Behavior of mitochondria from normal and tumor cells in isopycnic sucrose gradients.

Rat and mouse liver mitochondria, when centrifuged in a sucrose density gradient (25--50%, w/w), showed the presence of heavy (H) and light (L) subfractions with buoyant densities 1.185 and 1.170--1.165 g/ml, respectively. Mild treatment with digitonin or EDTA (30 mM) shifted H-subfraction of mitochondria into the lighter zone of the gradient and as a result of this the mitochondria were distributed as a homogenous band with buoyant density 1.170--1.165 g/ml. Mitochondria isolated from both MD hepatoma and Zajdela rat hepatoma were characterized by a homogenous banding with buoyant density 1.160--1.165 g/ml. Regarding to this, the content and patterns of polyribosomes bound to outer membranes of mouse tumor mitochondria were studied. Analysis of polyribosomes as well as the results of RNA polyacrylamide gel electrophoresis indicate that the content of these polyribosomes in tumor mitochondria is less than that in normal liver ones. However, the decrease of cancer cell membrane-bound polyribosomes cannot account for the differences in buoyant densities of mitochondria from normal and tumor tissues.

Isolation and partial characterization of poly (A)-containing 7.5S messenger RNA from rat liver mitochondria.

Poly(A)-containing low molecular weight (7.5S) messenger RNA was isolated in a highly purified form from both polyribosomes and post-polysomal supernatant of rat liver mitochondria. Both mRNA's contain rather short poly(A) tracts (40-70 mononucleotides) according to a profile of their elution from poly(U)-Sepharose column with a gradient of formamide concentration. Both mRNA's when added to a preincubated mitochondrial lysate programmed the synthesis of a hydrophobic polypeptide of a molecular weight about 9000 daltons which was soluble in the neutral chloroform-methanol mixture.

On the cell-free system of protein synthesis from mammalian mitochondria.

Some properties of a submitochondrial cell-free system for protein synthesis are described. The system was prepared from rat liver mitochondria lysed with Triton X-100, and the lysate was characterized by a linear rate of [14C]amino acid incorporation for 15-20 min with subsequent decline in activity. The incorporation reaction was inhibited by chloramphenicol and was insensitive to cycloheximide. Poly(U) addition stimulated [14CA1phenylalanine incorporation by the preincubated submitochondrial system. Upon the addition of 7.5S mRNA that was isolated from mitochondria the major translation product was identified as a hydrophobic poly-peptide which in some properties (solubility in chloroform-methanol mixture) was similar to one of polypeptides synthesized by the sub-mitochondrial system on endogeneous mRNAs.

Polyribosomes and messenger RNA from rat liver mitochondria.

Rat liver mitochondrial polyribosomes were isolated free from cytoplasmic ribonucleoprotein contaminations in a number of criteria (sedimentation and buoyant density patterns, ribosomal RNA composition). Heterogeneous poly A containing RNA from mitochondrial polysomes was purified by two-stage cellulose chromatography. This RNA was in vitro labelled with 125I up to specific activity approximately 10(6)-10(7) cts.min-1.microng-1 and used for hybridization experiments with separate complementary strands of mitochondrial DNA and nuclear DNA fragments. The proportions of mitochondrial poly A containing RNA that is complementary to heavy and light strands of mtDNA were respectively 31.5% and 8.3%. Besides, a significant RNA fraction was complementary to unique sequences of nuclear DNA (2-3 copies per haploid genome). The hybrids that were formed possessed a high Tm indicative of a perfect base pairing. A dual intracellular origin of mitochondrial messenger RNA is discussed.

Nucleic acids from subcellular fractions of N-nitrosodiethylamine-induced hepatoma in mice.

During eight successive isologous passages of hepatoma induced in male C3HA mice by N-nitrosodiethylamine, no common features of tumor progression were observed, although both the mitotic pattern and ploidy differed from generation to generation. These additional cytologic criteria allowed the biochemical examination of material least changed due to tumor progression. Tumor nDNA's were characterized by greater actinomycin D (AD)- and acridine orange (AO)-binding abilities than were normal nDNA's; this could have resulted from a higher proportion of double-stranded regions in tumor DNA. Isolated tumor deoxyribonucleoprotein had both lower template activity in an RNA polymerase system and fewer AD- and AO-binding sites, when compared with the activity and sites from normal mouse liver. RNA-DNA hybridization data with the above-mentioned findings showed that in hepatoma, part of the nuclear genome was repressed. Also, RNA "new classes" appeared and a certain proportion of nuclear genes controlling mitochondrial protein biosynthesis were derepressed in tumor mitochondria. The hybridization of mitochondrial RNA (mtRNA) and DNA revealed new classes of pulse-labeled RNA's in in vitro-incubated liver mitochondria that were absent from intact cell organelles; the hybridization properties of in vivo- and in vitro-formed hepatoma mtRNA's were similar. Competition and hybridization experiments demonstrated that in tumor mitochondria in vivo, some new classes of RNA existed. Hepatoma mitochondrial mRNA had a higher metabolic stability than did normal mRNA.

