Showing papers on "RNA published in 1974"

Identification of Methylated Nucleosides in Messenger RNA from Novikoff Hepatoma Cells

Abstract: The poly(A) tract found in eukaryotic mRNA was used to study methylation in mRNA obtained from Novikoff hepatoma cells. Methyl labeling of RNA was achieved with L-[methyl-3H]methionine under conditions that suppress radioactive incorporation into the purine ring. RNA that contains a poly(A) segment was obtained from polysomal RNA by chromatography on oligo(dT)-cellulose. Sucrose density gradient centrifugation of this RNA revealed a pattern expected for mRNA. The composition of the methyl-labeled nucleosides in the RNA was analyzed after complete enzymatic degradation to nucleosides. By use of DEAE-cellulose (borate) chromatography, which separates 2′-O-methylnucleosides from normal and base-methylated nucleosides, about 50% of the radioactivity was recovered in the 2′-O-methylnucleoside fraction and 50% in the base-methylnucleoside fraction. High-speed liquid chromatography (Aminex A-5) of the 2′-O-methylnucleoside fraction produced four peaks coincident with the four 2′-O-methylnucleoside standards. Analysis of the base-methylnucleoside fraction revealed a unique pattern. While ribosomal RNA and tRNA possessed complex base-methylnucleoside patterns, the distribution in mRNA was quite simple, consisting predominantly of N6-methyladenosine. These results demonstrate a unique distribution of methylated nucleosides in mRNA. By analogy to ribosomal RNA synthesis, the presence of methylnucleosides in mRNA may reflect a cellular mechanism for the selective processing of certain mRNA sequences.

Female steroid hormones and target cell nuclei.

Abstract: The data discussed herein demonstrate the great variation in target-tissue response that can occur after administration of steroid hormones. The female sex steroids can exert regulatory effects on the synthesis, activity, and possibly even the degradation of tissue enzymes and structural proteins. Each response, nevertheless, appears to be dependent on the synthesis of nuclear RNA. In many instances, the steroid actually promotes a qualitative change in the base composition and sequence of the RNA synthesized by the target cell, implying a specific effect on gene transcription. Most important is our direct quantitative evidence that sex steroids cause a net increase in the intracellular amounts of specific mRNA molecules in target tissues. It thus appears that we are discovering a pattern of steroid hormone action which includes (Fig. 1): (i) uptake of the hormone by the target cell and binding to a specific cytoplasmic receptor protein; (ii) transport of the steroid-receptor complex to the nucleus; (iii) binding of this "active" complex to specific "acceptor" sites on the genome (chromatin DNA and acidic protein); (iv) activation of the transcriptional apparatus resulting in the appearance of new RNA species which includes specific mRNA's; (v) transport of the hormone-induced RNA to the cytoplasm resulting in synthesis of new proteins on cytoplasmic ribosomes; and (vi) the occurrence of the specific steroid-mediated "functional response" characteristic of that particular target tissue. To elucidate fully the mechanism of steroid hormone action we must study the biochemistry of the process by which information held by the steroid hormone-receptor complex is transferred to the nuclear transcription apparatus. If our assumptions are correct, we should ultimately be able to discover how this hormone-receptor complex exerts a specific regulatory effect on nuclear RNA metabolism. Such regulation might be achieved (i) by direct effects on chromatin template leading to increased gene transcription and thus RNA synthesis; (ii) by activation of the polymerase complex itself; (iii) by inhibition of RNA breakdown; or (iv) by intranuclear processing of large precursor molecules so that smaller biologically active sequences are produced, and (v) by transport of RNA from the nucleus to the cytoplasmic sites of cellular protein synthesis.

The dna of caenorhabditis elegans

Abstract: Chemical analysis and a study of renaturation kinetics show that the nematode, Caenorhabditis elegans, has a haploid DNA content of 8 x 10(7) base pairs (20 times the genome of E. coli). Eighty-three percent of the DNA sequences are unique. The mean base composition is 36% GC; a small component, containing the rRNA cistrons, has a base composition of 51% GC. The haploid genome contains about 300 genes for 4S RNA, 110 for 5S RNA, and 55 for (18 + 28)S RNA.

