Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The alkoxybenzophenanthridine alkaloids (coralyne acetosulfate, fagaronine chloride, and nitidine chloride) have been reported to possess antileukemic activity in mice. These compounds were tested for inhibition of reverse transcriptase activity of an RNA tumor virus and DNA polymerase, RNA polymerase, and polyadenylic acid polymerase activities of NIH-Swiss mouse embryos. Reverse transcriptase and DNA polymerase activities were strongly inhibited by these antileukemic alkaloids, whereas RNA polymerase and polyadenylic acid polymerase activities were only moderately affected. Viral and cellular DNA polymerase activities were potently diminished by the alkaloids when poly[d(A-T)], poly(dA)-oligo(dT), and poly(rA)-oligo(dT) template primers were used in the reaction mixture; however, no inhibition of enzyme activity was obtained with poly(rC)-oligo(dG) as template primer. These results suggest that alkoxybenzophenanthridine alkaloids inhibit DNA polymerase activity by interaction with A:T base pairs of the template primer.
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PMID:Inhibition of mammalian and oncornavirus nucleic acid polymerase activities by alkoxybenzophenanthridine alkaloids. 5 19

Procedures were established for the isolation and partial purification of DNA polymerase, RNA polymerase and poly(A) polymerase activities from the cytoplasm and nuclei of NIH-Swiss mouse embryos. Based on the elution pattern of these enzyme activities from DEAE-cellulose and phosphocellulose columns in Tris-HCl buffer, pH 8.0, the apparent basicities of the enzymes can be arranged as follows: cytoplasmic(C) poly(A) polymerase greater than (C)DNA polymerase beta greater than (C)DNA polymerase alpha and nuclear(N) poly(A) polymerase greater than (N)DNA polymerase greater than (N)RNA polymerase I greater than (N)RNA polymerase II. Twenty rifamycins, including rifamycin B, rifamycin S, rifamycin SV, and rifamycin SV derivatives, were examined for their ability to inhibit the above mentioned nucleic acid polymerizing enzymes and Simian sarcoma virus type I (SSV-1) reverse transcriptase. Rifamycin SV 3'-formyldiphenylhydrazone, rifamycin SV 3'-formyl-n-octyloxime (AF/013) and rifamycin SV 3'-formyldiphenylmethyloxime (AF/05) inhibited all the tested enzyme activities. Rifamycin SV 3'-formylpropylphenyloxime (AF/015) inhibited cellular nucleic acid polymerase activities but not SSV-1 DNA polymerase activity. Rifamycin SV 3'-formyldinitrophenylhydrazone (AF/DNFL) strongly inhibited reverse transcriptase activity but did not inhibit cellular DNA polymerase activities. AF/DNFI slightly inhibited RNA and poly(A) polymerase activities. Rifamycin SV 3'-formyldipropylhydrazone (AF/DPI) and 2,6-dimethyl-4-N-benzyldemethyl-rifampicin (AF/ABDMP) slightly inhibited reverse transcriptase activity but did not inhibit cellular nucleic acid polymerase activities. Active rifamycin derivatives inhibited enzyme reactions by interacting with the enzyme proteins. Nascent polynucleotide chain elongation continued although at a reduced rate in the presence of inhibitor. The addition of increasing concentrations of nonionic detergent (Triton X-100) to rifamycin-inhibited enzyme reactions fully restored enzyme activities. The presence of highly lipophilic 3'-side chains on active rifamycins and the reversibility of enzyme inhibition by Triton X-100 suggest that the tested nucleic acid polymerizing enzymes may have hydrophobic regions with which inhibitory rifamycins interact.
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PMID:Interaction of rifamycins with mammalian nucleic acid polymerizing enzymes. 6 93

Catalytic properties of terminal riboadenylate transferase from Escherichia coli and the products of the enzymic reaction were investigated. The kinetic analysis revealed that the reaction obeys the sequential ordered bi-bi mechanism. The application of conditions elaborated in this study resulted in the synthesis of products of defined size and efficient primer utilization. The tRNA(rA)n obtained was a good template for the synthesis of complementary DNA with reverse transcriptase.
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PMID:Terminal riboadenylate transferase from Escherichia coli. Characterization and application. 6 60

