No abstract

The initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mitochondrial DNA have been investigated by mapping experiments utilizing in vitro "capped" mitochondrial RNA molecules or nascent RNA chains. Mitochondrial poly(A)-containing RNA molecules were labeled at their 5' ends with [a-32P]GTP and guanylyltransferase ("capping" enzyme) and mapped on the mitochondrial genome by DNA transfer hybridization and S1 nuclease protection experiments. A mapping site for the capped 5' ends was found on the H strand very near to the 5' terminus of the 12S rRNA gene, and another site was found on the L strand very near to the 5' terminus of the 7S RNA coding sequence. In parallel experiments, the 5' ends of the nascent chains isolated from mitochondrial DNA transcription complexes were similarly mapped very near to the 5' termini of the 12S rRNA gene and of the 7S RNA coding sequence. The in vitro capped RNA molecules and the nascent chains thus presumably identify the same transcriptional initiation sites on the H strand and the L strand. The occurrence of a second possible initiation site for H-strand transcription 90-110 nucleotides upstream of that described above-i.e., 20-40 nucleotides upstream of the tRNAPhe gene-had been previously indicated by a mapping analysis of the nascent RNA chains and has been confirmed in the present work. The presence of two initiation sites for H-strand transcription can be correlated with other types of evidence that point to two different transcription events leading to the synthesis of a polycistronic molecule corresponding to the almost entire H strand and to the synthesis of the rRNA species.Previous studies on mtDNA transcription in HeLa cells have shown that both strands are completely or almost completely transcribed (1,2). This conclusion has been recently confirmed for the heavy (H) strand by the observation that the discrete transcripts of this strand form a continuum extending over its entire length, with the exception of a relatively small region from coordinate 98/100 to coordinate 5/100 [relative to the origin ofreplication taken as 0/100 (3-5) (Fig. 1)]. In the present work, the initiation sites for heavy (H) and light (L) strand transcription in HeLa cell mtDNA have been investigated by an approach that has been used successfully for identifying the transcriptional initiation sites in yeast mtDNA (8,9). In particular, the 5' ends oftotal poly(A)-containing mitochondrial RNA possessing a di-or triphosphate, and therefore presumably resulting from transcriptional initiation, have been identified by taking advantage of the capacity of the guanylyltransferase ("capping" enzyme) to transfer GMP from GTP to di-or triphosphate-terminated polynucleotides (10- (6) The publication costs ofthis article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisenent" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Actinomycin inhibits DNA-dependent RNA synthesis of intact cells' and enzymes from mammalian2 and bacterial' sources but not the growth of RNA-viruses in

The phospholipid composition of various strains of the yeast, Saccharomyces cerevisiae, and several of their derived mitochondrial mutants grown under conditions designed to induce variations in the complement of mitochondrial membranes has been examined . Wild type and petite (cytoplasmic respiratory deficient) yeasts were fractionated into various subcellular fractions, which were monitored by electron microscopy and analyzed for cytochrome oxidase (in wild type) and phospholipid composition . 90% or more of the phospholipid, cardiolipin was found in the mitochondrial membranes of wild type and petite yeast . Cardiolipin content differed markedly under various growth conditions . Stationary yeast grown in glucose had better developed mitochondria and more cardiolipin than repressed log phase yeast . Aerobic yeast contained more cardiolipin than anaerobic yeast . Respiration-deficient cytoplasmic mitochondrial mutants, both suppressive and neutral, contained less cardiolipin than corresponding wild types . A chromosomal mutant lacking respiratory function had normal cardiolipin content . Log phase cells grown in galactose and lactate, which do not readily repress the development of mitochondrial membranes, contained as much cardiolipin as stationary phase cells grown in glucose . Cytoplasmic mitochondrial mutants respond to changes in the glucose concentration of the growth medium by variations in their cardiolipin content in the same way as wild type yeast does under similar growth conditions. It is concluded that cardiolipin content of yeast is correlated with, and is a good indicator of, the state of development of mitochondrial membrane .