Three abundance classes in HeLa cell messenger RNA

Abstract: Approximately 35,000 different poly(A)-containing RNA sequences are present in HeLa cell cytoplasm. The sequences are grouped in three distinct abundance classes.

Role of DNA-Dependent RNA Polymerase III in the Transcription of the tRNA and 5S RNA Genes

Abstract: Mouse myeloma cells have previously been shown (L. B. Schwartz, V. E. F. Sklar, J. A. Jaehning, R. Weinmann & R. G. Roeder, submitted for publication) to contain two chromatographically distinct forms of RNA polymerase III (designated IIIA and IIIB). The enzymes are unaffected by low α-amanitin concentrations which completely inhibit RNA polymerase II, but they exhibit characteristic inhibition curves (identical for IIIA and IIIB) at higher toxin concentrations. RNA polymerase I was unaffected at all α-amanitin concentrations tested. Myeloma RNA polymerases II, IIIA, and IIIB appear to be inhibited by the same mechanism, since the toxin rapidly blocks chain elongation by each enzyme. The characteristic α-amanitin sensitivity of RNA polymerase III has been employed in studies of the function(s) of the class III RNA polymerases. Isolated myeloma nuclei and nucleoli continue to synthesize RNA via the endogenous RNA polymerases when incubated in vitro. With nuclei, newly synthesized 4S precursor (pre-4S) and 5S RNA species were detected by electrophoretic analysis either of the total nuclear RNA or of the RNA released into the supernatant during incubation. The synthesis of both pre-4S and 5S RNA species was inhibited by α-amanitin, but only at high concentrations; and the α-amanitin inhibition curves for these RNAs were identical to those obtained for solubilized RNA polymerases IIIA and IIIB. In control experiments it was shown that the endogenous RNA polymerase II activity of isolated nuclei was inhibited by α-amanitin concentrations similar to those required to inhibit purified enzyme II. However, 40-50% of the endogenous activity of nuclei and 100% of the endogenous activity of purified nucleoli was completely resistant to the high α-amanitin concentrations necessary to inhibit the RNA polymerase III activities. These experiments rule out nonspecific inhibitory effects in the endogenous systems. These results unequivocally demonstrate the role of RNA polymerase III (IIIA and/or IIIB) in the synthesis of (pre) 4S RNAs and a 5S RNA species.

A biochemical mechanism for the delayed cytotoxic reaction of 6-mercaptopurine.

Abstract: 6-Thioguanosine-35S and β-2′-deoxythioguanosine-35S were identified in venom hydrolysates of RNA and DNA isolated from 6-mercaptopurine (MP)-35S-treated L5178Y cell cultures. Digestion with purified phosphodiesterases demonstrated that MP is incorporated as thioguanine (TG) nucleosides in 3′,5′-phosphodiester linkages in DNA and RNA. 3′-Nucleotides of TG-35S were released from 35S-labeled DNA and RNA by digestion with micrococcal nuclease plus spleen phosphodiesterase. The 5′-ribonucleotide of TG-35S was released from 35S-labeled RNA by digestion with venom phosphodiesterase, and the 5′-deoxyribonucleotide of TG-35S was released from 35S-labeled DNA by digestion with pancreatic deoxyribonuclease plus venom phosphodiesterase. A relationship between delayed cytotoxicity and incorporation of MP as TG into nucleic acids was apparent in the following observations. Mycophenolic acid protected L5178Y cells against the delayed cytotoxic effect of MP and suppressed MP incorporation into nucleic acids. A spontaneous development of partial tolerance to MP in two lines of L5178Y cells was associated with a reduced capacity for incorporation of MP as TG into DNA and RNA. 6-Methylthioinosine potentiated cytotoxic effects of MP in a partially tolerant cell line and stimulated incorporation of MP into DNA and RNA. Nucleic acid-incorporated TG was the major thiopurine derivative in MP-sensitive cells at the time of the delayed cytotoxic reaction to MP exposure. Pulse exposure of cells to equitoxic concentrations of MP-35S and TG-35S resulted in similar levels of incorporation of TG-35S in nucleic acids. The delayed cytotoxic activity of MP is probably a consequence of MP anabolism to TG nucleotides followed by incorporation of the latter into DNA. It is likely that the same biochemical mechanism is responsible for delayed cytotoxic effects of both MP and TG.

