Transcription of yeast mitochondrial deoxyribonucleic acid

The transcription of yeast mitochondrial DNA (mtDNA) was examined by analysis of the kinetics of hybridization of mitochondrial RNA (mtRNA) with highly labeled mtDNA. The RNA was purified by guanidium chloride-CsCl equilibrium centrifugation, and the DNA was labeled in vitro by nick translation. Hybridization levels at apparent saturation were consistently about 35%. After preannealing of the RNA, it was found that approximately 30% hybridication remained. Hybridization levels were 10 to 15% at a R/sub 0/t (thr product of RNA concentration and time) of 1 mol s L/sup -1/ and reached apparent saturation at R/sub 0/t values of 10/sup 2/ to 10/sup 3/ mol sL/sup -1/. Hybridization with a restriction fragment containing sequences of 21S rRNA also reached an apparent saturation level of about 35%, but hybridization was elevated at low R/sub 0/t values (10/sup -1/ mol s L/sup -1/). Hybridization with separated strands of cloned EcoRI fragment 7-pMB9 recombinant DNA molecules was carried out. One DNA strand hybridized to approx. 42% and the other hybridized only to 2%. When a correction for pMB9 sequences was made, more than 95% of one mtDNA strand was transcribed. There is extensive (at least 60%) transcription of one strand-equivalent of yeast mtDNA. The extent of transciption and the frequency distribution of total mtRNA populations were also evaluated in yeast grown anaerobically. The results could not be distinguished from those obtained in yeast grown under aerobic conditions. Finally, sequence differences between mitochondrial poly(A+) and poly(A-) RNA were investigated. the hybridization profiles were similar, except for the absence of an initial low level of hybridization at low R/sub 0/t values for poly(A+) RNA.