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)

We surveyed the occurrence of unique restriction sites on the cDNAs of viroids, virusoids, and plant viral satellite RNAs that have a circular RNA as an intermediate of replication and found that four such sites would linearize their circular cDNAs. A rapid and simple method was then developed for cloning a naturally occurring viroid from Nematanthus wettsteinii plants. First-strand cDNA was synthesized using random hexanucleotide DNA primers and M-MuLV reverse transcriptase (Superscript RT). Second-strand DNA was synthesized by employing the replacement synthesis method using Escherichia coli RNase H, E. coli DNA polymerase I, E. coli DNA ligase, and beta-NAD+. The circular double-stranded DNA was analyzed for the presence of commonly available, unique restriction sites and subsequently linearized with a selected restriction enzyme. The linear cDNA was ligated to dephosphorylated plasmid vector pGEM 3Z f(+) and cloned in E. coli strain DH5 alpha. This cDNA cloning procedure is suitable for cloning sequence variants of well-characterized viroids, virusoids, certain plant viral satellite RNAs, and new such pathogens of unknown sequence.
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PMID:A rapid and versatile method for cloning viroids or other circular plant pathogenic RNAs. 138 86

Cytosine arabinoside (araC) is a potent antileukemic agent that is misincorporated into DNA in the course of its action. We have developed a chemical synthetic method that allows site-specific introduction of araC into synthetic DNA oligomers. We describe here the utilization of these oligomers as primer/template substrates for in vitro DNA synthesis reactions and as fragments for DNA ligation. These studies were undertaken to investigate the manner in which sites of araC misincorporation constitute sites of DNA dysfunction. AraCMP at the primer terminus dramatically reduced the rate of next nucleotide addition for Escherichia coli polymerase I (Klenow fragment) (Pol I), T4 polymerase, HeLa cell polymerase alpha 2 (Pol alpha 2), and AMV reverse transcriptase. Polymerases with associated 3'-5' exonuclease activity preferentially excised araCMP from the primer terminus prior to chain elongation. AraCMP-terminated fragments were ligated more slowly than control fragments by T4 DNA ligase. AraCMP located at an internucleotide site in the template markedly slowed replicative bypass for Pol I, T4 polymerase, and Pol alpha 2, but not for reverse transcriptase. Synthesis was partially arrested after insertion of the correct nucleotide opposite the lesion site. These results suggest a complex mechanism for the inhibition of DNA replication by araC when it is misincorporated into DNA.
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PMID:Functional consequences of the arabinosylcytosine structural lesion in DNA. 245 56

We have developed a technique for synthesis of single stranded complementary DNA (ss cDNA) using specifically designed phage ssDNA as vector primer. This vector (pPBS27) was constructed by introducing a poly(dT) tail adjacent to the XbaI site of pTZ18R, which can exist either as a plasmid in Escherichia coli or as a ssDNA phage. The pPBS27 phage vector is linearized with XbaI using a restriction-site-directed fragment and used to anneal a mixture of poly(A) + RNA for cDNA synthesis by reverse transcriptase. The RNA is then hydrolysed with NaOH and a poly(dG) tail added to the 3' end of the vector-cDNA with terminal transferase. The linear hybrid ssDNA is then closed by annealing with a 15-mer site-directed fragment oligodeoxynucleotide molecule and ligated with T4 DNA ligase. Almost 10(5) E. coli transformants per microgram of vector primer can be obtained in two days.
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PMID:Use of a phage vector for rapid synthesis and cloning of single-stranded cDNA. 303 56

We have developed conditions for efficient cDNA cloning of nanogram amounts of purified mRNAs coding for cystathionine beta-synthase [L-serine hydro-lyase (adding homocysteine), EC 4.2.1.22] and for the cytosolic precursors of mitochondrial ornithine transcarbamylase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) and the beta subunit of propionyl-CoA carboxylase [propanoyl-CoA: carbon-dioxide ligase (ADP-forming), EC 6.4.1.3]. The three mRNAs, prepared by sequential immunoselection from the same batch of rat liver polysomes, were pooled (20 ng each), and cDNA was synthesized by using avian reverse transcriptase. The second DNA strand was prepared by "nick-translation repair" of the cDNA . mRNA hybrid with RNase H, polymerase I, and DNA ligase from Escherichia coli. The double-stranded (ds) DNA was tailed with deoxycytidine residues, annealed with Pst I-cut/dG-tailed pBR322, and used to transform E. coli. The library generated by this three-step procedure contained 5000 independent colonies. A 550-base-pair (bp) cDNA clone of the beta subunit of propionyl-CoA carboxylase was detected by hybrid-selected translation; it was then used to screen the library for longer cDNAs. Two hybridizing cDNAs, 1200 and 1000 bp long with a 200-bp overlap, representing together a full-length copy of the coding region and 446 bp of 3' untranslated sequence, were recovered. Each plasmid mapped to the region q13.3----q22 of human chromosome 3. Cystathionine beta-synthase clones were obtained by screening the library with a single-stranded [32P]cDNA prepared directly from the highly purified synthase mRNA by reverse transcriptase. The longest hybridizing cDNA of 1700 bp was used in hybrid-selected translation and detected a polypeptide of 63 kDa, identical in size to rat liver synthase. In situ hybridization of this cDNA to q22 of human chromosome 21 confirmed two previous tentative assignments of the synthase locus to this chromosome.
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PMID:Cloning and screening with nanogram amounts of immunopurified mRNAs: cDNA cloning and chromosomal mapping of cystathionine beta-synthase and the beta subunit of propionyl-CoA carboxylase. 345 73

