Low RNA Content Research Articles

A rapidly sedimenting fraction of rat liver endoplasmic reticulum

ABSTRACT Balance-sheet experiments carried out to account for the distribution of endoplasmic reticulum fragments during subcellular fractionation of rat liver showed that a large proportion of these fragments are present in the pellets of low-speed centrifugation. Using glucose-6-phosphatase and RNA as markers we found that approximately 50 % of the fragments of endo-plasmic reticulum sedimented in the pellet of a 640-g centrifugation, 10 % in that of a 6000-g centrifugation and 35 % in the pellet of a 105000-g centrifugation. Starvation of the animals before use did not alter this distribution, nor did the use of more vigorous homogenization conditions. We have developed a procedure for removing nuclei and erythrocytes from the material sedimenting at 640 g to give a fraction (rapidly sedimenting ER fraction or RS-ER) similar to the standard microsomal preparation. Centrifugation of this RS-ER fraction over 1·3 M sucrose yields sub fractions of high and low RNA content analogous to the rough and smooth microsomal fractions. Electron-microscopic studies showed that, whereas the rough microsomal fraction consisted of ribosome-studded vesicles of varying size and content density, the rough RS-ER fraction contained a mixture of mitochondria and double lamellar membranes with ribosomes attached. These double lamellar membranes closely resemble the endoplasmic reticulum of intact rat liver. The double lamellar membranes are frequently observed grouped in stacks and in close association with the mitochondria. The significance of the association between endoplasmic reticulum and mitochondria of the RS-ER fraction and the relation between it and the standard microsomal preparation are discuased.

Degradation of ribosomal RNA during chicken lens development

During the development of the vertebrate lens it acquires a low RNA content. Evidence indicates the persistence of a low molecular weight RNA while rRNA is depleted. This low molecular weight RNA has been characterized by chromatography, centrifugation and electrophoresis. Data indicate that it is primarily tRNA with small amounts of 5-S RNA. The persistence of the low molecular weight component is shown to be due to the selective retention of tRNA and not the accumulation of rRNA breakdown products. The degradation of rRNA is shown to occur on sedimentable ribonucleoprotein particles.

Reduction of the nucleic acid content of single-cell protein concentrates.

AbstractMethods for reducing the content of nucleic acid in protein concentrates from disintegrated yeast and microalgae were investigated. Protein concentrates were prepared by acid precipitation of extracted protein after cell wall separation. The influence of alkaline protein extraction on the content of RNA in isoelectrically precipitated protein concentrates was studied. It was found that when a strong decrease in the RNA content was obtained, this was followed by a decrease in the yield of protein concentrate. Protein concentrates were also prepared without cell wall separation by precipitation with different agents after cell disintegration. In the precipitates from microalgae, a RNA reduction was obtained. Precipitation of yeast, protein gave no essential reduction with the precipitants used. Precipitation of yeast protein by heating at an alkaline pH gave a protein concentrate with a low content of RNA. A slightly lower RNA content was obtained when the precipitation was performed in the presence of NaCl. The yield of amino acid nitrogen was 70–80% and the RNA content was 1–2%. A process with precipitation at alkaline pH for the production of microbial protein concentrates with a low content of nucleic acid is suggested.

Isolation and Characterization of Epidermal DNA and RNA from Guinea Pig Skin

DNA and RNA were isolated from mammalian epidermis in a relatively small scale procedure. The high purity and native state of the DNA isolated is reflected by its molar absorptivity E (P), its thermal hyperchromicity and its hyperchromicity upon DNase treatment and by its sedimentation profile as well as by its profile in a cesium chloride density gradient. The very low content of protein and RNA, as well as the data of DNA determination, indicate that this method permits the isolation of a highly purified product. This is further substantiated by the determination of UV absorption spectra and by analysis of the base composition. The mammalian skin DNA showed the following properties: Mol. wt. = 3.7 × 10 6 ; melting point=80.7°C; E(P) = 6150; molar N/P ratio=3.67; thermal hyperchromicity=40.9%; hyperchromicity upon DNase treatment=38%; sedimentation coefficient=16.3; boyant density in CsCl=1.6969 g/cm 3 . The guanine, cytosine, adenine and thymine content was 19.0, 20.5, 29.0 and 28.5 moles% respectively. Epidermal RNA was isolated and characterized in terms of phosphorus content, RNA content, and hydrolysis by RNase. By our results, a minimum content of 3mg DNA per 1000mg epidermis (minus stratum corneum) appears reasonable.

Protein synthesis in non-growing yeast. The respiratory adaptation system

1. General aspects of protein and RNA synthesis in yeast during respiratory adaptation, in the absence of growth, are described and compared with those under growth conditions. 2. A procedure for the subcellular fractionation of yeast cells is reported. Four distinct cell fractions can be defined which differ in their chemical constitution and in their function in macromolecular synthesis in vivo. 3. Several classes of ribosomes have been characterized, and their protein-synthesizing capacity in vivo has been examined: only one class, firmly attached to lipoprotein particles (Fraction NM), is active; 80-S ribosomes found in the free state in cell extracts are inert. 4. Fraction NM has been isolated and partially purified. It has a characteristically high content of DNA and a low content of RNA. The largest part of pulselabelled RNA is detected in this fraction, which represents liponucleoprotein particles containing the sites of both RNA and protein synthesis.

