Mitochondria Of Mammalian Cells Research Articles

Demonstration and analysis of ribosomes and RNA associated with mitochondria of mammalian cells

Mitochondrial fraction prepared from mammalian cells (rat liver) by high and slow speed centrifugation and purified by sedimentation in an isopycnic sucrose gradient are shown to contain ribosomes.These ribosomes have a sedimentation coefficient of 83 S (measured in the absence of KCl) and a buoyant density of 1.54 g/cm3 as compared to 80S and 1.55 g/cm3 for cytoplasmic ribosomes.The sedimentation behaviour of mitochondrial ribosomes varies as a function of KCl concentration. The dissociation of cytoplasmic and mitochondrial ribosomes into sub‐units begins towards 0.1 M KCl and is complete in 1 M KCl. The sedimentation coefficient of the intact ribosomes and their dissociated units fall as the concentration of KCl is increased between 0 and 1 M.RNA prepared from rat liver mitochondrial fraction contains a low molecular weight component and two high molecular weight species whose sedimentation coefficients 26 S and 17 S correspond to molecular masses of 1.51 × 106 and 0.61 × 106 daltons. These mitochondrial RNA can be separated by sucrose gradient sedimentation from cytoplasmic ribosomal RNA species sedimenting at 28 S and 18 S.Mitochondrial RNA were also characterized by acrylamide gel electrophoresis. In 2.50% gels, the electrophoretic mobility of high molecular weight mitochondrial RNA was found to be slightly higher than that of their cytoplasmic counterparts. Moreover, the RNA corresponding to the smaller units was separated into two distinct peaks.Electrophoresis in 8% acrylamide gels revealved 5 S and 4 S RNA with a migration pattern similar to that of cytoplasmic 5 S and transfer RNA but present in different amounts and additional RNA with an electrophoretic mobility of 0.31 and 0.85 if that of 4 S RNA is equalled to 1.

Demonstration of the differentiation of ribosomal particles from cytoplasmic ribosomes by size and their nucleic acids in mammalian cell mitochondria

Evidence showing the presence of 61 S, 46 S and 35 S ribosome‐like particles in mitochondria of mammalian cells (a permanent line of mouse cells (LMTK‐) CI.1d, and mycoplasma‐free HeLa cells, is described.The two known techniques for isolating the mitochondira were used and compared: method I involves the use of EDTA and prevents large contamination by the cytoplasmic ribosomes; method II is based on the use of high concentration of magnesium and NH4Cl, and is thought to give better protection of the mitochondrial ribosomes. Method II applied to mammalian cells gives larege contaimination by cytoplasmic ribosomes, but it is shown that it does not prevent the study of the mitochondiral particles.The 61 S, 46 S and 35 S particle exhibit a ribosomalbehaviour at different magnesium conentrations: the 46 S and 35 S peaks increase at the expense of the 61 S peak at low magnesium concentration. The 80 S peak obtained by method II is shown to contain exclusively cytoplasmic ribosomes as indicated by base composition.RNA was extracted and purified from whole mitochondira isolated by method I and II. The RNA species obtained by method I were the already known 16 S, 12 S, and 4 S mitochondiral RNA species. The RNA species obtaiend by method II were the 28 S cytoplasmic RNA and 18 S RNA. The proporation of the latter was however large than expected. No clear 16 S and 12 S RNA were seen between the large 18 S and the 4–5 S RNA. Base composition of the 18 S RNA proved to be different from that the 18 S cytoplasmic RNA, with a higher content of cytosine and adenine, and lower of guanine. On the other hand, the RNA extracted from the mixture of 61 S and 46 S particles obtained from mitochondria isolated by method I, gave the 16 S, 12 S and 4 S species, the 12 S species predominating. In order to define more precisely the RNA molecules of the 61 S and 46 S particles (obtained from mitochondira isolated by method II) their RNAs were extractd using the RNase inhibitors polyvinylsulfate and diethylpyrocarbonate. The base compositions of these RNAs are very similar. They differ both from the 28 S and 18 S cytoplasmic RNAs and the 16 S and 12 S mitochondrial RNAs by the proportions of cytosine, adenine and guanine. The analysis on surcrose grdient of the RNA of the 61 S particle has shown that it comprises to RNA molecules: an 18.6 S molecule migrating a little farther than the 18 S cytoplasmic RNA, and a 16 S molecule. Essentially a single RNA species was found in the 46 S particle which is 18.6 S.Since a very strong nuclese activity in the mitochondrial lysates in the absence of nuclease inhibitors is observed the possible effect of nucleases on the measured size of ribosome‐like particles, and on the RNA species characterized, are discussed. Our data suggest that the 16 S and 12 S mitochondiral RNAs might be party degraded products of larger molecules.

The RNA Metabolism of Nucleoli and Mitochondria in Mammalian Cells

The RNA Metabolism of Nucleoli and Mitochondria in Mammalian Cells