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At least 253 records · Page 14Linked to original sources
PubMed1988

Chloroplast rpoA, rpoB, and rpoC genes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription.

The purpose of this study was to determine the relationship between putative chloroplast RNA polymerase subunit genes and known chloroplast transcriptional activities. We have prepared fusion polypeptide genes from fragments of chloroplast DNA homologous to bacterial RNA polymerase subunit genes and expression vectors carrying portions of the anthranilate synthetase gene (trpE). Fusion proteins for chloroplast homologs of the RNA polymerase alpha (rpoA), beta (rpoB), and beta' (rpoC) subunits were obtained from these genes. The fusion polypeptides synthesized by Escherichia coli in vivo were purified and used as antigens for production of rabbit polyclonal anti-RNA polymerase subunit-specific antibodies. The purified antibodies were able to immobilize chloroplast DNA-dependent RNA polymerases from spinach, pea, and Euglena gracilis. In addition, the soluble chloroplast RNA polymerase activity in tRNA and mRNA synthesis was strongly inhibited by these antibodies under conditions which had little effect on transcription by the chloroplast transcriptionally active chromosome that preferentially transcribed rRNA genes (Greenberg, B. M., Narita, J. O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K. A., and Hallick, R. B. (1984) J. Biol. Chem. 259: 14880-14887). From these data we conclude that the chloroplast genes homologous to bacterial RNA polymerase subunit genes are expressed in vivo and that the protein products specify at least three of the components of the chloroplast RNA polymerase(s) involved in tRNA and mRNA transcription.

Amino Acid Sequence↗
PubMed1998

RNA-dependent RNA polymerase activity of the soluble recombinant hepatitis C virus NS5B protein truncated at the C-terminal region.

The hepatitis C virus (HCV) NS5B protein encodes an RNA-dependent RNA polymerase (RdRP), which is the central catalytic enzyme of HCV replicase. We established a new method to purify soluble HCV NS5B in the glutathione S-transferase-fused form NS5Bt from Escherichia coli which lacks the C-terminal 21 amino acid residues encompassing a putative anchoring domain (anino acids 2990-3010). The recombinant soluble protein exhibited RdRP activity in vitro which was dependent upon the template and primer, but it did not exhibit the terminal transferase activity that has been reported to be associated with the recombinant NS5B protein from insect cells. The RdRP activity of purified glutathione S-transferase-NS5Bt and thrombin-cleavaged non-fused NS5Bt shares most of the properties. Substitution mutations of NS5Bt at the GDD motif, which is highly conserved among viral RdRPs, and at the clustered basic residues (amino acids 2919-2924 and 2693-2699) abolished the RdRP activity. The C-terminal region of NS5B, which is dispensable for the RdRP activity, dramatically affected the subcellular localization of NS5B retaining it in perinuclear sites in transiently overexpressed mammalian cells. These results may provide some clues to dissecting the molecular mechanism of the HCV replication and also act as a basis for developing new anti-viral drugs.

Amino Acid Sequence↗
PubMed2000

Antenatal dexamethasone administration increases fetal lung DNA synthesis and RNA and protein content in nitrofen-induced congenital diaphragmatic hernia in rats.

Antenatal glucocorticoids treatment has been shown to correct pulmonary immaturity. The thymidine analog bromodeoxyuridine (BrdU) is incorporated into S-phase cells and used as a marker of DNA synthesis. In this study, we investigated the effect of antenatal glucocorticoid administration on DNA synthesis and RNA and protein content in nitrofen-induced congenital diaphragmatic hernia (CDH) in rats to better understand the effect of antenatal glucocorticoids on CDH lung. The CDH model was induced in pregnant rats using nitrofen. Dexamethasone (0.25 mg/kg) was given on d 18.5 and 19.5 of gestation (term = 22 d). BrdU was administered 1 h before fetuses were killed on d 21, and detected by immunohistochemistry. DNA synthesis was evaluated by percentage of BrdU-incorporated nuclei (BrdU labeling index). Total RNA and soluble protein were extracted from another set of left lungs to measure RNA and protein content. BrdU labeling index and total RNA content were significantly decreased in CDH lung compared with control rats. Antenatal dexamethasone treatment significantly increased BrdU labeling index and RNA and protein content in the left CDH lung. Our findings of decreased DNA synthesis and decreased RNA and protein content in CDH lung suggest that lung growth and development are suppressed in hypoplastic CDH lung. Increased DNA synthesis and increased RNA and protein content in dexamethasone-treated CDH lung suggest that antenatal glucocorticoids may accelerate fetal lung growth and development in CDH.

