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PubMed1977

Killer double-stranded ribonucleic acid synthesis in cell division cycle mutants of Saccharomyces cerevisiae.

The synthesis of killer double-stranded ribonucleic acid (dsRNA) in Saccharomyces cerevisiae was examined in seven different cell division cycle mutants (cdc) that are defective in nuclear deoxyribonucleic acid replication and contain the "killer character." In cdc28, cdc4, and cdc7, which are defective in the initiation of nuclear deoxyribonucleic acid synthesis, and in cdc23 or in cdc14, defective in medial or late nuclear division, an overproduction of dsRNA at the restrictive temperature was observed. In contrast to the above mutants, the synthesis of killer dsRNA is not enhanced at the restrictive temperature in either cdc8 or cdc21, which are defective in deoxyribonucleic acid chain elongation. Examination of killer sensitive strains (cdc7 K- and cdc4 K-) has shown that the complete killer dsRNA genome is essential for the overproduction of dsRNA at the restrictive temperature.

Cell Division↗
PubMed1977

Zeatin ribonucleosides in the transfer ribonucleic acid of Rhizobium leguminosarum, Agrobacterium tumefaciens, Corynebacterium fascians, and Erwinia amylovora.

Until recently, the presence in transfer ribonucleic acid (tRNA) of the hydroxylated cytokinin ribosylzeatin [N6-(4-hydroxy-3-methylbut-2-enyl)adenosine]was thought to be unique to higher plants. This extension of work from several laboratories indicates the presence of 2-methylthioribosylzeatin in the tRNA of the plant-associated bacteria Rhizobium leguminosarum, Agrobacterium tumefaciens, and Corynebacterium fascians, but not in that of Erwinia amylovora. This cytokinin has the cis configuration, as is normally found in the tRNA's of plants. The tRNA thionucleotide patterns in these bacteria are different from those of Escherichia coli, Bacillus subtilis, and Salmonella typhimurium, which contain the unhydroxylated analogs of ribosylzeatin or 2-methylthioribosylzeatin.

Corynebacterium↗
PubMed1977

Genetic mapping of the F plasmid gene that promotes degradation of stable ribonucleic acid in Escherichia coli.

F+ Escherichi coli cells that contain an srnA mutant allele degrade their stable ribonucleic acid (RNA) extensively after RNA synthesis is blocked at 42 degrees C. The relevant gene promoting degradation of stable RNA, srnB+, or its promoter was mapped between 1.7 and 2.8 kilobases on the F plasmid by using deleted F' plasmids and chimeric plasmids composed of pSC101 and fragments of F plasmid.

Chromosomes, Bacterial↗
PubMed1968

Involvement of protein and ribonucleic acid in the association of deoxyribonucleic acid with other cell components of Escherichia coli.

Cells of Escherichia coli were labeled with precursors of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein, lysed with detergent, and examined by starch-block electrophoresis and CsCl density gradient centrifugation. A large amount of the DNA was seen to remain at positions of low electrophoretic mobility and light density along with tryptophan and arginine-containing proteins and some RNA. Addition of labeled, phenol-extracted DNA to unlabeled cells prior to lysis and electrophoresis showed that only a small amount of the DNA became associated during or after lysis. Sonic treatment of a lysate removed most of the DNA to a position of electrophoretic mobility and density similar to that of free DNA, whereas pronase and ribonuclease released only a part of the DNA. We concluded that binding of DNA to cell membranes or other cell components occurs in the cell prior to lysis and involves protein and probably a specific type of RNA.

Arginine↗
PubMed1968

Protein synthesis during fungal spore germination. II. Aminoacyl-soluble ribonucleic acid synthetase activities during germination of Botryodiplodia theobromae spores.

The specific activities of 13 aminoacyl-soluble ribonucleic acid (sRNA) synthetases were measured at various time intervals during the germination of Botryodiplodia theobromae conidiospores. The enzyme activities were low or absent in ungerminated spores, and they increased rapidly as germination proceeded. When extracts of the ungerminated spores were prepared with mortar and pestle, very little or no enzyme activity was detected. When the extracts were prepared with a mechanical homogenizer, however, they exhibited some enzyme activity, although less than did the extracts from germinated spores. Enzyme activities from germinated spores were approximately the same, regardless of the method of preparation. The enzyme fraction from ungerminated spores prepared with a mechanical homogenizer could also stimulate incorporation of phenylalanine into polyphenylalanine in the presence of ribosomes, polyuridylic acid, and sRNA, although the activity was approximately only 15 to 20% that of a similar enzyme fraction from germinated spores. It is concluded that ungerminated spores of B. theobromae contain active aminoacyl-sRNA synthetases and transfer enzymes, although the activities are low when compared to germinated spores.

Adenosine Triphosphate↗
PubMed1972

Reovirus replicase-directed synthesis of double-stranded ribonucleic acid.

After the incubation of reovirus replicase reaction mixtures (containing labeled ribonucleoside triphosphates), partially double-stranded ribonucleic acid (pdsRNA) products were isolated by cellulose column chromatography followed by precipitation with 2 m NaCl. The pulse-labeled reaction product contained a significantly large amount of pdsRNA that became complete dsRNA as reaction time increased, indicating that pdsRNA was an intermediate of the replicase reaction. The newly synthesized RNA strand ((3)H-labeled) of the pdsRNA was resistant to ribonuclease digestion, suggesting that single-stranded RNA regions were part of a preexistent unlabeled RNA template. These observations, together with the electrophoretic behavior of the pdsRNA in polyacrylamide gel, are consistent with the hypothesis that dsRNA is synthesized by the elongation of a complementary RNA strand upon a preexistent template of single-stranded RNA (i.e., messenger RNA). The direction of the RNA strand elongation was determined by carrying out the replicase reaction in the presence of (3)H-cytidine triphosphate (or (3)H-uridine triphosphate) and adenine triphosphate-alpha-(32)P followed by a chase with excess unlabeled cytidine triphosphate (or uridine triphosphate). The dsRNA product was digested with T1 ribonuclease and the resulting 3'-terminal fragments were isolated by chromatography on a dihydroxyboryl derivative of cellulose. Examination of the ratio of (3)H to (32)P in these fragments indicated that RNA synthesis proceeded from the 5' to 3' terminus.

Animals↗
PubMed1970

Membrane binding of input arbovirus ribonucleic acid: effect of interferon or cycloheximide.

By 1 hr after infection, 36% of input Semliki Forest virus ribonucleic acid (RNA) which was cell associated was found in a membrane structure. This structure had many similarities to the membrane-associated replication complex (MRC) which had previously been identified in arbovirus infections. Interferon treatment did not affect the association of viral RNA with the MRC structure, but cycloheximide treatment inhibited it.

Animals↗
PubMed1971

Inhibition of replication of ribonucleic acid bacteriophage f2 by superinfection with bacteriophage T4.

Superinfection by phage T4 of cells infected by the ribonucleic acid (RNA) phage f2 results in inhibition of further f2 production. Experiments using rifampin show that the exclusion of f2 requires T4 gene function soon after T4 infection. By using a sensitive new peptide-mapping procedure to identify f2 coat protein in infected cells, we show that synthesis of the f2 coat occurs at a reduced level until 4 min after T4 superinfection and then ceases abruptly. Within 4 min after T4 superinfection, there are also several changes in f2 RNA metabolism, all of which require T4 gene function: preexisting f2 replicative intermediate RNA and f2 single-stranded RNA are degraded to small but still acid-precipitable fragments, and most f2-specific RNA is released from polyribosomes. We favor the hypothesis that T4 induces the synthesis of a specific endoribonuclease which degrades f2 RNA and that the inhibition of f2 protein synthesis may be a consequence of this degradation, rather than a direct effect of T4 upon translation.

Autoradiography↗
PubMed1977

Ribosomal ribonucleic acid cistron homologies among Hyphomicrobium and various other bacteria.

The extent of hybrid formation between the ribosomal ribonucleic acid (r-RNA) of Hyphomicrobium strain B-522 and deoxyribonucleic acid (DNA) from bacteria of 21 different genera was examined. Three generalized groupings were formed. Group I (72-100%) consisted entirely of other strains of Hyphomicrobium. Representatives of the genera Rhodopseudomonas, Chromatium, Caulobacter, Prosthecomicrobium, Rhodomicrobium, Hyphomonas, and Hyphomicrobium made up group II (49-69%). The remaining Gram-negative, Gram-positive, and cell wall--less bacteria fell into group III (12-40%). The taxonomic implications of these results are discussed.

Bacteria↗
PubMed1994

Negative regulation of hypothalamic growth hormone-releasing factor messenger ribonucleic acid by growth hormone and insulin-like growth factor I.

Increased growth hormone-releasing factor messenger ribonucleic acid (GRF mRNA) and decreased somatostatin (SRIF) mRNA levels have been reported in the hypothalamus of hypophysectomized rats as well as of dwarf mice. In order to elucidate the effect of the growth hormone-insulin-like growth factor I (GH-IGF-I) axis on hypothalamic GRF and SRIF synthesis, we measured levels of mRNA coding for GRF and SRIF and for pituitary GH in pubertal male rats treated for 3 weeks with antirat GRF gamma-globulin (GRF-ab), anti-SRIF gamma-globulin (SRIF-ab) or both. Immunoneutralization of circulating endogenous GRF resulted in a marked decrease in serum IGF-I and pituitary GH mRNA levels in Northern blot analysis, whereas it caused a significant increase in GRF mRNA levels in the arcuate nucleus as assessed by both Northern blot and in situ hybridization analysis. SRIF mRNA levels in the periventricular nucleus were slightly decreased by GRF-ab treatment when analyzed by in situ hybridization, but not significantly after Northern blot analysis. Immunoneutralization of circulating endogenous SRIF failed to affect mRNA levels of hypothalamic GRF and SRIF but caused a slight reduction in pituitary GH mRNA levels. Levels of mRNA coding for hypothalamic GRF and pituitary GH were also measured by Northern blot analysis in young male rats treated with rat GRF-ab for 2 weeks and replaced with rat GH or IGF-I for the second 1 week. Replacement with either rat GH or IGF-I suppressed the increased hypothalamic GRF mRNA levels. These data indicate that endogenous GRF is essential for normal synthesis of pituitary GH and that both GH and IGF-I negatively regulate the synthesis of hypothalamic GRF.