We describe a simple method for joining the 5'-protruding, single-stranded DNA ends generated by restriction enzymes. The method allows ends with different sequences to be joined and prevents identical ends from being joined. This is accomplished by partially filling the single strands in a controlled reverse transcriptase reaction. Partial filling can create new single-stranded ends that can be ligated to different, partially filled ends. In almost all useful cases, partial filling simultaneously eliminates the self-complementarity of identical ends and thus prevents them from being joined by DNA ligase. Although all possible combinations of partially filled ends were not tested, the tests performed indicate that the method is fairly general. We demonstrate that ends of the same length can be ligated with useful efficiency if they are: 1) one nucleotide long and complementary; 2) two nucleotides long and complementary or have a mismatch (dA:dC) at one position; or 3) three nucleotides long and, in our test, have a dT:dC mismatch at the middle position.
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PMID:Different restriction enzyme-generated sticky DNA ends can be joined in vitro. 619 44

DNA amplification systems are powerful technologies with the potential to impact a wide range of diagnostic applications. In this study we explored the feasibility and limitations of a modified ligase chain reaction (Gap-LCR) in detection and discrimination of DNAs that differ by a single base. LCR is a DNA amplification technology based on the ligation of two pairs of synthetic oligonucleotides which hybridize at adjacent positions to complementary strands of a target DNA. Multiple rounds of denaturation, annealing and ligation with a thermostable ligase result in the exponential amplification of the target DNA. A modification of LCR, Gap-LCR was developed to reduce the background generated by target-independent, blunt-end ligation. In Gap-LCR, DNA polymerase fills in a gap between annealed probes which are subsequently joined by DNA ligase. We have designed synthetic DNA targets with single base pair differences and analyzed them in a system where three common probes plus an allele-specific probe were used. A single base mismatch either at the ultimate 3' end or penultimate 3' end of the allele specific probe was sufficient for discrimination, though better discrimination was obtained with a mismatch at the penultimate 3' position. Comparison of Gap-LCR to allele-specific PCR (ASPCR) suggested that Gap-LCR has the advantage of having the additive effect of polymerase and ligase on specificity. As a model system, Gap-LCR was tested on a mutation in the reverse transcriptase gene of HIV, specifically, one of the mutations that confers AZT resistance. Mutant DNA could be detected and discriminated in the presence of up to 10,000-fold excess of wild-type DNA.
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PMID:Detection of point mutations with a modified ligase chain reaction (Gap-LCR). 753 8

DNA topoisomerase I (topo I) is a member of a group of essential nuclear enzymes which control and modify the topological state of DNA and is recognized as the target for anticancer drugs. During the course of the catalytic activity of topo I, a covalent bond is formed between a tyrosine group at the active site of the enzyme and a 3' phosphate group along the DNA backbone. This chemical reaction resembles the protein kinase-mediated tyrosine phosphorylation process. We assumed, therefore, that tyrphostins, potent and selective blockers of protein tyrosine kinases, might affect topo I activity. We found that of three derivatives of tyrphostins (AG-555, AG-18, and AG-213) that inhibited topo I activity in an in vitro assay, AG-555 was the most active. Examination of the mechanism by which these compounds act as topo I inhibitors revealed that AG-555 blocked the binding of this enzyme to the DNA due to its interaction with the topo I enzyme. We showed that its mode of action differed from that observed for camptothecin, a known topo I inhibitor. However, AG-555 did not affect the activity of other major DNA binding enzymes (i.e., DNA ligase, DNA polymerase I, and reverse transcriptase). This study suggests that tyrphostins may serve as a new class of topo I inhibitors, and these results also present additional explanations for their antiproliferative effect.
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PMID:Inhibition of topoisomerase I activity by tyrphostin derivatives, protein tyrosine kinase blockers: mechanism of action. 792 31