Animals↗
PubMed1993

Structure of the maize mitochondrial replicon RNA b and its relationship with other autonomously replicating RNA species.

The mitochondria of maize plants with S-type cytoplasm possess a family of single and double-stranded RNA molecules, termed RNA plasmids, that replicate in a DNA-independent manner typical of RNA viruses. We have determined the sequence of the smallest and most abundant member of this family, a single-stranded RNA termed RNA b. The 719 nucleotide sequence of RNA b lacks open reading frames of significant length. Probes complementary to the determined (+) strand sequence identified two larger RNA species of 2900 and > 4000 nucleotides in S-type maize mitochondria, the smaller of which corresponds to a previously identified member of the RNA plasmid family designated RNA a. These probes also unexpectedly identified a low abundance form of RNA b in N-type maize mitochondria. (+) strand probes identified corresponding (-) strand forms in S cytoplasm that likely represent components of replication intermediates. Primer extension experiments demonstrated that RNA b is an internally deleted form of the larger RNA plasmid, RNA a, and that it has a discrete and homogeneous 5' terminus. The solubility of RNA b in 2 M-LiCl as well as structural modelling studies indicated that it has a very high degree of secondary structure. Analysis of specific cleavage products generated by treatment of RNA b-oligonucleotide duplexes with RNase H indicated that the only identifiable forms of RNA b were linear molecules with relatively homogeneous 3' termini; no circular forms were detected. RNA b was found to share a 13 nucleotide sequence with the circular cadang-cadang viroid; 11 of these nucleotides fall within the central conserved region of viroid RNAs. In addition, RNA b shares certain structural similarities with phage Q beta and "variant" RNAs that serve as in vitro templates for the Q beta replicase. Collectively, our results indicate that RNA b is a "defective-interfering" member of an RNA family representing a new and apparently distinct class of RNA replicons.

Base Sequence↗
PubMed1968

Double-stranded Ribonucleic Acid from Cytoplasmic Polyhedrosis Virus of the Silkworm.

Ribonucleic acid (RNA) was extracted by phenol treatment from cytoplasmic polyhedrosis virus isolated from the midgut of infected silkworms. This RNA appears as threads when precipitated in alcohol. Two components having different sedimentation constants were observed. The molecular weight of the RNA preparation obtained by sedimentation coefficient (weight-averaged) and intrinsic viscosity was about 2 x 10(6) to 3 x 10(6). It was one-half to one-third the size of the calculated molecular weight for an entire RNA molecule in a virion. Electron micrographs of this RNA preparation showed two peaks in the distribution of contour length, at 0.4 and 1.3 mum, which would correspond to molecular weights of 10(6) and 3 x 10(6), respectively. The extracted RNA seemed to split into segments at a preferential breaking point. This RNA was soluble in concentrated salt solution, differing from single stranded high-molecular-weight RNA. The base composition of this RNA was complementary in the ratios of adenosine to uridine and guanosine to cytosine. It contained 43% guanosine plus cytosine. Based on its filamentous appearance by electron microscopy, typical pattern of optical rotatory dispersion and circular dichroism, sharp transition of the optical properties on heating, great hyperchromicity on degradation, nonreactivity with formaldehyde, and resistance to ribonucleases, it is concluded that this RNA is double-stranded and has regular base pairings of guanosine-cytosine and adenosine-uridine.

Journal Article↗
PubMed2001

[The expression of HCV RNA polymerase in E.coli and the study of its solubility and antigenicity].

OBJECTIVE: To express HCV RNA polymerase and study its soluble condition and antigenicity. METHODS: We constructed expression vectors pQE-5B-Fl and pQE-5B-C21 and expressed them in E.coli (M15). We analyzed their solubility in different conditions and purified soluble pQE-5B-C21 protein by Ni-NTA column, then detected its antigenicity by ELISA and western blot. RESULTS: We obtained the purified soluble pQE-5B-C21 protein in the induction conditions of 18 degrees C and the protein was proved to be of good antigenicity by ELISA and western blot. CONCLUSIONS: The RNA polymerase of HCV expressed in E.coli has good solubility and antigenicity.

Blotting, Western↗
PubMed2002

Genes for a nuclease and a protease are involved in the drastic decrease in cellular RNA amount in fission yeast cells during nitrogen starvation.

