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PubMed1961

Experimentally induced changes in the base composition of the ribonucleic acids of isolated nerve cells and their oligodendroglial cells.

The effect of tricyano-amino-propene, a dimer of malononitrile, on the base composition of the RNA in isolated Deiters' nerve cells and their oligodendroglial cells has been studied using a microelectrophoretic method. Tri-a-p in a dose of 20 mg/kg has the effect of increasing the RNA and protein content per nerve cell by 25 per cent and decreasing the glia RNA by 45 per cent. The RNA base composition of the nerve cells from the control animals differs from that of their glial cells. The guanine of the nerve cell is significantly higher than that of the glia, but the content of cytosine is higher in the glia than in the RNA of nerve cell. The cytosine of nerve cells decreased significantly after tri-a-p administration. In the glial cells the cytosine showed a 20 per cent increase, and the guanine a 25 per cent decrease. Tri-a-p sharpened the difference in RNA composition already existing between the control nerve cells and their glial cells by almost 300 per cent for the guanine and by 400 per cent for the cytosine. The chemical and functional relationship between the nerve cell and its oligodendroglial cells is discussed.

Animals↗
PubMed1961

The intracellular distribution and heterogeneity of ribonucleic acid in starfish oocytes.

A study has been made of the content and composition of RNA in cytoplasm, nucleoplasm, and nucleoli from growing oocytes of the starfish Asterias rubens. The determinations were carried out, using ultramicrochemical methods, on units isolated by microdissection from fixed sections. Macrochemical and interferometric control experiments show that RNA can be quantitatively evaluated in this way. The results show that the growing oocyte represents a system in which the relations between the quantities of nucleolar, nucleoplasmic, and cytoplasmic RNA undergo great changes. These changes are continuous for nucleolar and cytoplasmic RNA so that their amounts may be predicted from the size of the cell. Nucleoplasmic RNA, on the other hand, shows great variations among different cells, independent of cell size. Purine-pyrimidine analyses show that each cell component contains an RNA which differs significantly from that of the other two. Cytoplasmic and nucleolar RNA are closely related, the only difference being a slightly higher guanine/uracil quotient for the nucleolar RNA. They are both of the usual tissue RNA type, i.e., they show a preponderance of guanine and cytosine over adenine and uracil. Nucleoplasmic RNA deviates grossly from the RNA of the other two components. Here the concentrations of adenine and uracil are higher than those of guanine and cytosine, respectively. This RNA consequently shows some resemblance to the general type of animal DNA although the purine/pyrimidine ratio is far from unity. Our data favor a nucleolar origin for the stable part of the ribosomal RNA and a nucleoplasmic one for the unstable part (the messenger RNA).

Adenine↗
PubMed1960

Studies on the function of intracellular ribonucleases. II. The interaction of ribonucleprotein and enzymes.

To determine the possible significance of in vivo or in vitro enzyme action in ribonucleoprotein systems, rat liver microsomes and ribonucleoprotein particles (RNP) prepared from them by deoxycholate treatment were incubated for 1 hour at 37 degrees C. with crystalline pancreatic ribonuclease (RNase) or various RNase-free crystalline proteolytic enzymes. The extent of the degradation of the RNA of the microsomes and RNP was determined and the protein degradation estimated in both cases. With either microsomes or RNP, RNase (0.5 to 1.0 mg. per ml.) degraded from 75 to 95 per cent of the RNA, with little protein breakdown being apparent when microsomes were used but with significant protein degradation in the RNP. When microsomes were treated with proteolytic enzymes approximately 40 to 50 per cent of the original microsomal protein became nonsedimentable while at the same time 60 to 80 per cent of the RNA was also found to be non-sedimentable. Of the non-sedimentable RNA, approximately one-third was in the form of acid-precipitable RNA while the remainder was in the form of acid-soluble nucleotides. When RNP was treated with proteolytic enzymes, about 95 per cent of the RNA could no longer be sedimented. About half of this appeared as acid-precipitable RNA and half as acid-soluble nucleotides. Both microsomes and RNP contained significant RNase activity with RNP exhibiting about 10 times the specific activity of microsomes. Some of the characteristics of this RNase activity were determined and the results with proteolytic enzymes interpreted in light of this activity.

Animals↗
PubMed1960

Cytoplasmic ribonucleoprotein components of the Novikoff hepatoma.

Prominent nucleoprotein sedimentation boundaries were demonstrable in cytoplasmic extracts of Novikoff hepatoma. Fractionation of the homogenates by differential centrifugation or a density gradient method revealed that 65 to 75 per cent of the cytoplasmic ribonucleic acid was present in the form of free ribonucleoprotein particles. After purification by differential centrifugation in dilute buffer, the particles contained 37 per cent RNA, very little lipid, and no demonstrable membrane material. Ultracentrifugal boundaries corresponding to those seen in the original extracts were present, the main component having an s20, (w) of 81 S. Upon exposure to chelating agents, the particles dissociated through an intermediate component with sedimentation rate of 56 S to a final stage in which 46 and 28 S subunits were present in a weight ratio of 2:1. ATP and pyrophosphate were equally effective in causing dissociation. ADP was considerably less effective. Treatment of the purified particles with deoxycholate removed one-third of the protein and significantly altered the ultracentrifugal pattern. The particles now dissociated directly to the 46 and 28 S subunit when exposed to chelating agents. Upon electron microscopy, the 81 S particle appeared as an oblate spheroid 24 mmicro in diameter. The 46 and 28 S subunits also appeared spheroidal.

Animals↗
PubMed1960

A cytochemical study on the pancreas of the guinea pig. 6. Release of enzymes and ribonucleic acid from ribonucleoprotein particles.

