Studies on mitochondria. II. Mitochondrial DNA of thoracic muscles of Schistocerca gregaria.

The buoyant densities of the nuclear and mitochondrial DNA from the thoracic muscles of Schistocerca gregaria were found to be 1 702 and 1.689 g/cm(3), respectively, corresponding to guanine plus cytosine (G + C) content of 42.2 and 30% A preliminary treatment of the mitochondrial pellet with DNase (25 degrees C, 20 min) is necessary to eliminate the contaminating nuclear DNA. The mitochondrial DNA renatures readily after heat denaturation and incubation at 65 degrees C. The DNA released from the mitochondrial pellet by osmotic shock consists of circular open and closed molecules with a contour length around 5 micro The instability of insect mitochondria in in vitro preparations is discussed.

Brain transfer RNA. II. Analysis of modified nucleosides.

Transfer RNAs were isolated from rat and calf brains and their nucleosides were analysed by tritium derivative technique. Qualitative changes in the minor nucleoside components were compared on the fluorograms which showed differences in the intensities of spots. Cerebellar and cortical tRNAs were also compared, but revealed no significant quantitative differences in their methylated constituants despite 60% higher methyltransferase activity observed in cerebellum compared to cerebral cortex. An overall similarity was noticed between the relative proportions of the major and minor nucleosides of tRNAs derived from rat or calf brain, expressed as mol%. Brain tRNA was also analysed by two-dimensional polyacrylamide gel electrophoresis which showed qualitative and quantitative changes during postnatal development.

Labelling kinetics of ribosomal RNA's in rat brain.

Slices of cerebral cortex and cerebellum from two-week-old rats were incubated in the presence of [14C] uridine and [methyl-3H] methionine. Incorporation of 14C- and 3H-radioactivity into 18S and 28S RNA's of the two tissues was analysed by sucrose-density-gradient centrifugation. The results show that the rates of synthesis of ribosomal RNA's as well as the pattern of methylation in the two tissues were different. Some of these differences may be ascribed to factors such as pool sizes, intracellular rate of transport of the precursors by other pathways, etc. Examination of the results indicates that some differences may consist in the actual biosynthesis and maturation of ribosomal RNA's.

Brain transfer RNA. I. Methylation of newly synthesized tRNA.

Present study demonstrates differences between the labeling patterns of rat cerebral cortex and cerebellum. Although the initial rates of synthesis and methylation of tRNA from these two regions of brain are different, their final degree of methylation is the same in both the cases. Differences in the rates of incorporation of radioactivities in both the tissues can be explained by the differences in the rates of equilibrium of the labeled precursors with the pools. Increased incorporation of labeled uridine into cerebral cortical tRNA following dibutyryl cAMP treatment probably resulted from higher uptake of the labeled precursor due to permeability changes.

Derepression of the glutamine synthetase in neuroblastoma cells at low concentrations of glutamine.

Regulation of the biosynthesis of glutamine synthetase was studied in neuroblastoma cells (Neuro-2A) by use of a recently developed, sensitive radioisotopic assay. The removal of glutamine from the culture medium of these cells for 24 h resulted in a 10-fold increase in glutamine synthetase specific activity (15-fold after 2 weeks) compared with the basal level found in cells grown in the presence of 2 mM glutamine. Following the growth of these cells for 2 weeks in the presence of various concentrations of glutamine, a negative linear correlation was observed between the specific activity of glutamine synthetase (from 1.7 to 0.14 unit/mg) and the concentration of glutamine in the growth medium (from 0.5 to 2 mM). Cycloheximide or actinomycin D blocked the increase in glutamine synthetase activity observed in the absence of glutamine. These results suggest that the removal of glutamine led to the induction of glutamine synthetase by stimulating new enzyme synthesis. The enzyme was not degraded, but only diluted, by growth upon readdition of glutamine to the medium. The influence of glutamine depletion is also reported for C-6 glioma cells and glial cells in primary cultures.