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The dissection of RNA editing mechanisms in PHYSARUM: mitochondria has been hindered by the absence of a soluble in vitro system. Based on our studies in isolated mitochondria, insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs is closely linked to transcription. Here we have fractionated mitochondrial lysates, enriching for run-on RNA synthesis, and find that editing activity co-fractionates with pre-formed transcription elongation complexes. The establishment of this soluble transcription-editing system allows access to the components of the editing machinery and permits manipulation of transcription and editing substrates. Thus, the availability of this system provides, for the first time, a means of investigating roles for cis-acting elements, trans-acting factors and nucleotide requirements for the insertion of non-encoded nucleotides into PHYSARUM: mitochondrial RNAs. This methodology should also be broadly applicable to the study of RNA processing and editing mechanisms in a wide range of mitochondrial systems.
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We assessed the value of HIV-1 RNA level compared to soluble immune activation markers, namely neopterin, beta2-microglobulin and soluble TNF receptor 75 (sTNFR-75), to predict the change in the number of CD4+ T cells over a 1-year period, the development of AIDS, and survival (median follow-up 54 months). The study population comprised a cohort of 47 individuals for the analysis of the change in CD4+ T cells and survival (20 died), and a subgroup of 31 individuals with a baseline CD4+ T cells above 200 x 10(6)/l for the development of AIDS (11 developed AIDS). HIV-1 RNA was measured from stored sera by quantitative PCR. The CD4+ T cell count obtained at study entry strongly correlated with baseline serum HIV-1 RNA levels (r=-0.47), and to a lesser extent with neopterin (r=-0.41) and beta2-microglobulin (r=-0.29). The percentage change in CD4+ T cells over a 1-year period correlated with HIV-1 RNA levels (r=-0.32, p=0.03), however, stronger correlations were found for neopterin, beta2-microglobulin and sTNFR-75 (r=-0.51, r=-0.41, r=-0.42; p< 0.01). No progression to AIDS or death was observed in individuals with baseline HIV-1 RNA levels below 20,000 copies/ml (10 of 31 and 15 of 47 individuals, respectively). A Cox's proportional hazard model to predict AIDS revealed that HIV-1 RNA, the change in CD4+ cells over a 1-year period and sTNFR-75 levels independently predict AIDS; the change in CD4+ cells, the absolute CD4+ T cell count and neopterin were jointly significant to predict death. All results were adjusted for nucleoside monotherapy. In conclusion, to improve the predictive power, quantitation of HIV-1 RNA as a 'natural history marker' may be supplemented by measurement of sTNFR-75 for 'early'-stage disease progression and neopterin for 'late'-stage disease progression.
The role of ribonucleic acid (RNA) synthesis in the development of sporangia in the saprolegniaceous mold Achlya was studied. Methods were developed for growing and treating large populations of mycelia so that the hyphal tips would differentiate into sporangia with considerable synchrony. Under the starvation conditions imposed for the differentiation of sporangia, net RNA, deoxyribonucleic acid (DNA), and protein synthesis ceased. However, incorporation of radioactive precursors into RNA continued at a high rate throughout the period of differentiation, showing that the enzymatic mechanism for RNA synthesis was still in an active state. Actinomycin D inhibited the differentiation of sporangia and the incorporation of labeled precursors into RNA. The level of actinomycin used did not inhibit the normal outgrowth and branching of the mycelia that occurred during differentiation. Thus, DNA-dependent RNA synthesis was required for the differentiation of sporangia. Sucrose gradient analysis of newly synthesized RNA showed that only the ribosomal and soluble fractions of RNA were labeled during vegetative growth. During the differentiation of sporangia, ribosomal and soluble RNA fractions were also labeled, and, in addition, a heterodisperse fraction of labeled RNA which was heavier than ribosomal RNA appeared; this fraction was not evident in the newly synthesized RNA from vegetative mycelia.
Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BDeltaCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.
The cytotoxicity of 5-FU and 5-FUrd, given via the hepatic artery, was measured by its incorpotation into the acid soluble fraction, RNA and DNA in normal tissues and an adenocarcinoma transplanted into the liver in rats. Drugs inhibiting the membrane transport of, especially, nucleosides were simultaneously administered by a femoral vein to modulate the cytotoxicity. None of them (dipyridamole, lidoflazine nor dilazep) had any statistically significant influence on the tumour. Dipyridamole and lidoflazine decreased the incorporation of 5-FU into the acid soluble fraction, RNA and DNA of the intestine. Dipyridamole probably decreased the incorporation of 5-FUrd into the acid soluble fraction and RNA of the intestine. Lidoflazine has not been tested with 5-FUrd. Dipyridamole increased the incorporation of 5-FU into the acid soluble fraction of liver, bone marrow and kidney, and of 5-FUrd into the acid soluble fraction of liver and bone marrow and liver RNA. Lidoflazine had fewer adverse effects. Both dipyridamole and lidoflazine increased the combined peak of UTP and FUTP in the liver, and dipyridamole also in the intestine of 5-FU treated rats. Dipyridamole which undergoes an enterohepatic circulation increased the combined peak of UDP-glucuronic acid and FUDP-glucuronic acid in 5-FU and 5-FUrd treated rats, as well as UDP-glucuronic acid in rats given neither 5-FU nor 5-FUrd in the liver. Membrane transport inhibitors seem to offer the opportunity to protect normal tissues from the cytotoxicity of 5-fluoropyrimidines, but the tissues can also be more exposed.