On the synthesis of viral ribonucleic acids and ribonucleoproteins in the submitochondrial system completely free of interfering cytoplasmic contaminations.

The synthesis of virus-specific macromolecules was studied in the reconstituted system containing inner membrane-matrix fraction from rat liver mitochondria and infectious RNA of Venezuelian equine encephalomyelitis (VEE) virus. In a series of preliminary experiments it was shown that isolated submitochondrial fraction was completely free of interfering cytoplasmic contaminations and particularly, of cytoplasmic 80S ribosomes. VEE RNA when added to submitochondrial system caused significant stimulation of RNA and protein synthesis. These processes were resistant to actinomycin D which inhibited profoundly the synthesis of proper mitochondrial macromolecules. The stimulating effect of VEE RNA in experiments with submitochondrial system was about three times higher than that with intact mitochondria. The stimulation of 14C-amino acid incorporation increased as a function of incubation time; a certain lag-period being observed. The newly formed virus-specific RNA's and ribonucleoproteins were identified with the aid of sedimentation analysis. In particular, radioactive RNA's with sedimentation coefficients 40S and 26-18S were isolated from the incubated system. These RNA's are similar respectively to VEE genome RNA and double-stranded VEE replicative RNA. In double labelling experiments with 3H-uridine and 14C-amino acids it was shown that VEE RNA induced synthesis of ribonucleoproteins containing newly formed RNA and protein. These RNP possessed sedimentation coefficients 60-80S, 140S and 300S in sucrose gradient and buoyant densities 1.32 and 1.50 g/cm3 in cesium chloride gradients. These properties of ribonucleoproteins synthesized de novo in submitochondrial system are close to those of RNP intermediates of VEE virus reproduction in the infected cells. We concluded that viral RNA could program virus-specific synthesis in the submitochondrial system under conditions that eliminated the contribution of cytoplasmic ribosomes.

On the defect of synthesis ceruloplasmin in the liver polyribosomes in Wilson's disease.

Comparative immunochemical analysis of ceruloplasmin-synthesizing polyribosomes in liver biopsies from control subjects and homozygous carriers of the Wilson's mutation was performed. According to I125-antibody binding data, the amount of ceruloplasmin-forming liver polysomes in patients with Wilson's disease was 10--20 times lower than that in non-Wilson patients. Correspondingly, the pulse labeling of ceruloplasmin polypeptides was decreased several-fold in the cell-free liver preparations from patients with Wilson's disease.

Messenger RNA-containing ribonucleoprotein from mitochondrial polyribosomes of rat liver.

A ribonucleoprotein was released from carefully purified rat liver mitochondrial polyribosomes after dissociation with 1 M potassium chloridepuromycin. This ribonucleoprotein was characterized by a sedimentation coefficient ranging from 10-14 S and buoyant density of 1.48 g cm(-3) in cesium chloride equilibrium centrifugation differing in these parameters from the subunits of mitochondrial ribosomes. Poly(A)-containing RNA constituted more than 30% of the total RNA content in this non-ribosomal ribonucleoprotein.

Letters to the editor: On the transfer of nuclear RNA into isolated mitochondria. Further evidence for template properties of nuclear RNA taken up by isolated mitochondria.

Some properties of nuclear RNA taken up by isolated mitochondria during aerobic incubation were studied. Nuclear RNA fraction re-isolated from incubated mitochondria was characterized by homogeneous sedimentation distribution in sucrose gradient (7-9S) and by absence of extended secondary structure. In this respect the RNA fraction differed from the nuclear RNA added to mitochondria and was similar to in vivo rapidly labelled mitochondrial RNA. The hybridization pattern of re-isolated RNA with nuclear DNA is consistent with the presence of transcripts from unique DNA sequences in this RNA fraction.