Characterization of a highly efficient protein synthesizing system derived from commercial wheat germ.

Abstract: A crude extract of commercial wheat germ is capable of translating mRNAs from widely different sources with high efficiencies. Of six wheat germs analyzed only one was found capable of a high level or incorporation with natural mRNAs. Under optimum conditions at a saturating level of Tobacco Mosaic Virus (TMV) RNA (4.5 mug) and labeled amino acid, 68% of all the available (14)C leucine is incorporated in 70 min. at 30 degrees C with a stimulation of 425 fold above background (with an efficiency of 252 moles leucine/mole TMV RNA). Thus this system which is 30 fold more efficient for TMV translation than previous reported wheat germ cell free systems is capable of yielding 568 pmoles of (14)C leucine incorporated into protein in a 50 mul assay. 80% of the proteins produced have a molecular weight greater than TMV coat protein (17,400). This level of incorporation requires optimization of extract concentration, pH, Mg(+2), K(+) and spermine concentration as well as the method of extract preparation. Samples of crude polysomal RNA from hen oviducts (3% mRNA) and chorionating moth follicular cells (1% mRNA) are also translated in the wheat germ cell free system with high efficiency.

Heterogeneity of RNA protein antigens reactive with sera of patients with systemic lupus erythematosus. Description of a cytoplasmic nonribosomal antigen.

Abstract: A soluble cytoplasmic RNA protein antigen (La) is described, which precipitates with sera of patients with systemic lupus erythematosus and a small number of patients who lack antinuclear factors but who have various connective tissue symptoms. Antibodies to the cytoplasmic RNA protein are almost invariably (8 of 9 cases) accompanied by antibodies to a non-nucleic acid cytoplasmic antigen, Ro. Description of this antigen enlarges the spectrum of nucleic acid antigens reactive with the sera of patients with systemic lupus erythematosus.

Total Reconstitution of Functionally Active 50S Ribosomal Subunits from Escherichia coli

Abstract: Total reconstitution of 50S subunits from E. coli was achieved by a two-step incubation procedure. In the first step, 23S RNA, 5S RNA, and the total proteins from 50S subunits were incubated for 20 min at 40° in the presence of 4 mM Mg++ and 400 mM NH4Cl. In the second step, the Mg++ concentration was raised to 20 mM and the incubation was performed for 90 min at 50°. No requirement for 30S subunits or other components (e.g., polyamine) was found. The reconstituted particle has the same sedimentation coefficient as the native 50S subunit and is highly active in protein synthesis with natural (R17 RNA) and artificial [poly(U)] messengers as well as in tests for peptidyltransferase (fragment assay) and for binding of antibiotics (chloramphenicol).

Introduction to Modern Virology

Abstract: Preface Towards a definition of a virus How to handle animal viruses The structure of viruses Viral nucleic acids The process of infection I: Attachment and penetration The process of infection IIA: The Baltimore classification The process of infection IIB: The replication of viral DNA The process of infection IIC: RNA synthesis by RNA viruses The process of infection IID: RNA viruses with a DNA intermediate and vice versa The process of infection III: The regulation of gene expression The process of infection IV: The assembly of viruses Lysogeny Interactions between viruses and eukaryotic cells The immune system and interferon Virus-host interactions Vaccines and chemotherapy: the prevention and treatment of virus diseases Carcinogens and tumour viruses The evolution of viruses HIV and AIDS Trends in virology The classification and nomenclature of viruses

Structural Proteins of Mammalian Oncogenic RNA Viruses: Multiple Antigenic Determinants of the Major Internal Protein and Envelope Glycoprotein

Abstract: The antigenic determinants of two purified protein constituents of mammalian C-type RNA viruses, the major structural protein of about 30,000 daltons, and the membrane glycopeptides of about 70,000 daltons were examined by competition radioimmunoassay. By the appropriate choice of antiserum and competing proteins, it was possible to distinguish type-specific, group-specific, and interspecies determinants. Both of the viral constituents were found to contain each of these three classes of antigens. The results suggested that the majority of the determinants of the major structural protein were group specific, 5% to 30% were interspecies, and a small fraction were type specific. In the case of the envelope glycopeptides, the chief determinants were type and group specific, and a small fraction were interspecies.