Cellular RNA in Schizosaccharomyces pombe cells drastically decreases in amount during nitrogen starvation. Previously, we found and purified a soluble RNA-degrading enzyme whose activity drastically increased in the cells of S. pombe undergoing nitrogen starvation. The enzyme was a nuclease encoded by pnu1(+). In this study, the increase in the RNA-degrading activity and the decrease in cellular RNA level are examined in a null-mutant of pnu1(+) (pnu1Delta). During nitrogen starvation, wild-type cells show an apparent increase in RNA-degrading activity, whereas the pnu1Delta cells do not. The wild-type cells show a drastic decrease in cellular RNA amount, whereas the pnu1Delta cells show only a slight decrease. These results suggest that Pnu1 nuclease is implicated in the decrease in cellular RNA amount during nitrogen starvation, probably via the RNA-degrading activity. The increase in the RNA-degrading activity is independent of both the Wis1 stress-activated MAP kinase cascade and Tor1 signaling pathway, but it is strongly dependent on isp6(+), a gene for a possible protease, whose expression is induced during nitrogen starvation. A disruption mutant for isp6(+) (isp6Delta) is deficient in both the increase in the RNA-degrading activity and the drastic decrease in the cellular RNA amount during nitrogen starvation, which suggests that isp6(+) is involved in the RNA degradation via regulating the RNA-degrading activity of Pnu1.

Endopeptidases↗
PubMedDate not supplied

[Incorporation in vivo of 14C-precursors into pyrimidine nucleotides of chicken liver and spleen tissues].

Maximum incorporation of the label after administration of 14C-bicarbonate to free pyrimidine nucleotides begins by the 60th minute. Radioactivity of cytidine nucleotides at all time points is four-eight times as low as that of uridine nucleotides. 2-14C-orotic acid is incorporated into acid-soluble nucleotides and RNA of the liver and spleen tissues. 60 min after administration of the isotope 20% of radioactivity was found in the liver and 0.3% in the spleen, in both tissues radioactivity of RNA is 20% of 14C-radioactivity in the acid-soluble fraction. The formation rate for cytidine nucleotides from uridine ones in the liver and spleen tissues is low, about 2-6% of uridine nucleotides radioactivity is found in them only, that may be due to a low activity of CTP-synthetase. The content of cytidine nucleotides in these organs is three-life times as low as compared to uridine nucleotides.

Animals↗
PubMed1993

Relation between the incorporation of 5-fluorouracil into liver carcinoma and normal tissue RNA at hepatic arterial administration in the rat is altered by overnight starvation.

The cytotoxicity of 5-FUra has been related to its incorporation into RNA. In a model of secondary liver cancer in the rat, the incorporation of 5-FUra into the acid-soluble fraction, RNA and DNA of several normal tissues and an adenocarcinoma of the colon transplanted to the liver was determined. A therapeutic labelled dose of the drug was infused via the hepatic artery for 2 hr and the rats killed 1 hr later. Half of the rats were starved overnight before treatment. The incorporation of 5-FUra into liver and intestinal RNA increased at starvation. It was unchanged in kidney and bone marrow. The incorporation into tumor RNA decreased insignificantly. The incorporation into tumor RNA was significantly higher than in hepatic, intestinal, and renal RNA at ad libitum feeding. This difference disappeared at overnight starvation.

Adenocarcinoma↗
PubMed1988

Effects of dosage and infusion time on the incorporation of 5-fluorouracil into DNA and RNA of normal tissues and an adenocarcinoma transplanted to rat liver.

In a model of secondary liver cancer in Wistar rats the incorporation of 5-FUra into the acid soluble fraction, RNA, and DNA of several normal tissues and of an adenocarcinoma of the colon transplanted to the liver was determined; 300, 1,200 or 24,000 nmoles of 5-FUra were infused via the gastroduodenal artery for 0.5, 2, or 24 hr. The rats were killed 1.5, 3, or 24 hr after the beginning of the infusions. In general, higher doses resulted in a higher labelling. However, the ratio of incorporation into tumor RNA compared to normal tissue RNA was higher at the 1,200 than at the 24,000 nmole dosage. There was a decreased RNA/DNA ratio in the tumor at 24 hr after 24,000 nmoles of 5-FUra had been infused over 0.5 or 2 hr, indicating decreased synthesis and/or increased breakdown of RNA.

Adenocarcinoma↗