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 poly(A)-containing mitochondrial mRNAs of rat liver were tested for their ability to serve as templates for the DNA synthesis by means of reverse transcription in the presence of the oligo(dT) primer and the RNA-directed DNA-polymerase from avian myeloblastosis virus. The mT-mRNA does not support the DNA synthesis in the standard conditions sufficient for effective reverse transcription of rabbit globin mRNA and of poly(A) in the presence of oligo(dT) primers. After a mild alkaline treatment of the mRNA and subsequent polyadenylation of the 3'-termini of the generated fragments with ATP:RNA adenyltransferase from E.coli the poly(A) (+) polyribonucleotides are able to serve as templates for reverse transcription in the presence of oligo(dT) and the reverse transcriptase. A conclusion is made that a "structural stop" exists in mitochondrial mRNA non-translable regions adjacent to the poly(A) terminal sequence. The "structural stop" is suggested to be caused by post-transcriptional modification of mRNA (methylation, etc.) or by a particularly stable secondary structure in this region of the mRNA molecules.
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PMID:Enzymatic synthesis of DNA complementary to mitochondrial mRNA via reverse transcription. 7 7

Human immunodeficiency virus 1 (HIV-1) nucleocapsid protein p15 was produced as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Rapid purification of GST::p15 in an active form by one-step glutathione-agarose chromatography was accomplished in the presence of an antioxidant. Recombinant p15 fused to GST was shown to stimulate the dimerization of viral RNA. HIV-1 reverse transcriptase-catalyzed in vitro synthesis of minus-strand cDNA from synthetic human tRNA(Lys3UUU) and natural bovine tRNA(Lys3SUU) primer molecules was enhanced by GST::p15. GST produced in E.coli revealed no effect with respect to RNA dimerization and cDNA synthesis, demonstrating that both activities reside in the p15 portion of the fusion protein.
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PMID:Recombinant HIV-1 nucleocapsid protein p15 produced as a fusion protein with glutathione S-transferase in Escherichia coli mediates dimerization and enhances reverse transcription of retroviral RNA. 128 Feb 40

RNase D was recently reported as a new enzymatic activity associated with HIV-1 reverse transcriptase (RT), cleaving RNA at two positions within the double-stranded region of the tRNA primer-viral RNA template complex (Ben-Artzi et al., Proc. Natl. Acad. Sci. USA 89 (1992) 927-931). This would make RNase D a fourth distinct activity of HIV-1 RT, in addition to RNA- and DNA-dependent DNA polymerase and RNase H. Using a specific substrate containing tRNA(Lys,3) hybridized to the primer binding site, we were able to detect the reported RNase D activity in our preparations of recombinant HIV-1 RT. This activity was also present in several active-site mutants of RT, suggesting that it is independent of the RNase H and polymerase functionalities of RT. Furthermore, we found that the cleavage specificity of RNase D is the same as that of RNase III isolated from E.coli. A likely explantation of these results--that the observed RNase D activity is attributable to traces of RNase III contamination--was further strengthened by the finding that the recombinant preparations of HIV-1 RT can specifically cleave a phage T7-derived double-stranded RNA processing signal, which has been used as a model substrate for detection of E.coli RNase III. Moreover, RT purified from an RNase III- strain of E.coli displayed no cleavage of the tRNA primer-RNA template complex.
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PMID:RNase D, a reported new activity associated with HIV-1 reverse transcriptase, displays the same cleavage specificity as Escherichia coli RNase III. 128 Aug 10

By screening of an Escherichia coli plasmidic library using antibodies against aspartyl-tRNA synthetase (AspRS) several clones were obtained containing aspS, the gene coding for AspRS. We report here the nucleotide sequence of aspS and the corresponding primary structure of the aspartyl-tRNA synthetase, a protein of 590 amino acid residues with a Mr 65,913, a value in close agreement with that observed for the purified protein. Primer extension analysis of the aspS mRNA using reverse transcriptase located its 5'-end at 94 nucleotides upstream of the translation initiation AUG; nuclease S1 analysis located the 3'-end at 126 nucleotides downstream of the stop codon UGA. Comparison of the DNA-derived protein sequence with known aminoacyl-tRNA sequences revealed important homologies with asparaginyl- and lysyl-tRNA synthetases from E.coli; more than 25% of their amino acid residues are identical, the homologies being distributed preferencially in the first part and the carboxy-terminal end of the molecule. Mutagenesis directed towards a consensus tetrapeptide (Gly-Leu-Asp-Arg) and the carboxy-terminal end showed that both domains could be implicated in catalysis as well as in ATP binding.
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PMID:Aspartyl-tRNA synthetase from Escherichia coli: cloning and characterisation of the gene, homologies of its translated amino acid sequence with asparaginyl- and lysyl-tRNA synthetases. 212 59

