Isolation of a cloned DNA segment containing a ribosomal protein gene of Drosophila melanogaster

A Drosophila genomic DNA library in the vector Charon 4 was screened using cDNA derived from the small (6S-12S) poly(A)+ mRNA of 2-6-h-old Drosophila embryos. This fraction of mRNA is enriched for ribosomal protein-coding sequences. The selected recombinants were hybridized to total mRNA under conditions which allowed for isolation of homologous mRNAs. The mRNA from these RNA/DNA hybrids was eluted and translated in vitro. The translation products were analyzed by one- and two-dimensional electrophoresis with authentic ribosomal proteins as standards. One cloned DNA segment was found to contain a ribosomal protein gene, and a sequence which hybridizes strongly to at least 5 other ribosomal protein mRNAs.     

Comparison of the levels of the 21S mitochondrial rRNA in derepressed and glucose-repressed Saccharomyces cerevisiae.

A cDNA preparation, synthesized by using Saccharomyces cerevisiae mitochondrial RNA as template and oligodeoxythymidylic acid as primer, was found to specifically hybridize to the mitochondrial 21S rRNA by the following criteria: (i) it hybridizes only to the 21S RNA species in mitochondrial RNA and not to RNA from a [rho0] mutant, and (ii) it hybridizes to fragments in restriction digests of mitochondrial DNA that contain the 21S rRNA gene but not to nuclear DNA. This cDNA was used as a probe to demonstrate that a 2.6-fold decrease in the cellular level of the mitochondrial large rRNA is associated with glucose repression of mitochondrial function in S. cerevisiae. A corresponding decrease in the level of mitochondrial DNA was not observed.     

Intracisternal A particles from FLOPC-1 BALB/c myeloma: presence of high-molecular-weight RNA and RNA-dependent DNA polymerase.

Intracisternal A particles from the FLOPC-1 line of BALB/c myeloma have been shown to contain high-molecular-weight RNA (60 to 70S) that is sensitive to RNase, alkali degradation, and heat but resistant to Pronase treatment. The intracisternal A-particle RNA contains tract of poly (A) approximately 180 nucleotides long. As shown in a reconstitution experiment, by antigenic analysis of A-particle preparation and the SC cytopathogenicity assay, the 70S RNA was not due to contamination by type C virus particles. The FLOPC-1 intracisternal A particles also possess an endogenous RNA-dependent DNA polymerase. The enzyme required Mn2+ or Mg2+, dithiothreitol, detergent, and four deoxyribonucleoside triphosphates for maximum activity. Enzymatic activity was maximally stimuated by poly (rC)-oligo (dG)12-18 and less with poly (rG)-oligo (dC)10 or poly (rA)-oligo (dT)12-18 as compare with synthetic DNA/DNA duplex templates such as poly (dA)-oligo (dT)12-18. The enzyme can utilize the A-particle endogenous RNA as template as shown by analysis of the early and late DNA products of the endogenous reaction by CsSO4 isopycnic gradient centrifuation and hybridization of purified 70S or 35S A-particle RNA with the purified complementary DNA product. Approximately 50% of the A-particle complementary DNA also hybridized with oncornavirus RNA.     

Properties of Intracytoplasmic A Particles Isolated from Oncornavirus-Producing Human Cells

A particles with the diameter of 70 to 80 nm were isolated from the cytoplasm of HEp-2, HeLa, and AO cells producing oncornavirus of Mason-Pfizer-like type. Most of the A particles banded at 1.23 to 1.24 g/ml, whereas 3 to 10% banded at 1.29 g/ml in equilibrium sucrose gradients. They banded at 1.30 g/ml in CsCl gradients suggesting that they contained 8% RNA. Individual A particles sedimented at 200 to 250S in velocity sucrose gradients, but their significant part was found aggregated and sedimented at more than 300S. They were resistant to RNase digestion. A particles possessed polymerase activity which was preferentially activated by Mn(2+) rather than by Mg(2+), the RNA template being 60S RNA. Cross-hybridization with two DNA products and immunoassay showed that A particles and Mason-Pfizer-like oncornavirus produced by the same cells contained neither homological RNA sequences nor common antigens, suggesting that A particles are not intracellular precursors of Mason-Pfizer-like oncornavirus but represent an independent oncornavirus. Hybridization of A particle RNA with excess of cellular DNA revealed about 20 proviral copies per HEp-2 cell genome and no proviral copies in human embryo and placenta cell genomes.     