The cytotoxicity of 5-FU, given by the hepatic artery, was measured by its incorporation into the acid soluble fraction, RNA and DNA in normal tissues and an adenocarcinoma transplanted into the liver in rats. Other substances were simultaneously administered by the portal vein to modulate the cytotoxicity. None of them had any significant influence on the incorporation of 5-FU into tumor. Dipyridamole decreased the incorporation of 5-FU into liver RNA and increased the nucleotide/DNA and RNA/DNA ratios so that the incorporation into mg RNA per liver cell was unchanged. Dipyridamole decreased the incorporation into the acid soluble fraction, RNA and DNA of the small intestine and also into RNA per mg DNA. It increased the nucleotide/RNA and RNA/DNA ratios in the bone marrow. Orotate decreased the incorporation into liver and intestinal RNA. Uridine increased the incorporation into liver RNA. The results obtained with dipyridamole were the most pronounced. Studies are continuing with this and other membrane transport inhibitors.
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The properties of a site, on Escherichia coli ribosomes, which binds peptidyl-s-RNA (where s-RNA refers to ;soluble' or transfer RNA) have been investigated. The binding is stable both in low Mg(2+) concentration (0.1mm), and in high Mg(2+) concentration (10mm) in the absence or presence of potassium chloride (86mm). Puromycin has been used to break the bond between the s-RNA and the polypeptide, and in the absence of further protein synthesis this technique exposes free s-RNA molecules on the ribosomes. The s-RNA exposed remains bound in high Mg(2+) concentration, but the binding is unstable in high Mg(2+) concentration with potassium chloride and the s-RNA can be freed completely from the ribosomes by lowering the Mg(2+) concentration. It can also be displaced by s-RNA in the medium. It is suggested that this ribosomal binding site for peptidyl-s-RNA is the site for peptide bond formation. Further, it is proposed that it is the same site that can be demonstrated on ribosomes not engaged in protein synthesis and that, in high Mg(2+) concentration, will bind s-RNA molecules charged or uncharged with amino acids.
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When incubated with a soluble extract from large oocytes of Xenopus laevis, recombinant DNA plasmids containing either X. laevis oocyte 5S DNA or X. borealis oocyte 5S DNA direct the synthesis of discrete 5S RNAs, which by size and sequence analysis are similar or identical to the corresponding 5S RNAs synthesized in vivo. Synthesis of the 5S RNAs is mediated by a soluble endogenous RNA polymerase III (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6), which presumably recognizes specific initiation and termination sites in the 5S genes. Optimal conditions for accurate synthesis and the kinetics of the reactions have been determined. A soluble postchromatin supernatant fraction has also been isolated from immature oocytes. Although devoid of a functional endogenous RNA polymerase III, this extract contains a component(s) that effects the accurate transcription of 5S genes (in a plasmid) by a purified RNA polymerase III.
1. Anaesthetized rats were given [3H]orotic acid either intraperitoneally or via a catheter into the hepatic artery with or without degradable starch microspheres. 2. The radioactivity in the acid soluble and RNA fractions of five pieces of the left lateral liver lobes was determined. 3. A variation of the distribution of the precursor into the different parts of the same liver lobe was shown. 4. This variation was most pronounced (3000-17,000 cpm/micrograms in the acid soluble fraction) when the precursor was administered via the artery and without microspheres. 5. The correlation between the radioactivity in the acid soluble and RNA fractions within each liver piece was 0.85, 0.90 and 0.75 in the three groups respectively. 6. It is suggested that the variation of the distribution depends on circulatory differences within the liver.
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Rats were fed a 0% casein diet for 1 week, with or without enteral or parenteral administration of essential amino acids, or a 25% casein diet, in one group supplemented with 5-fluorouracil treatment. Ninety minutes before sacrifice the rats were given a tracer of [3H]orotic acid. Incorporation into the acid soluble fraction, RNA, and DNA was determined in liver, small intestine, bone marrow, and kidney. Nucleotide profile was examined in liver and intestine. Protein deficiency caused inter alia a decrease in body weight; a decrease in RNA/DNA ratio and an increase in the specific RNA labeling in liver and kidney; an altered nucleotide profile in the liver; an increase in the nucleotide/DNA and RNA/DNA ratios and a decrease in the specific labeling of the acid soluble fraction, RNA, and DNA in the bone marrow. These changes were prevented to the same extent by giving essential amino acids, either orally or intravenously. The minor changes in intestinal nucleotide profile in protein deprivation were prevented to a slightly larger extent by amino acids orally than parenterally. 5-Fluorouracil treatment gave a decrease in the RNA/DNA ratio in the liver and kidney but an increase in the nucleotide/DNA and RNA/DNA ratios in the bone marrow. Nucleotide profiles were unaltered. The amount of DNA per gram of tissue decreased in bone marrow and increased in kidney. Parenteral administration per se resulted in almost no changes.
The gene 1 of the Klebsiella phage K11 encoding the phage RNA polymerase was amplified using the polymerase chain reaction of the Pfu DNA polymerase, cloned and expressed under the control of tac promoter in Escherichia coli. Although the gene was efficiently expressed in E. coli BL21 cells at 37 degrees C, most of the K11 RNA polymerase produced was insoluble, in contrast to soluble expression of the cloned T7 RNA polymerase gene. Coexpression of the bacterial chaperone GroES and GroEL genes together did not help solubilize the K11 RNA polymerase. When the temperature of cell growth was lowered, however, solubility of the K11 RNA polymerase was increased substantially. It was found much more soluble when expressed at 25 degrees C than at 30 and 37 degrees C. Thus, the cloned K11 RNA polymerase gene was expressed in E. coli mostly to the soluble form at 25 degrees C. The protein was purified to homogeneity by chromatography using DEAE-Sephacel and Affigel-blue columns and was found to be active in vitro with the K11 genome or a K11 promoter. The purified K11 RNA polymerase showed highly stringent specificity for the K11 promoter. Low-level cross-reactivity was shown with the SP6 and T7 consensus promoters, while no activity shown with the T3 consensus promoter at all.