Phosphorylation of purine and pyrimidine nucleosides by isolated rat liver mitochondria

Formation of 5'-AMP, 5'-GMP, 5'-CMP and 5'UMP was confirmed in isolated rat liver mitochondria incubated with alpha-ketoglutarate, inorganic phosphate, purine nucleoside and pyrimidine nucleoside. Increased incorporation of 32Pi into ATP, GTP and UTP was observed by adding purine- and pyrimidine nucleosides. The phosphorylation of nucleosides was inhibited severely by arsenite and affected slightly by the addition of nuclear or post-mitochondrial fraction.</p

Mitochondrial DNA from hamster tumors induced by adenovirus type 12

1. Mitochondria isolated from hamster tumors induced by adenovirus type 12 possessed circular DNA fibers. 2. The mean value of the length of the highest frequency group of the circular DNA molecules was 4.92 ±0.38 &#956;. 3. Catenated dimer DNA molecules and small (less than 2 &#956; in length) circular DNA molecules were observed.</p

Fractionation and characterization of euchromatin isolated from mouse ascites sarcoma cells

Euchromatin specimen prepared by the usual method formed large clumps and had various shapes under electron microscopy. A method of separation of the euchromatin specimen into chromatin fractions having relatively homogeneous form was examined and partial characterization of these fractions was carried out. The heavy euchromatin fraction was a large network of thin fibrils (about 100 A in diameter) and various thick fibers. The intermediate euchromatin fraction consisted of relatively homogeneous networks of thick knobby fibers (about 250 A in diameter). The light euchromatin fraction had metworks of thick fibers. These chromatin fractions were quantitatively prepared from sonicated nuclei of mouse ascites sarcoma cells. Twenty-one or twenty-two bands of non-histone proteins besides histones were detected in these chromatin fractions by SDS-polyacrylamide gel electrophoresis. There were significant differences in the electrophoretic patterns of non-histone proteins among these chromatin fractions.</p

Localization of TCA cycle dehydrogenases in the mitochondria

The site of localization of TCA cycle dehydrogenases in mitochondria has been investigated by observing the dehydrogenase activities and fine structure of the fractionated samples after freezing and thawing or sonication of beef heart and rat liver mitochondria. 1. In the sonicated mitochondria, activities of malic and isocitric dehydrogenases were highest in the supernatant fraction centrifuged at 198,000 x g for 60 minutes, while the specific activity of a-ketoglutaric dehydrogenase was higher in the fluffy or residue fraction. The distribution of the activity of pyruvic dehydrogenase was similar to that of a-ketoglutaric dehydrogenase. 2. In a sucrose density gradient fractionation of the fluffy fraction obtained by centifugation of sonicated mitochondria at 198, 000 x g for 60 minutes, the activities of malic and pyruvic dehydrogenase were observed in the top (or low density) layer in the form of fine particles, while that of a-ketoglutaric dehydrogenase was observed in the middle (or medium density) layers in the form of aggregates of fine particles and membranous fragments. 3. In the samples fractionated after freezing and thawing of mitochondria, which were considered to be a relatively mild disruption, the specific activity of a-ketoglutaric dehydrogenase was higher in the residue (submitochondria) fraction than that in the supernatant fraction (centrifuged at 144,000 x g, 30 minutes), and the activity of malic dehydrogenase still remained significantly high in the residue fraction. 4. It was deduced that the TCA cycle dehydrogenases could be localized in the matrix of the mitochondria by a loose binding to the inner membrane.</p

Characterization of RNA polymerases from Rous sarcoma virus-induced mouse ascites sarcoma cells.

RNA polymerase was extracted from the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV)-induced C3H/He mouse ascites sarcoma cells (SR-C3H). RNA polymerase was separated into RNA polymerases I and II by DEAE-Sephadex chromatography. RNA polymerase I was separated into Ia and Ib fractions by phospho-cellulose chromatography. In SR-C3H cells RNA polymerase Ib was the main component of RNA polymerase I. At 0.05--0.1 M ammonium sulphate RNA polymerase I transcribed native DNA most actively, and RNA polymerase II transcribed denatured DNA most actively. Partial digestion of DNA by DNAase I enhanced RNA synthesis by RNA polymerases I and II. At ionic strength over 0.2 M ammonium sulphate, the initiation reaction of RNA polymerases I and II was inhibited. The initiation complexes of RNA polymerases I and II with native DNA were more stable against high salt concentration than with denatured DNA.</p

Studies on nucleic acids in Rous sarcoma virus-induced mouse ascites sarcoma cells. Distribution and electron microscopy of nucleic acids in subcellular fractions and circular DNA in mitochondrial fraction