The 5S ribosomal RNA has been isolated, pure and intact, from rat liver (5 mg of 5S RNA from 150g of liver). The 5S RNA serves as a primer for calf thymus poly(A) polymerase with 20% of the efficiency of (Ap)3A. Bacterial 5S RNA and transfer RNA also serve as primers; rat liver 18S and 28S ribosomal RNAs support poly(A) synthesis poorly. Neither the 5S RNA primer nor the appended poly(A) tract is nicked or degraded by poly(A) polymerase, and initiation of poly(A) tracts on 5S RNA primers continues throughout the reaction period. The rate of initiation is dependent on the enzyme concentration; the ATP concentration affects the rate of elongation. The polyadenylated material increases in size over time, with the largest material reaching a size of 6.8 S in 5 h, corresponding to an appended poly(A) tract of 140 nucleotides. Using polyadenylated 5S RNA, oliog(dTY as primer, and avian myeloblastosis virus reverse transcriptase, we synthesized DNA complementary to 5S RNA. The complementary DNA has an apparent molecular weight (in alkaline sucrose gradients) of 4.3 X 10(4). Base composition analysis and nearest-neighbor analysis of the DNA are as expected for a complement of 5S RNA, indicating that the entire 5S sequence is copied. The complementary DNA hybridizes to 5S RNA with a R0t1/2 of 8.9 X 10(-4) mol.s.L-1. No hybrid is formed with Escherichia coli 16S and 23S ribosomal RNA, E. coli 5S ribosomal RNA, yeast transfer RNA, rat liver transfer RNA, or rat liver 18S and 28S RIBOSOMAL RNA. The Tm of the 5S RNA:5S DNA hybrid in 15 mM NaCl containing 1.5 mM sodium citrate is 74 degrees C, 2.5 degrees C below the theoretical melting temperature of a DNA duplex of 60% G + C. Analysis of the hybrid in buoyant density gradients also indicates that hybridization is both specific and precise. The complementary DNA anneals to calf thymus, rat liver, and salmon sperm DNAs but not to E. coli DNA. Annealing of 5S cDNA to calf thymus DNA with a C0t1/2 of 2.1 suggests that there are several thousand 5S RNA genes in the calf thymus genome (haploid). At least that number of 5S RNA genes is present in the salmon sperm genome.
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PMID:Enzymic polyadenylation of 5S ribosomal ribonucleic acid and synthesis of a complementary deoxyribonucleic acid. 8 57

Conditions are described under which poly(A) polymerase from Escherichia coli ribosomes will catalyse the addition of AMP residues onto the 3'-ends of human 18 S and 28 S ribosomal RNAs at an average rate of 40 AMP residues per 1000 nucleotides in 20 min. Single-stranded complementary DNAs (cDNAs) can then be transcribed from the polyadenylated RNAs with RNA-directed DNA polymerase from avian myeloblastosis virus. All of the sequences in the RNAs are represented in the cDNAs; measurements of the rates at which the cDNAs hybridize with their template RNAs showed that, when appropriate adjustments for differences in lengths and G + C contents of the reacting sequences are taken into account, the Rot 1/2 values of homologous RNA-cDNA hybridization reactions are directly proportional to the base-sequence complexity of the RNAs.
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PMID:Synthesis of DNAs complementary to human ribosomal RNAs polyadenylated in vitro. 78 37

The influence of 9-beta-D-arabinofuranosyladenine (ara-A) and its 5'-triphosphate derivative on programmed synthesis was tested with an intact cell system as well as with isolated enzyme systems. The effect of ara-A was tested in mouse lymphoma cells (L5178Y). The compound reduces cell proliferation in low concentration by cytostasis; under high ara-A concentration of radioactive precursors into DNA, RNA, and protein showed that ara-A selectively inhibits DNA synthesis. Formation of a polysome complex is not affected by ara-A. [3H]ara-A is incorporated into DNA in an intact cell system; 1 molecule of ara-A is incorporated per 8000 molecules of deoxyadenosine. Most of the ara-A molecules appeared to be in internucleotide linkages. Incorporation of ara-A into RNA could not be detected. 9-BETA-D-Arabinofuranosyladenine 5'-triphosphate (ara-ATP) does not reduce the incorporation rate of the following enzymes, isolated from quail oviducts: DNA-dependent RNA polymerases I and II, polyadenylic acid polymerase, and poly(adenosine diphosphate ribose) polymerase. The compound was found to inhibit DNA synthesis catalyzed by DNA polymerases isolated from quail oviducts and from oncogenic RNA viruses (Rous sarcoma viruses). All the enzymes tested were inhibited by ara-ATP in a competitive way with respect to deoxyadenosine 5'-triphosphate. The highest affinity of ara-ATP, i.e., the highest inhibitory potency of the drug, was found in the assays with the eukaryotic low-molecular DNA-dependent DNA polymerase. The influence on the eukaryotic high-molecular DNA-dependent Dna polymerase was a litte less. Compared to the eukaryotic DNA polymerases, the viral enzymes (RNA-directed DNA polymerase and DNA-directed DNA polymerase) are affected to a smaller extent by ara-ATP. No effects of ara-A and ara-ATP are observed in a protein-synthesizing, cell-free system isolated from L5178Y cells.
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PMID:Mode of action of 9-beta-D-arabinofuranosyladenine on the synthesis of DNA, RNA, and protein in vivo and in vitro. 114 31

