Plasmids can stably transform yeast mitochondria lacking endogenous mtDNA.

Abstract

The mitochondrial gene oxil, carried on a bacterial plasmid, has been used to transform the mitochondria of a yeast strain lacking mtDNA (rhoo). The plasmid DNA behaved in a manner entirely consistent with the known properties of normal yeast rho-mtDNA after its introduction by high-velocity microprojectile bombardment. Like the mtDNA sequences retained in natural rho-strains, the plasmid DNA in the transformants was reiterated into concatemers whose size was indistinguishable from that of wild-type mtDNA. The oxil sequences in the transformants were surrounded by restriction sites derived from the plasmid that were not present in wild-type mtDNA. oxil genetic information in these "synthetic rho"-strains could be expressed in diploids either after "marker rescue" by recombination with rho+ mtDNA carrying an appropriate oxil point mutation or in trans during the growth of diploids heteroplasmic for both the plasmid-derived oxil sequences and rho' mtDNA with oxil deleted. The ability to generate such "synthetic rho-" strains by transformation will allow transfer of mutations generated in (7), where it became incorporated into preexisting organellar DNA (rho+ in the case ofyeast) by recombination.We report here that yeast mitochondria lacking mtDNA can be transformed by bombardment of a rho' strain with microprojectiles carrying a mitochondrial gene, oxil (8-10), on a plasmid. Genetic and molecular analysis of the transformants demonstrates that they exhibit all the expected properties outlined above for a rho-strain carrying reiterated copies of the plasmid. The ability to generate such "synthetic rho-" strains by transformation will now allow transfer of mutations generated in vitro to wild-type rho+ mtDNA as well as examination of the function of altered genes in trans. MATERIALS AND METHODSMedia, Yeast Strains, and Genetic Methods. Minimal medium (SD), complete medium containing glucose (YPD), complete medium containing the nonfermentable carbon sources ethanol and glycerol (YPEG), and standard genetic manipulations were as described (8,11). Key strains and their genotypes are listed in Table 1. TD28rho°was generated by treatment of TD28 (12) with ethidium bromide (13). TF138 and TF140 were progeny of crosses between DAU2 (MATa, ade2, ura3, [rho+]) and the oxil mutants M8-171 and M11-30, respectively (8,14,15). TF145 was similarly derived from a spontaneous oxil deletion isolated in this laboratory. All other strains have been described previously (8,16,17).Plasmids and Transformations. The plasmid pMT36 (18) carries the wild-type oxil gene inserted at the Pst I site of pBR322 (19) after GC-tailing such that the mtDNA insert could be cleaved out by using either the Pst I sites derived from the plasmid or the Msp I (Hpa II) sites derived from mtDNA (8) (see Fig. 1). pCGE137 carries the wild-type URA3 gene and a 1.56-kb fragment containing the origin of replication of the 2-,um plasmid of yeast in pBR322 and has been used previously (20). Equal amounts of these plasmid DNAs were mixed and precipitated o...

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