Incorporation of labelled precursors into the electrophoretic fractions of rat-liver ribonucleic acid

1. Incorporation of [(32)P]orthophosphate and of [2-(14)C]orotic acid into rat-liver RNA was studied by agar-gel electrophoresis by using u.v.-densitometry and radioautography of dried agar electrophoretograms. 2. During the electrophoresis some low-molecular-weight contaminants, including inorganic phosphate present in the RNA preparations, were separated from the RNA fractions. Since nucleoside mono-, di- and tri-phosphates still interfered, the RNA preparations had to be subjected to a purification procedure [Sephadex G-25 or Dowex 1 (X8)]. 3. In RNA extracted from cytoplasm, isolated microsomes or ribosomes, whatever variations were made in the phenol procedure no special rapidly labelled RNA fraction was detected other than ;soluble' RNA and the ribosomal RNA components. 4. When the whole homogenate or cytoplasmic fraction was treated only with phenol (pH6) a considerable part of the cytoplasmic RNA was not extracted. The treatment of the cytoplasmic fraction with sodium dodecyl sulphate before the addition of phenol increased the yield of the high-molecular-weight RNA and at the same time a higher specific activity was found for the faster ribosomal RNA component. 5. The presence of four distinct rapidly labelled RNA fractions was established in the RNA not extracted by phenol, and they moved slower than the ribosomal RNA. They were extracted only with the use of phenol-sodium dodecyl sulphate at an elevated temperature.     

Studies on cytoplasmic ribonucleic acid from rat liver: IV. Distribution of odd nucleotides in ribonucleic acid fractions

• 1.1. Odd nucleotides are not distributed uniformly in microsomal RNA isolated from rat liver, but appear to be mainly present either in the terminal polynucleotide part of high-molecular-weight microsomal RNA or in microsomal RNA subfractions of lower molecular weight.
• 2.2. An atypical nucleotide, tentatively identified as 1-methyladenylic acid, has been found in a terminal position in a microsomal RNA fraction, isolated by chromatography on Ecteola resins.
• 3.3. Soluble RNA as obtained by conventional methods may in general be contaminated with free mononucleotides including pseudo-uridylic acid. The presence of large amounts of atypical nucleotides in soluble RNA, however, is not due to this type of contamination.
     

Characterization of rapidly labelled rat liver ribonucleic acid showing high affinity for columns of methylated albumin on kieselguhr

Most of the rapidly labelled RNA from rat liver submitted to column chromatography on methylated albumin on kieselguhr remains tightly bound to the column and can only be recovered by elution with m-ammonia. The tightly bound RNA is composed mainly of DNA-like RNA. The binding capacity is dependent not only on base composition but also on molecular size: the heavier RNA molecules show a greater affinity to the column than do the lower-molecular-weight components. Rapidly labelled mouse liver and Saccharomyces cerevisiae RNA show similar behaviour to rat liver RNA on columns of methylated albumin on kieselguhr.     

Ruminal degradability of 15N labelled ribonucleic acid in grass

The ruminal degradation of RNA in rye grass (Lolium perenne) was studied using the bag method. A non-lactating cow (BW 550 kg) fitted with a rumen cannula was used and fed twice daily at maintenance level with a chopped grass hay-based ration containing 30% ground barley. Rye grass, labelled during growth by fertilization with 15N2-urea (9.5 atom% 15N, 20 g N/m2), was cut at seven stages of growth and maturity and freeze-dried. RNA-N represented 6 to 17% of total N. Labelled grass samples (milled to 5.0 mm screen, 5.0+/-0.1 g DM) were incubated in polyester bags (100 x 200 mm, pore size 50 microm) in the rumen for periods of 1, 3, 6, 9, 12, 24, and 48 h. Data of N and RNA disappearances from the bags were fitted to an exponential equation to estimate parameters of degradation. The effective degradability of RNA in the rumen averaged 90+/-4%, for N it was 11% units lower (P < 0.001). Degradability of RNA was correlated to that of N (R2 = 0.92). Degradability of RNA (R2 = 0.96) and N (R2 = 0.93) decreased with increasing fibre content of grass. Increasing the fibre content by 1% diminished the degradability of RNA and N by 1.1% units and 2.4% units, respectively (P < 0.001). Assuming a microbial protein synthesis in the rumen of 150 g/kg DOM, a N: RNA ratio of 1:1.35 in rumen microbes and a rumen outflow rate of 0.06 h(-1), a model calculation indicates that about 9 to 19% of duodenal RNA are of dietary origin in animals fed grass. This should be taken into account for the calculation of microbial N on the basis of RNA as marker.     

Stability of rapidly labelled messenger ribonucleic acid in Bacillus amyloliquefaciens during the phases of minimum and maximum extracellular enzyme formation.

The stability of rapidly labelled hybridizable messenger RNA in both exponential and post-exponential phase cells of Bacillus amyloliquefaciens was measured in terms of the rate of loss of its radioactivity. In the exponential phase, where 96% of the mRNA was specific for cell proteins and only 4% was exoprotein mRNA, the label was lost exponentially from the rapidly labelled hybridizable mRNA fraction with a half-life of six minutes at 30 °C. The antibiotic rifampicin, at a concentration of 10 μg/ml, had no effect on the characteristics of decay of this exponential-phase mRNA. In the post-exponential phase, where there were equal amounts of cell protein and exoprotein-specific mRNA, rapidly labelled hybridizable mRNA decayed exponentially in the presence of rifampicin (10 μg/ml), with a half-life of six minutes at 30 °C. In the absence of rifampicin the characteristics of decay were more complex. The evidence available suggested that this was due to the superimposition of a component attributable to reincorporation of degradation products of radioactive RNA on the characteristic exponential decay pattern of the post-exponential mRNA.Measurement of the stability of active mRNA, by studying the loss of ability to incorporate l-[¹⁴C]leucine into protein in the presence of rifampicin (10 μg/ml), gave half-lives of 4.5 minutes and six minutes, respectively, for exponential and post-exponential material.     

Studies on the metabolism of virus-infected tissues. III. The effect of influenza virus on ribonucleic acid fractions of chick chorio-allantoic membranes

Chromatography on cellulose ion-exchange columns of ribonucleic acid of (RNA) extracted by phenol from isolated chick chorio-allantoic membranes yielded a number of RNA fractions which exhibited differential rates of incorporation of glycine-1-C¹⁴ and adenine-8-C¹⁴. Propagation of influenza A virus in this tissue was associated with an alteration both in the metabolic behavior and Chromatographic behavior of the RNA fractions isolated. The base composition of certain of the RNA fractions were considerably altered in the virus-infected tissue.