Identification of the products of mitochondrial transcription in the walker corcinosarcoma by the use of actinomycin D and ethidium bromide

Abstract

The major RNA species present in the purified mitochondrial fraction of the Walker carcinoma were investigated in order to determine which of them are located in the mitochondria and coded by the organelle DNA. The subcellular distribution of these RNA's and the in vivo sensitivity of the transcription process to selective inhibitors were examined. Among the different species separated by polyacrylamide gel electrophoresis, only the 21 and 16 Se RNA's were found exclusively in the purified mitochondria, approximately Se being the S value estimated from the relative electrophoretic mobility of the RNA. A bifid peak observed in the 16-15 Se region was shown to be an artifact caused by the ribonuclease inhibitor, naphthalene disulfonate. Ethidium bromide at high doses inhibited the incorporation in vivo of 32P into 21, 16, and 4 Se RNA, but the nuclear transcription of cytoplasmic RNA was also inhibited to the same extent. No significant effect was observed at lower doses. In contrast, actinomycin D exerted a differential inhibition of the synthesis of 28 and 18 Se RNA from both the cytoplasmic and the mitochondrial fractions, practically without affecting the transcription of the 21 and 16 Se species. The incorporation of 32P into mitochondrial 4 Se RNA was also considerably more resistant to the drug than the synthesis of the cytoplasmic tRNA. It is concluded that the 21, 16, and Se RNA's are the only major discrete species transcribed from mitochondrial DNA present in the Walker carcinoma.