Macromolecular Composition During Steady-State Growth of Escherichia coli B/r

Abstract: By using Escherichia coli B/r, the cellular amounts of ribonucleic acid (RNA) and protein were determined as a function of the steady-state growth rate (0.67 to 2.40 doublings per h) by a method which combines measurements of the RNA to deoxyribonucleic acid (DNA) ratio and the differential rate of ribosomal protein synthesis with the Cooper and Helmstetter theory of DNA replication. The results indicate that the ratios RNA/DNA, RNA/protein, and protein/DNA give linear relationships with the growth rate (above 1.2 doublings per h), whereas RNA/cell and protein/cell show a more complex growth rate dependency. The significance of these relationships is discussed. Finally, a detailed description of the growth parameters and composition of E. coli B/r is presented.

Mapping of adenovirus 2 RNA sequences in lytically infected cells and transformed cell lines.

Abstract: The strands of the six EcoRI fragments and the HpaI fragments E and C of Ad2 DNA were separated by electrophoresis in agarose gels. Using 32P-labeled fragment strands in solution hybridization experiments, the fraction of each strand complementary to RNA extracted from infected or transformed cells was assayed by chromatography on hydroxylapatite. In this manner, a tentative map of the cytoplasmic RNA sequences has been constructed for viral RNA extracted from cells both early and late during infection (see Fig. 16; in the map shown, the two strands of Ad2 are named the r and l strands following the bacteriophage convention). Since early cytoplasmic RNA anneals to four distinct regions of the genome, Ad2 probably codes for at least four early gene functions. Summation experiments have shown that all RNA sequences found in the cytoplasm of cells early during infection are also present in the cells' cytoplasm at late times. Viral RNA sequences in five independently isolated and cloned transformed rat cell lines were also mapped on the Ad2 genome. One class of Ad2-transformed rat cells contains RNA sequences complementary to only the segment of Ad2 DNA from 0.03-0.10 on the physical map, and this corresponds to one of the four regions of the genome expressed early during infection. If a viral gene product is necessary to maintain the transformed phenotype of the cell or codes for the virus-specific tumor (T) antigen, this genetic information must be at the left end of the genome (see Fig. 16). The two other classes of Ad2-transformed rat cells contain viral RNA sequences complementary to two or three of the regions of the genome transcribed into early cytoplasmic RNA. At both early and late times during the lytic cycle, the nucleus of the infected cell contains viral RNA sequences that are not transported to the cell's cytoplasm, suggesting that RNA processing and selection may play a role in the regulation of viral mRNA production.

Transcription of DNA from the 70S RNA of Rous Sarcoma Virus I. Identification of a Specific 4S RNA Which Serves as Primer

Abstract: The enzymatic transcription of DNA from the 70S RNA of Rous sarcoma virus (RSV) is initiated on the 3′ terminus of a molecule of 4S RNA which is hydrogen bonded to the viral genome. We labeled this primer with radioactive deoxynucleotides, and demonstrated that its release from 70S RNA by thermal denaturation was accompanied by a reduction in the template activity of the viral RNA. Two-dimensional electrophoresis in polyacrylamide gels separated the 4S RNAs associated with the 70S RNA of RSV into approximately eight fractions, each of which appeared to contain a discrete species of tRNA. The RNA in one of these fractions served as the principal primer for initiation of DNA synthesis by both detergent-disrupted virions of RSV and purified RNA-directed DNA polymerase with RSV 70S RNA as template.