Although it has been known for some time that bacterial mRNA molecules carry polyadenylate moieties at their 3' ends, nothing is known about the molecular structure of bacterial poly(A) RNA. To define the polyadenylylation site of a specific bacterial mRNA, we took advantage of the presence of elevated levels of poly(A) RNA in cells of Escherichia coli deficient in exoribonucleases and synthesized DNA complementary to polyadenylylated lipoprotein mRNA, encoded by the lpp gene, by using avian myeloblastosis virus reverse transcriptase and an oligo(dT)-containing primer. The 5'-terminal portion of the cDNA was amplified by the polymerase chain reaction and appropriate oligonucleotide primers, and the amplified DNA was cloned in pUC18 and subjected to nucleotide sequence analysis. Four clones were found to contain the entire 3'-terminal coding region of lpp mRNA, with poly(A) attached to either of two sites in the downstream untranslated region of the transcript. In one type of clone, the polyadenylate moiety was attached at the putative transcription termination site of lpp mRNA, whereas other clones lacked the stem-loop structure of the rho-independent transcription terminator and the polyadenylate moiety was attached to the residue just preceding the terminal stem-loop of the primary transcript. A model for the polyadenylylation of bacterial mRNA is proposed in which poly(A) polymerase and exonucleases compete for the 3' ends of mRNA molecules.
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PMID:Poly(A) RNA in Escherichia coli: nucleotide sequence at the junction of the lpp transcript and the polyadenylate moiety. 138 Jan 61

To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with decreased half-lives of the rpsO, trxA, lpp and ompA transcripts. In contrast, the transcripts for rne (RNase E) and pnp (polynucleotide phosphorylase, PNPase), enzymes involved in mRNA decay, were stabilized. rnb (RNase II) and rnc (RNase III) transcript levels were unaffected in the presence of increased polyadenylation. Long-term overproduction of PAP I led to slower growth and irreversible cell death. Differential display analysis showed that new RNA species were being polyadenylated after PAP I induction, including the mature 3'-terminus of 23S rRNA, a site that was not tailed in wild-type cells. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated an almost 20-fold variation in the level of polyadenylation among three different transcripts and that PAP I accounted for between 94% and 98.6% of their poly(A) tails. Cloning and sequencing of cDNAs derived from lpp, 23S and 16S rRNA revealed that, during exponential growth, C and U residues were polymerized into poly(A) tails in a transcript-dependent manner.
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PMID:Analysis of the function of Escherichia coli poly(A) polymerase I in RNA metabolism. 1059 33

We describe a fast and robust new assay format to measure poly(A) polymerase (PAP) activity in a microtiter plate format. The new assay principle uses only natural nucleotide triphosphates and avoids a labour-intensive filtration step. A coupled enzymatic system combining PAP and reverse transcriptase forms the basis of the assay. The PAP generates a poly(A) tail on a RNA substrate and the reverse transcriptase is used to quantify the polyadenylated RNA by extension of a biotinylated oligo-dT primer. We demonstrate the principle of the assay using influenza virus RNA polymerase and yeast PAP as examples. A specific increase in the K(m) value for ATP and the observation of burst kinetics in the polyadenylation dependent, but not in the polyadenylation independent, assay suggest that a rate limiting step of influenza polymerase activity occurs after transcription elongation. Yeast PAP was used to validate the assay as an example of a template independent PAP. The new yeast PAP assay was approximately 100-fold more sensitive than the conventional TCA precipitation assay for yeast PAP, but the kinetic analysis of the PAP reaction gave similar results in both assays. The two enzymes show important differences with respect to inhibition by 3'-deoxy-ATP. Whereas the K(i) value for 3'-deoxy-ATP (105-117 microM) is similar to the K(m) value for ATP (186 microM) in the case of influenza RNA polymerase, the K(i) value for 3'-deoxy-ATP (0.4-0.6 microM) is approximately 100-fold lower than the K(m) value for ATP (50 microM) in the case of yeast PAP.
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PMID:A sensitive, single-tube assay to measure the enzymatic activities of influenza RNA polymerase and other poly(A) polymerases: application to kinetic and inhibitor analysis. 1143 13

Cytoplasmic polyadenylation of mRNAs is involved in post-transcriptional regulation of genes, including translational activation. In addition to yeast Cid1 and Cid13 and mouse TPAP, GLD-2 has been recently identified as a cytoplasmic poly(A) polymerase in Caenorhabditis elegans and Xenopus oocytes. In this study, we have characterized mouse GLD-2, mGLD-2, in adult tissues, meiotically maturing oocytes, and NIH3T3 cultured cells. mGLD-2 was ubiquitously present in all tissues and cells tested. mGLD-2 was localized in the nucleus as well as in the cytoplasm of somatic, testicular, and cultured cells. Transfection of expression plasmids encoding mGLD-2 and the mutant proteins into NIH3T3 cells revealed that a 17-residue sequence in the N-terminal region of mGLD-2 probably acts as a localization signal required for the transport into the nucleus. Analysis of reverse transcriptase-polymerase chain reaction indicated the presence of mGLD-2 mRNA in the oocytes throughout meiotic maturation. However, 54-kDa mGLD-2 was found in the oocytes only at the metaphases I and II after germinal vesicle breakdown, presumably due to translational control. When mGLD-2 synthesis was artificially inhibited and enhanced by injection of double-stranded and polyadenylated RNAs into the germinal vesicle-stage oocytes, respectively, oocyte maturation was significantly arrested at the metaphase-I stage. These results suggest that mGLD-2 may act in the ooplasm on the progression of metaphase I to metaphase II during oocyte maturation.
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PMID:Possible role of mouse poly(A) polymerase mGLD-2 during oocyte maturation. 1632 97


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