Expression of petite mitochondrial DNA in vivo: zygotic gene rescue.

Abstract

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...

Location and structure of the var1 gene on yeast mitochondrial DNA: Nucleotide sequence of the 40.0 allele

“…Map of Petite Al 7-10 and Sequencing Strategy The mitochondrial DNA (mtDNA) of petite Al 7-10 was shown by Vincent et al (1980) to be a 5.3 kb headto-tail repeat. From transmission analysis and zygotic gene-rescue analysis (Strausberg and Butow, 1977;Vincent et al, 1980;Lopez et al, 19811, we know that Al 7-l 0 mtDNA contains the entire varl determinant region located within a 2.7 kb fragment corresponding to Hint II band 10 of the wild-type genome. The restriction map of Al 7-l 0 mtDNA has been extended with ten additional enzymes to show fine structure (Hudspeth et al, manuscript in preparation), and a summary of that map for Hint II band 10 is shown in Figure 1.…”

Specific translational activation by nuclear gene products occurs in the 5' untranslated leader of a yeast mitochondrial mRNA.

“…To avoid this problem, we tested transient expression of cytochrome b in zygotes with the desired genotype generated by mass mating, an approach developed by Strausberg and Butow (21 Genetics: Costanzo and Fox to wild-type PET494 by using the cloned gene, and the resulting transformant, MCC67, was also mass-mated with the double mutant, MSUcbsl-2 strain MCC64. Mitochondrial translation products of the mass-mated cells were radioactively labeled and inspected for the presence of cytochrome b (Fig.…”

“…A 4:1 ratio of mated to unmated cells was achieved. Mitochondrial translation products of mated or haploid cells were radioactively labeled and subjected to electrophoresis as described (9,21).…”

Oestrogen Influences on Mitochondrial Gene Expression and Respiratory Chain Activity in Cortical and Mesencephalic Astrocytes

“…To determine whether there would be a correlation between an increased expression of mitochondrial catalytic respiratory chain subunits and mitochondrial respiratory activity by E 2 , we measured the succinate‐stimulated respiration of isolated intact mitochondria from E 2 ‐treated in comparison to control astrocytes from cortex and mesencephalon. After E 2 treatment, cortical and mesencephalic astrocytes were incubated in NBM for an additional 4 h to allow translation of mRNA transcripts and protein targeting into mitochondria (28, 29). Subsequently, intact mitochondria were isolated from E 2 ‐treated and untreated cortical and mesencephalic astroglial cell cultures.…”