The inhibitory effects of hexasodium sym-bis(m-aminobenzoyl-m-amino-p-methylbenzoyl-1-naphthylamino-4,6, 8-trisulfonate)carbamide (trivial name: suramin) on the activities of various deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) polymerases from mammalian cells, bacteria and retrovirus were examined and compared with each other. Among the various DNA and RNA polymerases tested, the activities of DNA primase, DNA polymerase alpha, reverse transcriptase and Escherichia coli RNA polymerase were strongly inhibited by suramin, while the activities of other enzymes including DNA polymerases beta and gamma, terminal deoxynucleotidyl-transferase and DNA polymerase I were relatively resistant to inhibition by this drug. The inhibition by suramin of DNA polymerase alpha from KB cells and Rauscher murine leukemia virus (RLV) reverse transcriptase was due to competition with the respective template primer (activated DNA for alpha polymerase and (rA)n.(dT)12-18 for reverse transcriptase) for the template.primer-binding site of the enzyme, while the inhibition of DNA primase and E.coli RNA polymerase was due to competition with the ribonucleoside triphosphate substrate. The inhibition constants (Ki) of suramin were determined to be 2.6 microM, 0.35 microM, 0.54 microM and 0.70 microM for DNA primase, DNA polymerase alpha, RLV reverse transcriptase and E. coli RNA polymerase respectively. The observed inhibitions of these polynucleotide-synthesizing enzymes by suramin seem to explain, at least in part, an as yet unknown mechanism of trypanocidal action of this drug.
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PMID:Differential inhibition of various deoxyribonucleic and ribonucleic acid polymerases by suramin. 245 Jul 43

It is shown that dNTP(3'F) are terminators of DNA synthesis and may serve as very effective tools for DNA sequencing with E.coli DNA polymerase I and AMV reverse transcriptase. The dNTP(3'F) are found to be chain terminator substrates for calf thymus terminal deoxyribonucleotidyl transferase but not for calf thymus DNA polymerase alpha. The optimal dNTP(3'F) concentration for DNA sequencing by DNA polymerase I is found to be an order of magnitude lower than that of ddNTPs. dNTP(3'F) produce a more clear sequence pattern than do ddNTPs.
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PMID:3'-Fluoro-2',3'-dideoxyribonucleoside 5'-triphosphates: terminators of DNA synthesis. 258 Jul 38

Dp71, a C-terminal isoform of dystrophin, has been identified as the major DMD gene product in many nonmuscle tissues. In this report, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to clone and characterize four alternatively spliced Dp71 transcripts from cultured human amniocytes. The cDNAs encoding these Dp71 transcripts were shown to be alternatively spliced for exons 71 and/or 78. RT-PCR analysis also revealed that Dp71 transcripts alternatively spliced for exons 71 and/or 78 were expressed at varying levels in a number of adult human tissues, including muscle, heart, brain, kidney, lung, testis and liver. To investigate size heterogeneity at the translational level, Dp71 cDNAs isolated from amniocytes were expressed in E.coli to generate recombinant Dp71 fusion proteins. These fusion proteins were identified on immunoblots using antibodies specific for the C-terminal sequences of dystrophin that either included (antibody 1461) or excluded exon 78 (antibody 462B). The molecular masses of the Dp71 fusion proteins ranged from 71-75 kDa on SDS-PAGE, consistent with their predicted values. Immunoblot analysis using antibodies 1461 and 462B identified multiple Dp71 isoforms of approximately 70-75 kDa on SDS-PAGE in total protein lysates from amniocytes and various adult human tissues. This variation in molecular mass is consistent with the expression of Dp71 isoforms derived from transcripts alternatively spliced for exons 71 and/or 78. Total protein lysates from normal skeletal muscle, DMD muscle, amniocytes and brain were shown to contain beta-dystroglycan, a component of the dystrophin-associated glycoprotein complex (DGC).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cloning and characterization of alternatively spliced isoforms of Dp71. 854 29