Amino acid incorporation by cell fractions from the oviduct of the laying hen and the synthesis of egg-white proteins.

1. Homogenates of the magnum of the hen oviduct have been fractionated by differential centrifuging. 2. Centrifuging at 600g for 10min. gave a pellet containing most of the particulate material of the cell, but on washing this fraction some particles were removed from it. The washed 600g pellet contained most of the DNA of the homogenate. 3. Centrifuging the 600g supernatants at 10000g for 10min. gave particulate fractions (I particles) richer in RNA than other fractions which were active in incorporating amino acids into protein in isolation. When minced tissue was incubated with radioactive amino acids before homogenizing these particles were the most radioactive of the cell fractions. 4. The pellet obtained by centrifuging the 10000g supernatant at 105000g for 60min. was very small; its RNA/protein ratio was raised compared with that of the homogenate. It only incorporated amino acids in isolation to a small extent or not at all. 5. The 105000g supernatant contained a large proportion of the protein of the homogenate. 6. The I particles could be subfractionated by layering over denser sucrose to give a pellet with lower RNA content and incorporating activity, and also suspended material richer in both these properties. 7. Treatment of the I particles with deoxycholate gave rise to particles sedimenting at 105000g for 60min. containing most of the RNA of the original particles, but only about 34% of the protein, and with a high activity in incorporating amino acids. 8. The I particles, or particles derived from them by deoxycholate treatment, required GTP and phosphoenolpyruvate for the incorporation of free amino acids. The omission of ATP reduced the incorporation to varying extents. Chicken-liver cell sap stimulated the activity. 9. Radioactive amino acids linked to transfer RNA were transferred to protein by I particles. GTP and phosphoenolpyruvate were required for this transfer. The phosphoenolpyruvate requirement could not be replaced by increasing the GTP concentration. ATP partially inhibited the transfer. 10. After incorporation by the cell-free system, the hot-trichloroacetic acid extract, but not the lipid extract, was radioactive. Ribonuclease and puromycin inhibited at low concentrations. Lecithinase-C was much less inhibitory. Transfer of amino acid, from a radioactive lipid-amino acid complex prepared from hen oviduct, was not detected. 11. After short periods of incubation of minced tissue with [(14)C]lysine some of the radioactive protein of the isolated I particles behaved as ovalbumin. The distribution of radioactivity in the protein resembled that in ovalbumin in soluble extracts of the tissue obtained after longer periods of incubation.

The histology and histochemistry of oogenesis in the water strider, Gerris remigis say

AbstractIn each ovariole of Gerris remigis, nurse cells arise by mitotic divisions at the anterior end of the germarium. These cells enlarge as they move posteriorly. This size increase is possibly caused by fusion of cells, but probably by endopolyploidy as well. The nurse cells then establish connections with a central trophic core, which receives the products of subsequent nurse cell degradation. Two possible pathways of nuclear degradation are suggested: one involves the condensation of chromatin within the nucleus; the other, the release of DNA as fine granules into the cytoplasm. Cytoplasmic areas containing such DNA are also rich in proteinaceous granules, but have a meager content of RNA. The remainder of the cytoplasm of the mature nurse cells contains a high concentration of RNA, as do the nucleoli.Posteriorly the trophic core connects via nutritive cords with each developing oocyte in the prefollicular region and in the anterior vitellarium. RNA is apparently contributed to the ooplasm via the trophic stream. Patches of cytoplasmic DNA are present in the young oocytes; the origin and fate of this DNA is uncertain.During early oocyte maturation chromosomal stainability decreases, and the nucleolus enlarges. In previtellogenic stages, numerous proteinaceous bodies appear in association with the nucleolus‐chromosome complex. These bodies, like the nucleolus, have only a low RNA content. They may pass to the cytoplasm, but cannot be traced with certainty. During the latter part of this period a complex population of small proteinaceous and lipid preyolk bodies accumulates peripherally in the oocyte. Definitive protein and lipid yolk are probably derived by the enlargement and inward migration of these bodies.The oocytes are each surrounded by a layer of follicle cells proliferated in the prefollicular region. These become binucleate and enlarge as the enclosed oocytes grow and elongate. RNA also increases in the nucleoli and cytoplasm of the follicle cells as they move posteriorly in the vitellarium. There is no evidence of transfer of nucleic acids or protein from the follicle cells to the oocyte.The nurse cells are therefore implicated as the major source of nucleic acids for the maturing oocyte.

Isolation and composition of lens ribonucleic acid

Ribonucleic acid has been isolated from bovine and rabbit lens by extraction of the proteins with phenol. Fractionation by differential centrifugation showed that the RNA was mostly in the supernatant fraction. The low content of RNA in the bovine lens, and its distribution, are discussed. The base composition of the RNA has been determined.