoned that it might be possibl...