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, we found that approximately 30% hybridization remained. Hybridization levels were 10-15% at a R0t (the product of RNA concentration and time) of 1 mol s L-1 and reached apparent saturation at R0t values of 102-103 mol s L-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 R0t values (10-1 mol s L-1). We also carried out hybridization with separated strands of cloned EcoRI fragment 7-pMB9 recombinant DNA molecules. One DNA strand hybridized to ⋍ 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. We conclude that there is extensive (at least 60%) transcription of one strand-equivalent of yeast mtDNA. The extent of transcription 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 R0t values for poly(A+) RNA. © 1979, American Chemical Society. All rights reserved.