I. A Study by Electron Microscopy of the RNA and RNA-DNA Hybrids of Hela Mitochondria. II. Replication of Closed Circular DNA in Mouse L Cells

Year: 1972 DOI: 10.7907/JKBR-M955
The thesis consists of two parts. In Part I, electron microscope analysis of HeLa mitochondrial RNA-DNA hybrids isolated from CsSO₄ gradients demonstrates that more than 85% of the heavy strand of HeLa mitochondrial DNA is transcribed in vivo.

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 (rRNA) components. Length measurements on purified 12S and 16S mitochondrial rRNA, on mixtures of the two, and on mixtures of 12S with 18S cytoplasmic rRNA have given molecular lengths of 0.27 μ, 0.42 μ, and 0. 55 μ for the 12S, 16S, and 18S rRNAs, respectively. If these molecular lengths are proportional to molecular weight, and if the molecular weight of 18S cytoplasmic rRNA is taken as 0.71 x 10⁶, as determined by sedimentation equilibrium, the molecular weights of the 12S and 16S components are 0.35 x 10⁶ and 0.54 x 10⁶, 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.

Electron microscopy of hybrids between the heavy strand of HeLa mitochondrial DNA and HeLa mitochondrial rRNA demonstrates that the genes for 16S and 12S 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.

A coupling procedure has been developed for the efficient covalent attachment of periodate oxidized RNA to an insoluble matrix of hydrazide-derived sepharose, which is subsequently available for nucleic acid hybridization.

In Part II of the thesis, the properties of a new structure of mitochondrial DNA are discussed. In two strains of mouse L cells, it was found that approximately one half of the closed mitochondrial DNA molecules contain a short three-stranded DNA region in which a short single strand of progeny DNA containing about 350 nucleotides is inserted into the closed circular duplex with the displacement of a corresponding stretch of the still circular parental strand. The three-stranded region is called a D-loop because of its formal shape and because it appears to have been formed by displacement replication. Double-length mitochondrial DNA molecules often contain two D-loops which are always at diametrically opposed positions on the 20 megadalton circle. This latter result is taken to indicate that one of the forks in each D-loop marks a unique site for the initiation of replication.