Yosef Aloni

Expression of the mitochondrial genome in HeLa cells: II. Evidence for complete transcription of mitochondrial DNA☆

Abstract The fraction of mitochondrial DNA which is complementary to mitochondria-associated RNA in exponentially growing HeLa cells has been investigated by RNA-DNA hybridization experiments using mitochondrial DNA strands separated by alkaline CsCl density-gradient centrifugation and RNA from cells uniformly labeled with [5- 3 H]uridine. It has been found that mitochondrial RNA hybridizes almost exclusively with the heavy strand, in agreement with the complementarity of their base compositions. Less than 2% of the light strand, after purification by two cycles of alkaline CsCl gradient centrifugation, was found to form hybrids with mitochondrial RNA. In hybridization saturation experiments, the RNA sedimenting slower than 22 s or faster than 30 s gave about 100% saturation level, whereas the components in the intermediate range of sedimentation coefficients gave about 85% saturation level. An analysis in Cs 2 SO 4 gradients of the density of the RNA-DNA hybrids formed at saturation showed that the hybrids formed with the RNA of less than 22 s and greater than 30 s banded at a density of 1.491 g/cm 3 , i.e. the density expected for fully base-paired hybrids; whereas the hybrids formed with the components of intermediate sedimentation coefficients banded at a slightly lower density (1.485 g/cm 3 ), in agreement with the lower saturation level obtained with these components. An analysis by electron microscopy of the hybrids separated in a Cs 2 SO 4 density-gradient showed that essentially all the material from the 1.491 g/cm 3 band consists of nucleic acid duplexes. In view of the available evidence for the absence of major internal repetitions in mammalian mitochondrial DNA, the results obtained by the three approaches described here indicate that the whole or almost whole mitochondrial genome is transcribed in HeLa cells. The possibility that this transcription occurs in the form of a continuous RNA chain is discussed.

Expression of the mitochondrial genome in Hela cells: VI. Size determination of mitochondrial ribosomal RNA by electron microscopy☆☆☆

Abstract A modified basic protein, film method of spreading RNA in a strongly denaturing solvent for examination in the electron microscope has been developed and applied to determine the size of the HeLa mitochondrial specific ribosomal RNA components. Length measurements on purified 12 s and 16 s mitochondrial rRNA, on mixtures of the two, and on mixtures of 12 s with 18 s cytoplasmic rRNA, have given molecular lengths of 0.27 μ, 0.42 μ, and 0.55 μ for the 12 s, 16 s and 18 s rRNAs. If these molecular lengths are proportional to molecular weight, and if the molecular weight of 18 s cytoplasmic rRNA is taken as 0.71 × 10 6 , as determined by sedimentation equilibrium, the molecular weights of the 12 s and 16 s components are 0.35 × 10 6 and 0.54 × 10 6 , respectively. These molecular weight values are in good agreement with the relative values predicted from sedimentation velocity measurements, but not with the relative values based on gel electrophoresis.

Expression of the mitochondrial genome in HeLa cells: XIV. The relative positions of the 4 s RNA genes and of the ribosomal RNA genes in mitochondrial DNA☆☆☆★

Abstract HeLa mitochondrial 4 s RNA has been covalently coupled to the electron opaque label, ferritin, which is visible in the electron microscope. Mixtures of HeLa mitochondrial 12 s ribosomal RNA, 16 s rRNA and/or the 4 s RNA-ferritin conjugate have been hybridized to separated heavy (H) and light (L) strands of HeLa mitochondrial DNA, or to a mixture of H and L strands. The relative positions of the duplex regions corresponding to the 12 s and 16 s rRNA—DNA hybrids and of the ferritin-labeled 4 s RNAs have been mapped in the electron microscope after spreading the DNA strands by the formamide modification of the basic protein film technique. The 12 s and 16 s duplex regions have lengths of 0·-26 ± 0.04 μm and 0.46 ± 0.07 μm, respectively. They are separated by a single-strand region of length 0.047 ± 0.017 μm, corresponding to 160 ± 60 nucleotides. There are nine reproducible binding sites for 4 s RNA on the H strand. One such site lies within the spacer region between the 12 s and 16 s coding sequences, one site is immediately adjacent to the other side of the 12 s sequence and one is adjacent to the other side of the 16 s sequence. The other 4 s sites are rather evenly spaced along the DNA strand of total length 15,600 nucleotides, except that two of them are clustered with a spacing of 120 ± 30 nucleotides between them. There are three 4 s RNA coding sequences on the L strand, separated from one another by 2280 and 3900 nucleotides, respectively.