For the purpose to clarify the distribution of DNA in mouse ascites sarcoma cells (SR-C3H) induced by Rous sarcoma virus (Schmidt-Ruppin strain), quantitative assays were carried out by SCHMIDT-THANNHAUSERSCHNEIDER'S method using subcellular fractions isolated from SR-C3H cells and C3H mouse liver as a control tissue, and simultaneously electron microscopic observations were conducted with the rotary shadowed preparations of the SDS-phenol extracted nucleic acids by the protein monolayer technique. The results are briefly summarized as follows. 1. The RNA/DNA ratios in SR-C3H cells and liver cells were 2.3 and 3.7, while those in nuclear fraction of SR-C3H cells and liver cells were 0.34 and O. 56, respectively. The electron micrographs of nuclear nucleic acids revealed a DNA-RNA complex-like structure. 2. DNA and RNA contents of SR-C3H mitochondria were found to be 3.1 and 24 fl-g per mg of protein, respectiVely, which proved to be greater than those of liver mitochondria. The mean values of the contour length of circular DNA molecules in highest frequency group observed in the electron micrographs were 4.88 &#956;. in SR-C3H mitochondria and 5.08 &#956;. in mouse liver mitochondria. There could be observed circular molecules of duplicated-length in both mitochondrial DNA's and small circular molecules in SR-C3H mitochondrial DNA. 3. In the microsomal and supernatant fractions of SR-C3H cells and mouse liver cells, the ratios of DNA to RNA gave several percent by chemical analysis and this percentage was particularly high in the supernatant of SR-C3H cells. On the other hand, in the electron micrographs, the fibrous structure was significantly recovered in the supernatant nucleic acids of SR-C3H cells, but with difficulty in the other three fractions. This fibrous structure measured 1.13 &#956; in the mean value of the length and was considered to be DNA as it readily disappeared after the treat· ment with DNase.</p

Studies on human KC cell syncytia formation induced by Mason-Pfizer monkey virus

Human KC cell monolayer inoculated with concentraten Mason-Pfizer monkey virus (MPMV) showed syncytia formation within an hour. The cell fusion was blocked by the treatment of the MPMV with neutralizing antiserum. Treatment of the MPVM with beta-propiolactone resulted in the loss of infectivity although KC cell fusion ability of the virus still remained. KC cells inoculated with unconcentrated MPMV showed no cell fusion even after several transfers, although a chronic MPMV infection was established. The virus-producing KC cells were refractory to fusion by MPMV. Human embryonic lung cells (HEL) were infected by serially diluted MPMV harvested from virus-producing culture, transferred twice, then cultivated together with KC cells for syncytia formation to examine the end point dilution titer of the virus. HEL infected by 10(-4)-diluted MPMV still induced syncytia formation by cocultivation with KC cells.</p

Ultrastructure of the ribonucleoprotein and messenger-like ribonucleic acid of the polyribosomes isolated from Rous sarcoma virus-induced mouse ascites sarcoma cells

Electron microscopic observation was made on the length distibution of messenger RNA molecules in polyribosome pre· paration isolated from mouse ascites sarcoma cells, which was de· stroyed by ethylenediamine tetraacetate treatment in hypotonic solu. tion. The ribosomes appeared first to be a hollowed structure by swelling and then were destroyed to a rod·like structure consisting of ribonucleoprotein strand, which was clearly distinguishable from the linear structure of messenger RNA released from the polyribosomes. The length of messenger RNA was poly.dispersed measuring from 0.02 up to 6 &#956;, the majority (92%) of which was in the length less than 3 &#956; with a prominent peak between 0.6 to 0.8 &#956;.</p

Electron Microscopic Observations on the Ribonucleic Acid Molecules of Linear Structure in Rous Sarcoma Virus-induced Mouse Ascites Sarcoma Cells

The RNA extracted from Rous sarcoma virus (RSY)induced mouse ascites sarooma cells (SR·C3H, N. P.) by means of the cold SDS-phenol was examined by the electron microscopy on the specimens spread wi th or wi thout urea according to the protein mono· layer technique. The majority of RNA molecules was found in a collapsed agglomerated form, derived from matured ribosomal RNA. Using sucrose gradient, linear molecules of RNA were observed in the interspace of the agglomerated form of RNA at the region of high molecular weight of the band sedimentation. The histogram of the distribution in length of the linear molecules involved up to 6 /1 in length wi th a modal length of 2. 28 f1 and 2.0 to 2. 2 f1 in a pro. minent peak; longer molecules up to 18 f1 in length were scarcely observed. Species of the linear RNA molecules is not exactly known, although this is not mature ribosomal RNA and likely to be messenger RNA or nascent RNA molecules, some of which might associate with RSY·RNA.</p

Search for virus specific DNA sequences and viral particles in mitochondria of avian leukemic myeloblasts

The intracellular localization of the avian myeloblastosis virus (AMV) genome was studied. Nuclear and mitochondrial DNAs from myeloblasts were examined by hybridization with 32P labeled AMV-RNA of high molecular weight for the presence of virus specific DNA sequences. Nuclear DNA (nDNA) from myeloblasts specifically hybridized with viral RNA, whereas purified closed circular mitochondrial DNA (mtDNA) did not hybridize with viral RNA. It was therefore concluded that viral genome was present in nuclear DNA and not in mitochondrial DNA. Likewise, in normal chick cells, nDNA but not mtDNA hybridized with viral RNA.</p