Biochemical and electron microscopical evidence for the presence of oncorna viruses in spleen tissue from two patients with haematological malignancies

In extracts of spleen tissue from two patients with haemotological malignancies an RNA dependent DNA polymerase was found in particle with a density of 1.16, that is at the density of oncorna viruses. After treatment with noniomic detergents the enzyme activity was found in particles with a density of 1.23–1.24, similar to the density of oncorna viral cores. A simultaneous detection test with this core fraction material for 70 S RNA and RNA dependent DNA polymerase was positive for both patients.Electron microscopical inspection of the material with a density of 1.16 revealed immature C-type virus like particles, various stages of maturing particles and a number of particles resembling mature C-type oncorna viruses.In two normal spleens from patients with carcinoma of the colon and oesophagus respectively and in three spleens from patients with no history of malignancy no RNA dependent DNA polymerase was found. Material from one normal spleen was examined in the electron microscope and no virus-like particles were seen.     

Characterization of an Endogenous RNA-Dependent DNA Polymerase Associated with Murine Intracisternal A Particles

An RNA-dependent DNA polymerase associated with intracisternal A particles has been characterized. The enzyme required Mg(2+) or Mn(2+), dithiothreitol and the presence of all four deoxyribonucleoside triphosphates for the expression of maximal activity. Sensitivity of the endogenous RNA-dependent DNA polymerase activity to a low concentration of pancreatic ribonuclease in the presence of a high concentration of NaCl suggested that the enzyme might be utilizing the A particle endogenous RNA as template. Evidence in support of this was provided by analyses of early and late DNA products of the endogenous reaction by Cs(2)SO(4) isopycnic gradient centrifugation and hybridization of purified 60 to 70S and 35S RNAs of A particles with the purified DNA product.     

A new type of prosome-like particle, composed of small cytoplasmic RNA and multimers of a 21-kDa protein, inhibits protein synthesis in vitro

A large fraction of the translationally repressed non-globin messenger RNA in duck erythroblasts is present in non-polyribosomal free mRNP structures which sediment in the 30-40-S range ('35 S'). In 0.5 M KCl, they form core complexes which show a pronounced peak at about 32 S containing mRNA and a discrete spherical RNP particle with a diameter of about 12 nm and the typical morphology of a prosome [H.-P. Schmid et al. (1984) EMBO J. 3, 29-34]. Buoyant density measurements and chromatography on oligo(dT)-cellulose indicate that this particle is bound to mRNA; it can be released from the mRNA by treatment of the free mRNP fraction with SDS. This prosome-like particle inhibits the translation of mRNA in vitro. It is composed primarily of multimers of a single 21-kDa protein and at least one species of RNA of about 80-100 nucleotides. It is resistant to dissociation by 2 M CS2SO4 and 1% SDS; the 21-kDa protein is not attacked by proteinase K unless the particle is extracted with phenol prior to treatment with the protease. The small RNA moiety of the particle hybridizes to the poly(A)-rich mRNA derived from the free mRNPs, as well as to polyribosomal mRNA. These data indicate that prosomes may serve to regulate mRNA translation; they show furthermore that prosome-like particles (about 600 kDa mass) may be built of up to 25 molecules of a single specific protein, rather than of the entire set of about 20 prosomal proteins previously identified.     

Comparison of the Ribonucleic Acid Polymerases of Two Rhabdoviruses, Kern Canyon Virus and Vesicular Stomatitis Virus

A ribonucleic acid (RNA)-dependent RNA polymerase has been demonstrated in Kern Canyon virus (KCV) particles. The RNA product of the KCV polymerase hybridizes to KCV viral RNA. The properties of this viral enzyme have been characterized and compared with those of vesicular stomatitis virus (VSV). RNA polymerases from both viruses require similar conditions of temperature, pH, and detergent and magnesium concentrations for maximal synthesis of RNA. The RNA polymerase contained in the virion of KCV was more dependent on the presence of a sulfhydryl agent than was the VSV enzyme. Under optimal conditions, the specific activity of the VSV polymerase is about twenty-five times as great as that of KCV.     