Expression of the mitochondrial genome in HeLa cells: IV. Titration of mitochondrial genes for 16 s, 12 s and 4 s RNA☆☆☆

Abstract RNA-DNA hybridization experiments utilizing separated strands of HeLa mitoehondrial DNA and purified 16 s, 12 s and 4 s mitochondrial RNA species from HeLa cells have indicated the existence of one 16 s gene and one 12 s gene per mitoehondrial DNA molecule, which are located in the heavy mitoehondrial DNA strand, and of approximately eleven genes for 4 s RNA: of these about eight appear to be located in the heavy strand and about three in the light strand.

Studies of fractionated HeLa cell metaphase chromosomes: II. Chromosomal distribution of sites for transfer RNA and 5 s RNA☆☆☆

Abstract DNA extracted from HeLa cell metaphase chromosomes fractionated on the basis of sedimentation velocity in glycerol-sucrose gradients has been tested for the capacity to hybridize with highly purified tRNA and 5 s RNA. The results obtained indicate that the sites for these two RNA classes, in contrast to those for the high molecular weight rRNA, are distributed among chromosomes of all size ranges.

Expression of the mitochondrial genome in HeLa cells: XI. Isolation and characterization of transcription complexes of mitochondrial DNA

Abstract Transcription complexes of HeLa cell mitochondrial DNA have been isolated by sedimentation velocity in a sucrose gradient and buoyant density fractionation in a CsCl/ethidium bromide density-gradient and characterized in the electron microscope and by biochemical tests. Both open and closed circular mitochondrial DNA molecules carrying nascent RNA chains have been observed, with the open forms showing signs of a more intensive transcriptive activity in terms of the number and size of RNA chains attached to them. At least 25 RNA bushes were counted in the same transcription complex. The largest bushes in the transcription complexes have been estimated to correspond to RNA chains as long as the whole mitochondrial DNA molecule. The observation that treatment with a protein denaturant such as sodium dodecyl sulfate failed to cause the release from the transcription complexes of a good part of, if not all, the nascent chains, strongly suggests that base-paring, in addition to RNA polymerase, has a role in holding the RNA and DNA together. Measurement of the rate of labeling of the transcripts of the heavy and light mitochondrial DNA strands in the transcription complexes has indicated that the two mitochondrial DNA strands are transcribed at a similar rate.

Expression of the mitochondrial genome in HeLa cells: VIII. The relative position of ribosomal RNA genes in mitochondrial DNA

Abstract Electron microscopy of hybrids between the heavy strand of HeLa mitochondrial DNA and HeLa mitochondrial ribosomal RNA demonstrates that the genes for 16 s and 12 s HeLa mitochondrial rRNAs are situated adjacent or very close to each other on mitochondrial DNA. The length of the DNA segment separating the two genes is estimated to correspond to less than 500 nucleotide pairs.

Expression of the mitochondrial genome in HeLa cells: XII. Relationship between mitochondrial fast-sedimenting RNA components and ribosomal and 4 s RNA

Abstract About 40% of the mitochondrial RNA labeled in a five-minute [5- 3 H]uridine pulse in HeLa cells decays within one minute after blocking further RNA synthesis with ethidium bromide and actinomycin D. The decay involves a sharp decrease in the amount of fast-sedimenting pulse-labeled mitochondrial RNA molecules, which is only in minor part compensated by the accumulation of 16 s, 12 s and 4 s RNA. The data strongly suggest that these discrete species derive from larger precursors and, furthermore, that they are synthesized in a fairly co-ordinate fashion.