Ribonucleoprotein (RNP)(1) particles isolated by DOC treatment from pancreatic microsomes have a RNA content of 35 to 45 per cent of their dry weight. In the analytical ultracentrifuge about 85 per cent of the material has a sedimentation coefficient of approximately 85 S. These particles contain amylase, RNase, and trypsin-activatable proteolytic activities which cannot be washed off or detached by incubation in 0.44 M sucrose. The enzymes are released, however, by incubation in the presence of low concentrations of ATP, PP, or EDTA, and high concentrations of IP and AMP. At the same time, and at the same concentrations, approximately 80 per cent of the RNA and approximately 25 per cent of the protein of the particles becomes also non-sedimentable. The simultaneous addition of Mg(++) to the incubation medium prevents these losses. This finding, together with the observation that all the Mg(++) of the particles is released by the same agents, makes it likely that Mg(++) holds the particles together, and that its removal by the chelators used causes the particles to disintegrate. These findings are discussed in relation to the molecular structure of the RNP particles.

Amylases↗
PubMed1960

Immune cytolysis. 1. The release of ribonucleoprotein particles. 2. Membrane-bounded structures arising during cell fragmentation.

It has been shown that Krebs ascites tumor cells incubated in vitro with immune gamma globulin and complement lose the bulk of their cytoplasmic RNA to the suspending medium, although the cell membrane remains visibly intact. The present experiments show that about four-fifths of the lost RNA is sedimented by centrifugation of the cell-free medium at 105,000 g. Electron microscopic and chemical analyses of the pellets show them to consist of 150 A ribonucleoprotein particles. It is concluded that most of the RNA passes from the cells in this form. Antibody-complement action causes osmotic swelling of the tumor cells and they become quite fragile. Fragmentation of such preparations yields large numbers of membrane-bounded spheres which may be separated from the heavier nuclei by differential centrifugation. Electron microscopic study of the spheres provides evidence that they can arise from segments of the cell surface as well as from mitochondria and the endoplasmic reticulum.

Animals↗
PubMed1960

Extraction, hydrolysis, and electrophoretic analysis of ribonucleic acid from microscopic tissue units (microphoresis).

A procedure for the purine-pyrimidine analysis of RNA in the 100- to 1000 micromicrog. range is presented. It includes hydrolysis and electrophoretic analysis of RNA, which is extracted from single isolated tissue units, like single cells. The quantitative determination of the separated compounds is carried out by a photographic-photometric procedure in ultraviolet light. The determined values show a coefficient of variation of about +/-7 per cent on test substance. Microelectrophoretic analyses of RNA from different sources have been performed and are compared to macrochemical analyses. The agreement is good in those cases in which it is possible to get any information at all through macrochemical analyses.

Hydrolysis↗
PubMed1960

Composition of ribonucleic acid from various parts of spider oocytes.

Microphoretic purine-pyrimidine analyses of the ribonucleic acid (RNA) in nucleoli, nucleoplasm, cytoplasm, and yolk nuclei of spider oocytes have been carried out. The material necessary for the analyses was isolated by micromanipulation. Determinations of the amounts of RNA in the different parts of the cell were also performed. No differences between the composition of RNA in the nucleolus and the cytoplasm could be disclosed. Nucleoplasmic RNA was, on the other hand, distinctly different from that in the nucleolus and in the cytoplasm. The difference lies in the content of adenine, which is highest in nucleoplasmic RNA. The few analyses carried out on yolk nuclei showed their RNA to be variable in composition with a tendency to high purine values. The cytoplasm contains about 99 per cent of the total RNA in these cells, the nucleoplasm about 1 per cent, and the nucleolus not more than 0.3 per cent, although the highest concentrations are found in these latter structures. When considered in the light of other recent findings the results are compatible with the view that nucleolar RNA is the precursor of cytoplasmic RNA.

Adenine↗
PubMed1961

A comparative electron microscopical study of RNA from different sources.

Electron micrographs of ribosomal RNA from Escherichia coli, microsomal RNA from calf, rat, and chick liver, Bacillus cereus RNA and E. coli soluble RNA are presented. Filaments of about 10 A in diameter could be observed in preparations obtained from aqueous solutions of high molecular weight RNA. When ammonium acetate solutions were used a tendency for coiling and aggregation was observed. E. coli soluble RNA appears as small, sometimes elongated particles the smallest diameter being of about 10 A.

Animals↗
PubMed1961

Localization of macromolecules in Escherichia coli. II. RNA and its site of synthesis.

The distribution of RNA in cells of E. coli 15 T(-)U(-) labeled with uridine-H(3) was studied by methods involving the analysis of radioautographic grain counts over random thin cross-sections and serial sections of the cells. The results were correlated with electron microscope morphological data. Fractionation and enzyme digestion studies showed that a large proportion of the label was found in RNA uracil and cytosine, the rest being incorporated as DNA cytosine. In fully labeled cells the distribution of label was found to be uniform throughout the cell. The situation remained unchanged when labeled cells were subsequently treated with chloramphenicol. When short pulses of label were employed a localization of a large proportion of the radioactivity became apparent. The nuclear region was identified as the site of concentration. Similar results were obtained when cells were exposed to much longer pulses of uridine-H(3) in the presence of chloramphenicol. If cells were subjected to a short pulse of cytidine-H(3), then allowed to grow for a while in unlabeled medium, the label, originally concentrated to some extent in the nuclear region, was found dispersed throughout the cell. The simplest hypothesis which accounts for these results is that a large fraction of the cell RNA is synthesized in a region in or near the nucleus and subsequently transferred to the cytoplasm.

Cell Nucleus↗