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The nature of RNA coded by the only light-strand (L-strand) open-reading frame unidentified reading frame 6 (URF6) was studied by using a variety of single- and double-strand DNA subclones derived from the 3.6-kilobase (kb) cytochrome b (cyt b)-URF5 coding region of the mouse mitochondrial genome. Northern blot experiments using single-strand-specific M13 clones indicate that both the heavy (H) and L strands of this genomic region are symmetrically transcribed and processed into poly(adenylic acid) [poly(A)] RNAs of comparable size. The 1.2- and 2.4-kb RNAs coded by the H strand, putative mRNAs for cyt b and URF5 reading frames, respectively, are derived from a common precursor of 3.6-kb RNA. The L-strand-coded 1.15-kb RNA, on the other hand, is derived from a short-lived precursor of 3.6-kb RNA by a multiple-step processing involving a 2.4-kb intermediate RNA. The S1 nuclease protection experiments using both the 3'- or 5'-end-labeled DNA probes and also affinity-purified 32P-labeled RNA probes indicate that the 1.15-kb RNA maps between the start of the URF6 reading frame (3' end) and a region 590-600 nucleotides to the 5' end of this reading frame. The 1.15-kb RNA thus contains the entire URF6 coding sequence and an about 590-nucleotide-long 3' untranslated region. The molar abundance of the three mRNAs in the steady-state mitochondrial RNA varies markedly. The 1.15-kb URF6 mRNA is only one-tenth the level of 1.2-kb cyt b mRNA, although it is nearly as abundant as the 2.4-kb URF5 mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo treatment of sensitive tumor cells with nitrogen mustard (HN2) results in marked inhibitions of protein and nucleic acid synthesis by mitochondria subsequently isolated from these cells. This inhibition occurs at doses of drug which produce no apparent inhibition of total RNA synthesis. The inhibition gradually reverses itself in sensitive cells and is less severe and more rapidly reversed in resistant cells. The in vivo sensitivity of the mitochondria is in striking contrast to their in vitro behavior; isolated mitochondria resist 50 times the in vivo ID50 level of the drug. The sensitivity of protein and RNA synthesis in mitochondria is presumed to be related to the differences in organization between the nucleocytoplasmic processes and the mitochondrial. But the data also suggest that the in vivo effects on mitochondria are indirect, either acting via the cytoplasm or nucleus or via a carrier mechanism having mitochondrial affinity.

The mitochondrial poly(adenylic acid)-containing mRNAs in mouse ascites cells pulsed with radioactive precursors contain 35-55 nucleotide long poly(adenylic acid) sequences. These sequences are shortened with age in cells chased with cold medium. The possible relationship between the decay rates of mRNA and their poly(adenylic acid) sequences has been investigated by using mitochondrial RNA and protein synthesis inhibitors. The pattern of mRNA decay as determined by a new solid-phase-bound complementary DNA procedure indicates the presence of two classes of poly(adenylic acid) containing mRNA in mammalian mitochondria: one decaying with a t1/2 of 45 min and the other class with a t1/2 of 210 min. Inhibitors such as ethidium bromide and puromycin which accelerate the decay of mitochondrial mRNA also cause an enhanced decay of poly(adenylic acid) sequences. These results have been interpreted as evidence supporting the involvement of poly(adenylic acid) sequences in the mRNA stability.

We have prepared a submitochondrial fraction from Ehrlich ascites tumor cell mitochondria which shows transcription and translation activities. The antibiotic aurin tricarboxylic acid (ATA) at low concentrations induces both RNA and protein synthetic activities of the mitochondrial lysate by several fold. At high concentrations, however, ATA inhibits the translation activity but continues to stimulate the transcription activity in a dose dependent manner up to 0.5 mM concentration tested. The lysate system transcribes endogenous DNA yielding RNA species resembling control mitochondrial RNA and synthesizes authentic cytochrome oxydase I and cytochrome oxidase II subunits.

The steady-state mitochondrial mRNAs in Ehrlich ascites tumor cells and mouse liver were identified by the Northern blot analysis using nick-translated mtDNA and 32P-labeled cDNA probes. The steady-state mRNA species were compared with the poly(A)-containing RNA synthesized in vitro in isolated mitoplasts. The results show that the isolated mitoplast system can efficiently transcribe almost all of the poly(A)-containing RNAs detected in the steady-state RNA population. It is also seen that the mode of transcription and maturation of mitochondrial mRNAs in different mouse tissues are identical. The results of Northern blot analyses suggest that there may be at least two different modes of mRNA maturation depending upon if the reading frames are interrupted by tRNA cistrons or not. mRNAs for reading frames with adjacent tRNA cistrons downstream appear to be processed from very short-lived precursors. In contrast, mRNAs coded by adjacently located reading frames with no interrupting tRNA genes such as URF3 -cyt ox III and URF5 -cyt b are processed from relatively long-lived precursors. The in vitro pulse-labeling studies also show that almost all of the poly(A)-containing mRNAs are transcribed at nearly identical rates, suggesting that the major regulation of mt gene expression may occur at the level of translation or mRNA decay. The present experiments have also identified a 1.85-kb poly(A)-containing RNA as the putative URF5 mRNA.

The Mg2+ precipitation procedure of R. D. Palmiter ((1974) Biochemistry 13, 3606) has been used for preparative scale isolation of polysomes from Ehrlich ascites mitochondria. Digitonin-washed metochondria used for isolating the polysomes contain no detectable reduced nicotinamide adenine dinucleotide phosphate-cytochrome c reductase and over 200-fold reduced hexokinase activity. The mitochondrial polysomes exhibit a heterogeneous sedimentation and appear to contain highly aggregated particlses ranging over hexamers. These polysomes are sensitive to RNase, (ethylenedinitrilo)tetraacetic acid and puromycin. Mitochondrial polysomes are active in portein synthesis when supplied with supernatant enzymes from the homologous mitochondrial source or from Escherichia coli. Cytoplasmic enzymes, however, appear to be completely inactive. Protein synthesis by mitochrondrial polysomes is sensitive to chloramphenicol and resistant to cycloheximide and emetine. The procedure yields particles containing intact rRNAs. The extent of cytoplasmic RNA contaminating the total mitochondrial RNA or mitochondrial polysomal RNA has been estimated to be negligible.