Expression of the mitochondrial genome in HeLa cells

Lederman, Muriel; Attardi, Giuseppe

doi:10.1016/0022-2836(73)90116-2

Cited by 47 publications

(15 citation statements)

References 28 publications

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“…The sodium dodecylsulphate electrophoretic profiles of mitochondrial proteins labelled at 40 "C in tsHl cells are very similar to those obtained by others using cycloheximide-inhibited [30] or emetine-inhibited mammalian cells [31,32]. They are also similar to the profiles of proteins labelled in isolated mitochondria from HeLa cells [31], BHK-21 cells [32] or rat liver [33].…”

Section: Discussionsupporting

confidence: 83%

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Mitochondrial Protein Synthesis in a Mammalian Cell-Line with a Temperature-Sensitive Leucyl-tRNA Synthetase

Williams

Freeman

1975

Eur J Biochem

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The temperature-sensitive Chinese hamster ovary cell mutant, tsH1, has been shown previously to contain a temperature-sensitive leucyl-tRNA synthetase. At the non-permissive temperature of 40 "C cytosolic protein synthesis is rapidly inhibited. The protein synthesis which continues at 40 "C appears to be mitochondrial, since : (a) whole-cell protein synthesis at the permissive temperature of 34 "C is not inhibited by tevenel, the sulfamoyl analogue of chlordmphenicol and a specific inhibitor of mitochondrial protein synthesis; however, whole-cell protein synthesis at 40 "C is inhibited by tevenel. The inhibition of cytosolic, but not of mitochondrial protein synthesis in tsHl cells at 40 "C allows the selective labelling of mitochondrial translation products in the absence of inhibitors. The mitochondrial translation products labelled in tsHl cells at 40°C and at 34°C in the presence of cycloheximide have been compared by sodium dodecylsulphate -polyacrylamide gel electrophoresis. Both conditions of labelling give similar profiles. The mitochondrial translation products are resolved into two components, one with an apparent molecular weight range from 40000 to 20000 and a second with an apparent molecular weight range from 20000 to 10000.The study of mitochondrial protein synthesis and of the nature of the protein products synthesized within mitochondria has made rapid advances in the recent past (for review see [l]). These studies, mainly with yeast, Neurospora crassa, Locusta migratoria and mammalian cells, have used essentially two approaches 1 the analysis of protein synthesis by isolated organelles and the analysis of protein synthesis by whole cells in which cytosolic protein synthesis has been specifically inhibited by drugs, such as cycloheximide or emetine. This latter approach has been particularly important in the demonstration that mitochondria are the site of synthesis of some of the subunits of cytochrome c oxidase and the ATPase complex of yeast and Neurospora [2 -51 and of cytochrome h of Neurospora and Locusta [6,7].

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Section: Discussionsupporting

confidence: 83%

“…They are also similar to the profiles of proteins labelled in isolated mitochondria from HeLa cells [31], BHK-21 cells [32] or rat liver [33]. In general, the proteins are often found as two or three poorly resolved classes having apparent molecular weights of less than 50000.…”

Section: Discussionsupporting

confidence: 49%

Mitochondrial Protein Synthesis in a Mammalian Cell-Line with a Temperature-Sensitive Leucyl-tRNA Synthetase

Williams

Freeman

1975

Eur J Biochem

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“…The two discrete poly(A)-containing components identified here represent a substantial portion of the RNA from the region of functioning polysomes, selected for poly(A) content by two passages through poly(dT)-cellulose columns. Previous work from this laboratory (38) has shown that the in vivo and in vitro products of mitochondrial protein synthesis consist of a group of not well-resolved components with molecular weights of 12,000-25,000, and another group, more abundant, with molecular weights of 40,000-55,000. The larger components may be polymeric forms of smaller units, as in yeast.…”

Section: Discussionmentioning

confidence: 99%

Identification of Discrete Polyadenylate-containing RNA Components Transcribed from HeLa Cell Mitochondrial DNA

Ojala¹,

Attardi²

1974

Proc. Natl. Acad. Sci. U.S.A.

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Polyacrylamide gel electrophoresis and sedimentation analysis under denaturing conditions of poly(A)-containing RNA from the polysome region of the sedimentation pattern of a HeLa-cell mitochondrial lysate has revealed the occurrence of a discrete RNA component, which sediments in the native state with a sedimentation constant of about 7 S. From the sedimentation behavior under native and denaturing conditions and the poly(A) content, a molecular weight of about 9 X 104 has been estimated for this component. RNA-DNA hybridization experiments have indicated that this component is coded for by the light strand of mitochondrial DNA. Evidence for the occurrence of a poly(A)-containing RNA component sedimenting at about 9 S and coded for by the heavy strand has also been obtained.Poly(A) sequences of a size corresponding to 60-80 nucleotides occur in mitochondrial RNA from HeLa cells (1, 2); a poly(A)-synthesizing enzyme has been identified in rat-liver mitochondria (3,4). In HeLa cells, the majority of mitochondrial poly(A) is covalently linked to mitochondrial DNA (mit-DNA)-coded RNA (2). Inasmuch as poly(A) in eukaryotic cells appears to be associated only with cytoplasmic messenger RNA (mRNA) and its presumptive nuclear precursors (5-10), and with viral or viral-directed RNA with messenger function (5,(11)(12)(13)(14)(15), the above evidence strongly suggests that at least a portion of the mRNA translated by mitochondrial polysomes is coded for by mit-DNA.Previously (2), no clear-cut evidence of discrete components was observed in the sedimentation patterns of poly(A)-containing RNA from HeLa-cell mitochondria. The main source of difficulty was probably the tendency of poly(A)-containing molecules to aggregate and sediment with heavier molecules. Thus, RNA from the polysome region of the sedimentation pattern of a mitochondrial lysate, selected for poly(A) by two passages through poly(dT)-cellulose and analyzed in sucrose gradient in the native state, showed a fairly uniform distribution of material in the region from 6 S to about 16 S, with only a hint of a 7S component emerging over a background of heterogeneous RNA. After formaldehyde denaturation and sedimentation in the presence of formaldehyde, most of the poly-(A)-containing RNA sedimented more slowly than 12 S RNA, and a pronounced sharp peak sedimenting about 20% faster than denatured 5S RNA and 40% slower than denatured 12S RNA was observed. ,ug/ml of ethidium bromide (eih. brom.), and passed through poly(dT)-cellulose. The mitochondrial 12S RNA and cytoplasmic 5S RNA were treated as the experimental samples and run in parallel gels. RESULTSA Triton X-100 mitochondrial lysate from HeLa cells labeled for 2 hr with [8-8H]adenosine in the presence of 0.1 ug/ml of actinomycin D was run through a sucrose gradient, and RNA was extracted from the polysome region (74-180 S) of the gradient (21) and passed through a poly(dT)-cellulose column. It had been shown (1, 2) that poly(dT)-cellulose or poly(U) filters bind mitochondrial poly(A)-containing RNA, wh...

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“…Presumably there is a sufficient endogenous pool of the cytoplasmic subunits to allow post-translational assembly of cytochrome oxidase. Although such experiments have not been extended to coenzyme QHrcytochrome c reductase and the ATPase complex, there is substantial evidence from studies with yeast and mammalian cells (80,81) that the relative proportions and electrophoretic mobilities of the major mitochondrial products formed in vitro and in vivo are very similar.…”

Section: In Vitro Synthesis Of Mitochondrial Productsmentioning

confidence: 99%