The RNase H family of enzymes degrades RNA in RNA.DNA hybrids in a divalent cation-dependent manner. RNases H from diverse sources such as Escherichia coli and human immunodeficiency virus (HIV) share homologous metal-binding active sites, and the activity of the RNase H domain of reverse transcriptase (RT) is required for retroviral replication. The isolated RNase H domain from HIV RT, however, is inactive. In contrast, the RNase H domain of Moloney murine leukemia virus (MMLV) is active, enabling functional studies. Unlike both E. coli RNase HI and HIV RT, the RNase H activity of MMLV RT shows greater activity in Mn(2+) than Mg(2+). We investigated the effect of mutations in five conserved active-site residues of the isolated MMLV RNase H domain. Mutations in two carboxylates eliminate metal binding while mutations in other active-site residues allow retention of metal ion affinity. Mutations that inactivate E.coli RNase HI in Mg(2+) have similar effects on the Mn(2+)-dependent activity of MMLV RNase H. These results suggest a similar one-metal catalytic mechanism for the Mn(2+)- and Mg(2+)-dependent activities of both prokaryotic and retroviral ribonucleases H.
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PMID:Metal binding and activation of the ribonuclease H domain from moloney murine leukemia virus. 1058 3

Among the polymerases, DNA polymerase alpha-primase is involved in lagging strand DNA synthesis. A previous report indicated that DNA polymerase alpha-primase initiates primer RNA synthesis with purine bases on a single-stranded G-rich telomere repeat. In this study, we found that DNA polymerase alpha-primase precisely initiated with adenosine opposite the 3'-side thymidine in the G-rich telomere repeat 5'-(TTAGGG)(n)-3' under rATP-rich conditions. Then, DNA polymerase alpha-primase synthesized the nascent DNA fragments by extending the primer. It was remarkable that DNA polymerase alpha-primase further expanded the product DNA far beyond the length of the template DNA, as ladders of multiple hexanucleotides on polyacrylamide gel electrophoresis. Using an oligomer duplex 5'-A(GGGTTA)(5)-3'/5'-(TAACCC)(5)T-3' as a template-primer, we show that both the Klenow fragment of Escherichia coli DNA polymerase I and HIV reverse transcriptase could expand telomere DNA sequences as well, giving products greater than the size of the template DNA. The maximum product lengths with these polymerases were approximately 40-90 nt longer than the template length. Our data imply that DNA polymerases have an intrinsic activity to expand the hexanucleotide repeats of the telomere sequence by a slippage mechanism and that DNA polymerase alpha uses both the repeat DNA primers and the de novo RNA primers for expansion. On the other hand, a plasmid harboring a eukaryotic telomere repeat showed remarkable genetic instability in E.coli. The telomere repeats exhibited either expansions or deletions by multiple hexanucleotide repeats during culture for a number of generations, suggesting involvement of the slippage mechanism in the instability of telomeric DNA in vivo.
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PMID:In vitro expansion of mammalian telomere repeats by DNA polymerase alpha-primase. 1093 27

A site-specifically modified oligonucleotide containing a single 2'-deoxyribonolactone lesion was used as a template for primer extension reactions catalyzed by M-MuLV reverse transcriptase (RT) and by the Klenow fragments of Escherichia coli DNA polymerase proficient (KF exo(+)) or deficient (KF exo(-)) in exonuclease activity. Analysis of the extension products in the presence of the four dNTPs or of a single dNTP showed that the M-MuLV RT was completely blocked and did not incorporate any dNMP opposite 2'-deoxyribonolactone. KF exo(-) preferentially incorporated nucleotides opposite the lesion following the frequency order dAMP > dGMP >> dTMP approximately dCMP and thus appeared to obey the 'A rule' for preferential incorporation as has been shown previously for the 2'-deoxyribose abasic site. In the sequence context examined, the primer extension by KF exo(-) appeared to be less efficient when dAMP was positioned opposite the lesion as compared with dTMP or dGMP. These two nucleotides promoted a more efficient polymerization accompanied by nucleotide deletion through misalignment incorporations. We therefore predict that the sequence context may strongly influence the translesional synthesis by KF exo(-) and thus the miscoding and mutational potential of the 2'-deoxyribonolactone in E.coli.
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PMID:Translesional synthesis on DNA templates containing the 2'-deoxyribonolactone lesion. 1143 17


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