Plasmids can stably transform yeast mitochondria lacking endogenous mtDNA.
The mitochondrial gene oxi1, carried on a bacterial plasmid, has been used to transform the mitochondria of a yeast strain lacking mtDNA (rho0). 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 oxi1 sequences in the transformants were surrounded by restriction sites derived from the plasmid that were not present in wild-type mtDNA. oxi1 genetic information in these "synthetic rho-" strains could be expressed in diploids either after "marker rescue" by recombination with rho+ mtDNA carrying an appropriate oxi1 point mutation or in trans during the growth of diploids heteroplasmic for both the plasmid-derived oxi1 sequences and rho+ mtDNA with oxi1 deleted. The ability to generate such "synthetic rho-" strains by transformation will allow transfer of mutations generated in vitro to wild-type rho+ mtDNA as well as examination of the function of altered genes in trans.
Fox
T D
TD
Section of Genetics and Development, Cornell University, Ithaca, NY 14853.
Sanford
J C
JC
McMullin
T W
TW
D004272:DNA, Mitochondrial / Q000172:deficiency*; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D010957:Plasmids; D012234:Rho Factor; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014170:Transformation, Genetic
D004272:DNA, Mitochondrial; D012234:Rho Factor; D019003:Deoxyribonuclease HpaII; C024388:CTGCAG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific