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At least 145 records · Page 8Linked to original sources

PubMed1991

De novo generation of defective interfering RNAs of tomato bushy stunt virus by high multiplicity passage.

Defective interfering (DI) RNAs were generated de novo in each of 12 independent isolates of tomato bushy stunt virus (TBSV) upon serial passage at high multiplicities of infection (m.o.i.) in plants, but not in any of 4 additional isolates after 11 serial passages at low m.o.i. The DI RNAs were detected in RNA isolated from virus particles and in 2.3 M LiCl-soluble RNA fractions isolated from inoculated leaves. Symptom attenuation leading to persistent infections was closely correlated with the passage in which DIs first developed. Comparisons of nucleotide sequences of 10 cDNA clones from 2 DI RNA populations and with a previously characterized TBSV DI RNA revealed the same four regions of sequence from the TBSV genome were strictly conserved in each of the DI RNAs: the virus 5' leader sequence of 168 bases; a region of approximately 200-250 bases from the viral polymerase gene; approximately 70 bases from the 3' terminus of the viral p19 and p22 genes; and approximately 130 bases from the 3' terminal noncoding region. Conservation of the sequence motif present in all of the DIs suggests that there might be a common mechanism of DI formation as well as selection pressure to maintain sequences essential for replication and encapsidation.

Base Sequence↗

PubMed1979

Developmental restrictions on transcription: determinants of the developmental program and their role in aging.

A developing plant system, the soybean hypocotyl has been used to investigate early transcription events which restrict auxin induced cellular proliferation to the appropriate developmental stage. Auxin treatment of 4-day old seedlings resulted in an early (6 hour) activation of chromatin-bound RNA polymerase activity wihich approached 200% of control values by 18 hours. This occurred without a detectable alteration in chromatin template capacity (assayed with exogenous RNA polymerase) and resulted in the synthesis of "induced RNA transcripts" as determined in vitro by nearest neighbor analysis. In contrast, auxin treatment of unresponsive 8-day old seedlings did not alter the chromatin-bound RNA polymerase activity. Hormonal activation did, however, result in the exposure of "induced template" regions in chromatin which could only be transcribed in vitro if exogenous RNA polymerase was included in the transcription reaction. Isoelectric focusing of the endogenous chromatin-bound and soluble RNA polymerase enzymes from successive developmental stages revealed that the chromatin-bound enzymes at the 2-day stage were first released from the chromatin complex and could be recovered in the soluble pool (4-day stage). This was followed by a gradual disappearance of these subspecies (6-day stage) until only a limited ensemble of RNA polymerase subspecies remained bound to chromatin and free in the soluble pool (8-day stage). Similar analyses of both the bound and free enzymes at the 4 and 8-day stages following auxin treatment revealed that the 4-day soluble enzymes could be induced to rebind to the chromatin complex in a defined sequence after hormone treatment while those of the 8-day hypocotyl were unable to do so. These developmental events indicate that the select loss of certain RNA polymerase subspecies serves to restrict the hormone responsivness of this tissue to the early developmental stages. Such restrictions could thus commit the constituent hypocotyl cells to their terminal post-mitotic phase of development.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1981

Increased transcription activity of rat liver chromatin after protein restriction and limited digestion of nuclei with micrococcus nuclease.

Protein restriction has been shown to produce either an enhancement or a reduction of transcription activity in vitro. Conditions for an enhanced transcription activity were investigated. Young male rats were fed a complete diet containing either 20% or 3% casein for 6 days. Body weight changed +7.4 g/day and -0.5 g/day, respectively. Liver wet weights were 6.8 and 3.7 g and the DNA amounts/g were 1.31 and 1.67 mg. Liver nuclei were incubated without or with 1 unit micrococcus nuclease (EC 3.1.4.7) per milligram of nuclear DNA, and chromatin was fractionated into a 2,000 x g, a 102,000 x g pellet and a supernatant fraction. In the livers of rats fed a low protein diet, chromatin-bound RNA polymerase I plus III and II activity/mg of fractional and nuclear DNA and soluble RNA polymerase activity were increased, while heparin-stimulated RNA polymerase II activity remained unchanged. An increased number of chains was synthesized by RNA polymerase I plus III without change in chain length and incorporation rate per chain. The length and incorporation rate per chain increased, while the number of chains synthesized by RNA polymerase II did not. After stimulation by heparin, an increased number of short chains was synthesized at a lower rate of incorporation per chain. In the complex chromating structures the capacity for RNA synthesis was determined by specific enzyme activity, RNA chain number and length.

Animals↗

PubMed1980

Isolation and properties of an RNA fraction present in Brucella culture supernatants.

The supernatant fluids of batch and continuous cultures of Brucella strains contained up to 100 mg/l of soluble RNA which could be recovered by precipitation with lysozyme, This RNA fraction had many of the properties of ribosomal RNA and was single-stranded, sensitive to ribonuclease, with an approximate sedimentation constant of 5S, a molecular weight of about 35000 daltons and an adenine; guanine; cytosine; uracil content of 17.5; 26.5; 33; 23 mol% respectively. RNA fractions from lysozyme precipitates evoked high titres of Brucella agglutinins on injection into rabbits and induced acute inflammatory responses in guinea-pig skin. Highly purified RNA fractions prepared by phenol extraction of lysozyme precipitates did not evoke antibodies to Brucella abortus.

Animals↗

PubMed1976

Fate of rubella genome ribonucleic acid after immune and nonimmune virolysis in the presence of ribonuclease.

To determine whether rubella virion ribonucleic acid (RNA) becomes accessible to nuclease attack after immune lysis of the viral envelope, virions containing radioactively labeled RNA were examined in three ways with the following results. (i) Incubation of purified virus with heat-inactivated rubella convalescent human serum and guinea pig complement resulted in an increase in acid-soluble RNA. Antibody was required; the reaction was temperature dependent and was blocked by ethylenediaminetetraacetic acid. When exogenous nuclease was added prior to lysis, radioactivity in virions was reduced to 15% of that in unlysed control pellets (ii) Sucrose gradient sedimentation profiles of RNA released from lysed and unlysed virions under controlled conditions showed that the nuclease content of serum-virus mixtures was sufficient to eliminate all RNA of genome size, although degradation was not complete. (iii) Virions were also lysed by unheated human immune sera in the absence of guinea pig complement and by some, but not all, unheated antibody-negative sera.

Acetates↗

PubMed1961

The kinetics of the synthesis of ribosomal RNA in E. coli.

The kinetics of the synthesis of ribosomal RNA in E. coli has been studied using C(14)-uracil as tracer. Two fractions of RNA having sedimentation constants between 4 and 8S have kinetic behavior consistent with roles of precursors. The first consists of a very small proportion of the RNA found in the 100,000 g supernatant after ribosomes have been removed. It has been separated from the soluble RNA present in much larger quantities by chromatography on DEAE-cellulose columns. The size and magnitude of flow through this fraction are consistent with it being precursor to a large part of the ribosomal RNA.A fraction of ribosomal RNA of similar size is also found in the ribosomes. This fraction is 5 to 10 per cent of the total ribosomal RNA and a much higher proportion of the RNA of the 20S and 30S ribosomes present in the cell extract. The rate of incorporation of label into this fraction and into the main fractions of ribosomal RNA of 18S and 28S suggests that the small molecules are the precursors of the large molecules. Measurements of the rate of labeling of the 20, 30, and 50S ribosomes made at corresponding times indicate that ribosome synthesis occurs by concurrent conversion of small to large molecules of RNA and small to large ribosomes.

Escherichia coli↗

PubMed1976

Plant Desiccation and Protein Synthesis: III. Stability of Cytoplasmic RNA during Dehydration, and Its Synthesis on Rehydration of the Moss Tortula ruralis.

RNA species from the haploid gametophyte generation of the moss Tortula ruralis exhibit typical eukaryotic characteristics. The major ribosomal and soluble RNA species are stable during drying and rehydration. RNA synthesis occurs rapidly on reintroduction of the moss to water and incorporation into high molecular weight RNA fractions was detected after 20 to 30 minutes of rehydration and into low molecular weight fractions after 30-60 minutes. Newly synthesized ribosomal RNA was detected in ribosomes within 2 hours of rehydration, but not in polysomes. It is apparent that the ribosomal and transfer RNA conserved during desiccation is involved in the re-establishment of early protein synthesis during subsequent rehydration and that, initially, there is no requirement for newly synthesized material.

Journal Article↗

PubMed1969

Selective effect of hormones on nucleic acid metabolism during germination of pear embryos.

1. The effect of hormones on (32)P incorporation into various RNA fractions in germinating pear embryos was studied by fractionation on methylated albumin-kieselguhr columns. Abscisic acid inhibited labelling of soluble RNA, DNA-RNA hybrid and light-ribosomal RNA fractions with (32)P and this effect was reversed by both kinetin and gibberellic acid. 2. Kinetin reversed the inhibition by abscisic acid of (32)P incorporation into total ribosomal RNA and appeared to promote labelling of heavy-ribosomal RNA. Gibberellic acid was more active than kinetin in reversing the inhibition by abscisic acid of labelling of the DNA-RNA hybrid fraction with (32)P, but in contrast with kinetin appeared to increase further the inhibition by abscisic acid of labelling of total ribosomal RNA. 3. The percentage of radioactivity in various RNA fractions showed marked variation in response to hormones. 4. The pattern of labelling of RNA in pear embryos during reversal of inhibition by abscisic acid with a combination of kinetin and gibberellic acid was similar to that after cold-treatment of dormant pear embryos. This is suggestive of hormonal interplay in dormancy release by cold-treatment in pear embryos.

Chromatography↗

PubMed1978

Induction of hepatic RNA-and protein synthesis by pregnenolone-16alpha-carbonitrile in rats.

Repeated administration of pregenolone-16alpha-carbonitrile (PCN) induced hepatic microsomal RNA-synthesis in female rats. The RNA that appeared with 14C-orotate in the microsomes after a 12-h pulse had maximal specific activity. Deoxycholate-soluble RNA demonstrated a higher specific activity than did its ribosomal counterpart, and both were significantly more radioactive than in the respective controls. Examination of the rate of 14C-leucine incorporation into microsome-bound polypeptides showed that the enhanced RNA-synthesis after PCN treatment resulted in an augmentation of protein-synthesis. Administration of this cyanosteroid was also associated with a significant decrease in glucose-6-phosphatase activity and an increase in the cholesterol content of rat hepatocellular microsomes, which correlate well with the degranulation of rough endoplasmic reticulum and the synchronous proliferation of smooth endoplasmic reticulum.

Animals↗

PubMed1985

Identification and mutational analysis of the promoter for a spinach chloroplast transfer RNA gene.

A transcription extract from purified spinach chloroplast was used to test chloroplast DNA sequences for their function as promoter elements. Chloroplast tRNA genes are correctly transcribed in the extract by a soluble RNA polymerase, and precursor molecules are processed into mature tRNAs. Transcription of the spinach chloroplast tRNA2Met gene (trnM2) in vitro requires 5' upstream DNA sequences. Deletion of 5' DNA sequences with exonuclease Bal31 was used to establish the 5' boundary of the promoter region. This boundary is part of a DNA sequence with partial homology to the prokaryotic -35 region. Seventeen base pairs downstream from this sequence a DNA sequence occurs which is homologous to the prokaryotic -10 region. We used synthetic oligonucleotides fused to trnM2 5' deletion mutants to create insertions, deletions and base substitutions in these regions. Internal deletion mutants demonstrated that the -10 promoter element is also required for transcription in vitro. The arrangement of DNA sequences recognised by the chloroplast RNA polymerase resembles the prokaryotic promoter organization.

Base Sequence↗

PubMed1979

Poliovirus replicase: a soluble enzyme able to initiate copying of poliovirus RNA.

The soluble phase of the cytoplasm of poliovirus-infected cells contains an enzymatic activity able to copy RNA without an added primer. This replicase activity has been purified 60-fold; it is absent from uninfected cells. Poly(U) polymerase activity copurifies with replicase activity. Although less pure replicase fractions copy a variety of RNAs, purer fractions respond better to poliovirus RNA than to other viral RNAs. Even the less pure fractions make a specific copy of the added template, as shown by hybridization of the product to its template RNA but not to other RNAs. Among homopolymers only poly(A)-oligo(U) was copied by the replicase; other primed homopolymer templates were inactive.

HeLa Cells↗

PubMed1966

Effect of purine and pyrimidine analogues on growth and RNA metabolism in the soybean hypocotyl-the selective action of 5-fluorouracil.

The effects of several base analogues and cycloheximide on RNA synthesis, protein synthesis, and cell elongation were studied in excised soybean hypocotyl. None of the pyrimidine analogues tested affected growth or protein synthesis; only 5-fluorouracil appreciably inhibited RNA synthesis. 8-Azaguanine and 6-methylpurine markedly inhibited RNA and protein synthesis and cell elongation. Cycloheximide effectively inhibited both cell elongation and protein synthesis. The results show that 5-fluorouracil selectively inhibited ribosomal and soluble RNA synthesis without affecting the synthesis of D-RNA. These results indicate that the requirement for RNA synthesis to support continued protein synthesis and cell elongation is restricted to the synthesis of D-RNA.5-Fluorouracil was incorporated into all classes of RNA in a form believed to be 5-fluorouridylic acid. Cycloheximide markedly inhibited the accumulation of ribosomal RNA, but the results indicate that CH did not inhibit, per se, the synthesis of ribosomal RNA. The accumulation of newly synthesized D-RNA was only slightly affected by cycloheximide. These results show that the inhibition of cell elongation by cycloheximide correlates with the inhibition of protein synthesis, but not with the effect on RNA metabolism.

Cycloheximide↗

PubMed1977

RNA synthesis in isolated nuclei of Xenopus laevis emproyos.

Nuclei were isolated from tailbud embryos of Xenopus laevis and their RNA synthetic activity was studied. The activity was sensitive to actinomycin D, and required the presence of all four ribonucleotide triphosphates and a divalent cation (Mg2+ or Mn2+). The effect of the divalent cation varied depending on the presence of 0.4 M KCl: in its absence more RNA was synthesized with Mg2+ than with Mn2+, but in its presence the situation was reversed. About 50% of the RNA synthesized was sensitive to the presence of alpha-amanitin. Polyacrylamide gel electrophoresis of the labeled RNA revealed that the RNA synthesized had a very heterogeneous size distribution ranging from 40 to 4 S, with a broad peak at around 28--18 S. The RNA which was distributed at around 28--18 S and 4 S was resistant to the presence of a low dose of alpha-amanitin, suggesting its ribosomal and soluble RNA nature. The RNA which was sensitive to this drug and hence may be assumed to be messenger RNA was distributed mainly between 18 and 4 S. From these results it appears that the isolated embryonic nuclei retain their activity to synthesize all of the major classes of RNA.

Amanitins↗

PubMed1983

Selective in vitro transcription of chloroplast genes.

Transcription of Euglena gracilis chloroplast genes has been investigated by using in vitro transcription systems. A DNA-dependent RNA polymerase responsible for the transcription of rRNA genes has been isolated as a nucleoprotein complex (transcriptionally active chromosome). The RNA polymerase remains tightly bound to the chloroplast DNA template and does not initiate transcription with cloned chloroplast genes. A transcriptionally active extract has been prepared from intact Euglena chloroplasts. The soluble RNA polymerase in this extract recognizes cloned chloroplast tRNA genes and tRNA-sized products have been detected after transcription. The tRNA-sized molecules specifically hybridize to the tRNA genes in the plasmid DNA. At least five tRNA-sized products have been identified from transcription of a trnY1trnH1-trnM1-trnE1-trnW1-trnG1 cluster. Evidence is also presented that processing enzymes in the chloroplast-extract can recognize a polycistronic tRNAVal-tRNAAsn-tRNAArg precursor and process it into tRNA-sized molecules. Truncated templates have been used to demonstrate that the chloroplast tRNA genes are actively transcribed. From a comparison of 5' flanking sequences in chloroplast tRNA genes, a consensus sequence which might function as a promoter, has been identified. The properties of the RNA polymerase involved in the transcription of chloroplast rRNA genes and tRNA genes have been investigated and compared.

Chloroplasts↗

PubMed1968

RNA and protein in nucleolar structures of dragonfly oocytes.

Dragonfly and damselfly oocytes regularly contain two nucleolus like bodies of different sizes, a primary nucleolus and a secondary nucleolus. The primary nucleolus stains more deeply with RNA stains than with an arginine stain; the seconidary nuicleolus stains conzversely. The primary nulcleolus incorporates uridine under all conditions tested; the secondary, only after precautions have been taken to preserve soluble RNA. Both inicorporate lysine.

Animals↗

PubMed2006

Specialized transcription factories.

We have previously suggested a model for the eukaryotic genome based on the structure of the bacterial nucleoid where active RNA polymerases cluster to loop the intervening DNA. This organization of polymerases into clusters--which we call transcription 'factories'--has important consequences. For example, in the nucleus of a HeLa cell the concentration of soluble RNA polymerase II is approximately 1 mM, but the local concentration in a factory is 1000-fold higher. Because a promoter can diffuse approximately 100 nm in 15 s, one lying near a factory is likely to initiate; moreover, when released at termination, it will still lie near a factory, and the movement and modifications (e.g. acetylation) accompanying elongation will leave it in an 'open' conformation. Another promoter out in a long loop is less likely to initiate, because the promoter concentration falls off with the cube of the distance from the factory. Moreover, a long tether will buffer it from transcription-induced movement, making it prone to deacetylation, deposition of HP1 (heterochromatin protein 1), and incorporation into heterochromatin. The context around a promoter will then be self-sustaining: productive collisions of an active promoter with the factory will attract factors increasing the frequency of initiation, and the longer an inactive promoter remains inactive, the more it becomes embedded in heterochromatin. We review here the evidence that different factories may specialize in the transcription of different groups of genes.

Bacteria↗

PubMed1987

Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2).

In Streptomyces coelicolor, bldA mutants are defective in antibiotic production and the development of aerial hyphae and spores. Subcloning analysis showed that sequences spanning an NcoI site in cloned bldA+ DNA were needed to allow complementation of a bldA mutant. Nucleotide sequencing revealed a tRNA-like sequence 9 bp downstream from the NcoI site. Five independent bldA mutations all fell in a 16-bp region in the tRNA-like sequence, one of them changing the putative anticodon. In RNA dot-blot analysis, hybridization was detected with a probe specific for the tRNA-like transcript but not with a probe for "anti-tRNA-like" transcripts. The transcripts detected were all in the salt-soluble RNA fraction and accumulated relatively late in growth. It is postulated that bldA specifies a tRNA that would recognize the codon UUA (for leucine). This codon is very rare in Streptomyces genes [which generally contain greater than 70 mole% (G + C)], suggesting a possible role for bldA in translational control of development.

Base Sequence↗

PubMed1965

Effect of nucleoside 5'-di- and 5'-tri-phosphates on pancreatic ribonuclease activity.

1. ADP, ATP and GDP inhibited the phosphotransferase activity, the release of cyclic nucleotides from RNA, of ribonuclease. No significant inhibition was elicited by pyrimidine 5'-nucleoside diphosphates, CDP and UDP. 2. Inhibition by ADP, AMP, adenosine, adenine, NAD and NADP was insignificant at the concentrations tested. Small inhibition was observed with high concentrations of AMP and only when soluble RNA was the substrate. 3. Inhibition by ADP was found to be ;uncompetitive'. 4. Results seem to indicate that at least for optimum inhibition the polyphosphate of the purine nucleoside is essential. They further suggest that the inhibitor acts by combining with the enzyme only when the enzyme is bound to the substrate.

Journal Article↗