Animals↗
PubMed2005

Short double-stranded ribonucleic acid as inhibitor of gene expression by the interference mechanism.

The rapid development of the small interfering ribonucleic acid (siRNA)-induced inhibition of the gene expression at the RNA level offers to research groups a new strategy for the understanding of gene functions. The siRNA approach is close to antisense oligonucleotide technology and takes advantage of the progress of chemically synthesized oligoribonucleotides. This approach for the mammalian cells was described by Elbashir et al. at the beginning of 2001, and in this chapter we describe methods for the design of siRNA molecules, solutions for efficiently transfecting cells, and methods for analyzing the inhibition of targeted genes. Methods for in vivo approach are also proposed.

Base Sequence↗
PubMed1975

Posthypoxia stimulation of ribonucleic acid synthesis in the isolated perfused rat heart.

To determine whether hypoxia per se could initiate myocardial ribonucleic acid (RNA) synthesis, isolated rat hearts were subjected to varying degrees of hypoxic perfusion in a modified Langendorff apparatus. Following a 30-min aerobic stabilization period the hearts were perfused for 30 min with media gassed with mixtures containing O:96% O2, 4% CO2, balance N2. Immediately following the stress period, the hearts were perfused for an additional 30 min with oxygenated media containing radiolabeled uridine, and the incorporation into RNA was determined. Exposure of hearts to 48% O2 resulted in a slight but nonsignificant increase in uridine incorporation. Reduction of perfusate oxygen to 24% caused a significant increase in label incorporation into RNA over that of the aerobic controls. Further reductions in oxygen tension resulted in concomitant increases in uridine incorporation, with the highest levels seen following 30 min of anoxia. Performance was not significantly different from that of controls during the aerobic labeling period. Exposure of hearts to brief (5 min) anoxic perfusion also resulted in significant increases in RNA synthesis during the posthypoxic perfusion period. It is concluded that intermittent hypoxia can initiate the biochemical mechanisms which result in increased myocardial RNA synthesis.

Aerobiosis↗
PubMed1972

Sensitivity of ribonucleic acid and deoxyribonucleic acid viruses to different species of interferon in cell cultures.

Although two deoxyribonucleic acid (DNA) viruses, pseudorabies (PsRV) and vaccinia, are as susceptible as a ribonucleic acid (RNA) virus, vesicular stomatitis (VSV), to interferon when tested in chicken or mouse cells, they are refractory to inhibition in interferon-treated primary rabbit kidney cells and in a continuous line (RK-13) of rabbit kidney cells. Superinfection with VSV of RK-13 cells first infected with PsRV completely blocks the replication of PsRV with no effect on VSV yield. When the same experiment is carried out in RK-13 cells pretreated with 1,000 units of interferon, VSV replication is inhibited, which permits PsRV to replicate normally. These findings demonstrate that in the same cell one virus (PsRV) can be refractory to interferon and a second virus (VSV) can be susceptible. These experiments show that rabbit kidney cell cultures are deficient in the synthesis of resistance factors active against the DNA viruses tested and raise the possibility that separate resistance factors may exist for RNA and DNA viruses. In the case of sequential infection of interferon-treated RK-13 cells with vaccinia and VSV, it was found that not only was vaccinia replication refractory to inhibition by interferon, but also that prior infection with vaccinia was able to partially reverse the effect of the inhibitor on the replication of the VSV used for superinfection. On the basis of these and other data it is postulated that a vaccinia virion component or a replication product of vaccinia virus, or both, enables VSV to escape the inhibiting action of interferoninduced resistance factors.

Animals↗
PubMed1981

Simultaneous rates of ribonucleic Acid and deoxyribonucleic Acid syntheses for estimating growth and cell division of aquatic microbial communities.

A method for measuring rates of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) syntheses using a single radioactive precursor has been devised and tested using bacterial cultures and natural assemblages of marine and freshwater microorganisms. The procedure is based upon the uptake and incorporation of exogenous [H]adenine into cellular adenosine triphosphate and deoxyadenosine triphosphate pools which serve as the immediate precursors for the adenine incorporated into RNA and DNA, respectively. It is proposed that the DNA/RNA rate ratio is correlated with the specific growth rate of microorganisms and can be used as an index for estimating and comparing the productivities of microbial assemblages in nature. This technique can also be used to detect discontinuous growth and cell division processes which frequently occur in surface plankton populations. The DNA/RNA rate ratios measured in a variety of aquatic ecosystems ranged from 3.3 to 31.8% without significant correlation to total microbial biomass.

Journal Article↗