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Ribosomal-type ribonucleic acid from rodent mitochondria
Abstract:
1. Highly purified mitochondria containing 3.0mug of RNA/mg of mitochondrial protein were prepared from rat liver by differential centrifugation. 2. RNA, labelled with [(32)P]P(i) or [(3)H]orotate, was isolated from these mitochondria by a phenol extraction method. The RNA sedimented at 15S and 13S on sucrose density gradients. Its nucleotide composition was 23% uridylate, 30% adenylate, 22% guanylate and 25% cytidylate. 3. RNA from mouse L cells was labelled with [(3)H]-uridine in the presence of 0.1mug of actinomycin D/ml to suppress the synthesis of cytoplasmic rRNA. The RNA isolated from crude L-cell mitochondria by a cold-phenol-sodium dodecyl sulphate method had components sedimenting at 15S and 12.5S. These components had an electrophoretic mobility on agarose-acrylamide gels of 21 and 12S(E) compared with 28 and 18S(E) for cytoplasmic rRNA. The nucleotide composition was 26% uridylate, 34% adenylate, 18% guanylate and 22% cytidylate. 4. RNA extracted from crude L-cell mitochondria by a hotphenol-sodium dodecyl sulphate method had an additional component sedimenting at 21S and having an electrophoretic mobility of 18S(E). It was probably DNA because of its sensitivity to deoxyribonuclease and its insensitivity to ribonuclease and alkali. It was present in nuclear fragments contaminating the crude mitochondrial fraction and could be removed by deoxyribonuclease or isopycnic-gradient centrifugation.
Insights
Mitochondria contain distinct RNA molecules, including 15S and 13S RNA from rat liver and 15S and 12.5S RNA from mouse L cells, characterized by specific nucleotide compositions and electrophoretic mobilities.
Area of Science:
- Mitochondrial biology
- Molecular biology
- RNA biochemistry
Background:
- Mitochondria possess their own genetic material, distinct from nuclear DNA.
- Characterizing mitochondrial RNA (mtRNA) is crucial for understanding mitochondrial function and disease.
- Previous studies have identified various RNA species within mitochondria, but their precise nature and origins require further elucidation.
Purpose of the Study:
- To isolate and characterize RNA from purified rat liver mitochondria.
- To isolate and characterize RNA from mouse L cell mitochondria, distinguishing it from cytoplasmic RNA.
- To identify and differentiate RNA species from potential DNA contaminants in mitochondrial preparations.
Main Methods:
- Differential centrifugation for purifying rat liver mitochondria.
- Phenol extraction and sucrose density gradient centrifugation for rat liver RNA isolation and analysis.
- Actinomycin D treatment to suppress cytoplasmic rRNA synthesis in mouse L cells.
- Cold and hot phenol-sodium dodecyl sulfate methods for mouse L cell mitochondrial RNA isolation.
- Agarose-acrylamide gel electrophoresis and deoxyribonuclease/ribonuclease sensitivity assays for RNA characterization.
Main Results:
- Rat liver mitochondria yielded RNA sedimenting at 15S and 13S with a specific nucleotide composition.
- Mouse L cell mitochondria, under conditions suppressing cytoplasmic rRNA, yielded RNA components sedimenting at 15S and 12.5S, with distinct electrophoretic mobilities (21S(E) and 12S(E)).
- A 21S component in crude L-cell mitochondrial preparations was identified as DNA due to its nuclease sensitivity and was attributed to nuclear fragment contamination.
Conclusions:
- Mitochondria contain unique RNA species with defined sedimentation properties and nucleotide compositions.
- Distinguishing mitochondrial RNA from cytoplasmic RNA and DNA contaminants is essential for accurate molecular analysis.
- The methods employed successfully isolated and characterized mitochondrial RNA, providing insights into its nature and purity.