Further characterization of isolated presumptive euchromatin fractions of mouse hepatoma cells utilizing a cDNA probe complementary to the homologous poly(A)+ mRNA

Poly(A)+ mRNA from mouse hepatoma ascites cell cytoplasm is characterized by three frequency classes: an abundant frequency class of a limited number of different nucleotide sequences, a less abundant frequency class of a larger number of different nucleotide sequences, and a rare frequency class containing a high number of different nucleotide sequences. [3H]cDNA synthesized on this poly(A)+ mRNA template hybridizes with some of the DNAs of the putative transcribable euchromatin fraction at a significantly faster rate than with total DNA if residual contaminating RNA is not removed. Following NaOH incubation to remove such RNA, the cDNA probe hybridized with essentially the same rate to the euchromatin fractions and total DNA. Nick translation of the nuclease-sensitive sequences of chromatin demonstrated that, even with limited nuclease digestion, the excised sequences rapidly converted to small oligonucleotides. The nick-translatable, small chromatin segments showed no enrichment for transcribable sequences. Chromatin segments, which distribute to the 50S-70S glycerol gradient fractions and which satisfy several of the presumptive criteria for enrichment for transcribable sequences, therefore show no enrichment for sequences complementary to the cDNA for poly(A)+ mRNA.     

Isolation of intracisternal type A-particles and associated high-molecular-weight RNA after cell disruption by nitrogen cavitation.

The liberation of intracisternal A-type particles from the endoplasmic reticulum of mouse plasmacytoma cells (line MPC-11) without the use of detergents, has been achieved by the technique of nitrogen cavitation. By this method cell rupture is due to a sudden decompression of a cell suspension after being equilibrated with nitrogen at high pressure, and this results in a relatively efficient release of A-type particles from the cisternae as judged by electron microscopy and the amount of A-particle-associated RNA that may be isolated. Comparative studies showed that at least 50% of the particles are released from the endoplasmic reticulum during nitrogen cavitation. Since the release is achieved purely by mechanical means, the A-particle preparation obtained may be useful for assaying these particles for biological activity. A further release of particles from the endoplasmic reticulum may be achieved by a combination of detergent treatment and mechanical shearing. About 30% of the particle-associated RNA was polyadenylated. As determined by velocity centrifugation, the polyadenylated RNA in the preparation of A-particles released during nitrogen cavitation and that of the detergent-released A-particles were similar with respect to the high-molecular-weight RNA species present. A dominant 35 S RNA species with a 30 S shoulder was observed in addition to a 24 S component in both preparations, but no significant amount of 60–70 S RNA.     

Replication of double-stranded RNA in particles of Penicillium stoloniferum virus S.

RNA polymerase activity was assayed in different particle classes of Penicillium stoloniferum virus S. RNA polymerase activity was found to be associated with H particles, which contain double-stranded RNA and single-stranded RNA, but not with L particles, which contain only double-stranded RNA and not with M particles, which contain only single-stranded RNA. In H particles the reaction occurred with the formation of one new molecule of double-stranded RNA (or two complementary single strands of RNA) per virus particle and the production of product particles (P particles), which contained two molecules of double-stranded RNA (or its equivalent). This RNA polymerase is therefore a replicase, which catalyses the synthesis of the two complementary strands of double-stranded RNA in a single virus particle. This is the first report of this type of RNA polymerase system.     

Small reovirus-specific particle with polycytidylate-dependent RNA polymerase activity.

We previously reported that virus-specific particles with polycytidylate [poly(C)]-dependent RNA polymerase activity accumulated at 30 degrees C in reovirus-infected cells. These particles sedimented heterogeneously from 300 to 550S and traversed through a 40% glycerol cushion to the pellet in 3 h at 190,000 x g. In the present report, we found that smaller particles with poly(C)-dependent RNA polymerase activity remained in the glycerol cushion. These smaller, enzymatically active particles, when purified, sedimented at 15 to 1S. They were spherical or triangular with a diameter of 11 to 12 nm. They were comprised mostly, and likely solely, of one reovirus protein, sigma NS. No particles with poly(C)-dependent RNA polymerase activity were found in mock-infected cells. Chromatography on the cation exchanger, CM-Sephadex, ascertained that sigma NS was the poly(C)-dependent RNA polymerase and showed its existence in two forms. In one form, it was enzymatically active and eluted from the column at 0.5 M KCl. In the enzymatically inactive state, it did not bind to the column. Our results suggest that the enzymatically active form of sigma NS carries a greater net positive charge than the inactive form. They also suggest that both forms of sigma NS are associated with a particle which has poly(C)-dependent RNA polymerase activity.