Activation of the Ha-ras oncogene in N-methyl-N-nitrosourea (MNU)-induced rat mammary tumors has been well documented. Such Ha-ras activation is thought to be brought about by direct action of carcinogens resulting in a G-->A transition at the second nucleotide of codon 12. However, a DNA repair enzyme, O6-methylguanine-DNA methyltransferase (MGMT), can specifically remove methyl groups from O6-methylguanine, which is a major mutagenic and carcinogenic DNA lesion leading to the G-->A transition. In this study, we compared the amount of MGMT mRNA in MNU-induced rat mammary tumors with and without such Ha-ras activation. A single injection of MNU into 82 female Sprague-Dawley rats induced 80 mammary carcinomas. RNase protection analysis and subsequent sequencing revealed that 42 of 65 randomly selected tumors contained Ha-ras oncogenes activated by the G-->A transition. The amount of MGMT mRNA was then measured by means of reverse transcriptase-mediated polymerase chain reaction (RT-PCR) amplification and Southern hybridization. No obvious difference in the level of MGMT mRNA was detected between the two tumor groups. In addition, in the course of our experiment, five of 42 tumors classified as containing activated Ha-ras oncogenes proved to contain low percentages of tumor cells with the Ha-ras activation. These results suggest that Ha-ras activation in MNU-induced rat mammary tumors may not necessarily be influenced by differences in MGMT activity. They also raise the possibility that activation of other oncogenes and/or inactivation of unidentified tumor suppressor gene(s) may be involved in development of a certain proportion of tumors with activated Ha-ras oncogenes, as is suspected in the case of tumors without Ha-ras activation.
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PMID:Comparison of O6-methylguanine-DNA methyltransferase mRNA levels in Ha-ras mutated and non-mutated rat mammary tumors induced by N-methyl-N-nitrosourea. 811 29

The family Poxviridae contains two subfamilies: the Entomopoxvirinae (poxviruses of insects) and the Chordopoxvirinae (poxviruses of vertebrates). Here we present the first characterization of the genome of an entomopoxvirus (EPV) which infects the North American migratory grasshopper Melanoplus sanguinipes and other important orthopteran pests. The 236-kbp M. sanguinipes EPV (MsEPV) genome consists of a central coding region bounded by 7-kbp inverted terminal repeats and contains 267 open reading frames (ORFs), of which 107 exhibit similarity to previously described genes. The presence of genes not previously described in poxviruses, and in some cases in any other known virus, suggests significant viral adaptation to the arthropod host and the external environment. Genes predicting interactions with host cellular mechanisms include homologues of the inhibitor of apoptosis protein, stress response protein phosphatase 2C, extracellular matrixin metalloproteases, ubiquitin, calcium binding EF-hand protein, glycosyltransferase, and a triacylglyceride lipase. MsEPV genes with putative functions in prevention and repair of DNA damage include a complete base excision repair pathway (uracil DNA glycosylase, AP endonuclease, DNA polymerase beta, and an NAD+-dependent DNA ligase), a photoreactivation repair pathway (cyclobutane pyrimidine dimer photolyase), a LINE-type reverse transcriptase, and a mutT homologue. The presence of these specific repair pathways may represent viral adaptation for repair of environmentally induced DNA damage. The absence of previously described poxvirus enzymes involved in nucleotide metabolism and the presence of a novel thymidylate synthase homologue suggest that MsEPV is heavily reliant on host cell nucleotide pools and the de novo nucleotide biosynthesis pathway. MsEPV and lepidopteran genus B EPVs lack genome colinearity and exhibit a low level of amino acid identity among homologous genes (20 to 59%), perhaps reflecting a significant evolutionary distance between lepidopteran and orthopteran viruses. Divergence between MsEPV and the Chordopoxvirinae is indicated by the presence of only 49 identifiable chordopoxvirus homologues, low-level amino acid identity among these genes (20 to 48%), and the presence in MsEPV of 43 novel ORFs in five gene families. Genes common to both poxvirus subfamilies, which include those encoding enzymes involved in RNA transcription and modification, DNA replication, protein processing, virion assembly, and virion structural proteins, define the genetic core of the Poxviridae.
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PMID:The genome of Melanoplus sanguinipes entomopoxvirus. 984 59

Simian varicella virus (SVV) infection of non-human primates is used as a model to study the pathogenesis and latency of varicella-zoster virus (VZV), the etiological agent of chickenpox and shingles. Uracil DNA glycosylase (UDG) is a DNA repair enzyme responsible for excision of uracil residues misincorporated into DNA. UDG is conserved throughout the herpesvirus family and may play an important role in viral pathogenesis. This study identified a 300 amino acid SVV UDG that shares 53.9% amino acid identity with the VZV UDG. The SVV UDG is expressed in infected Vero cells as determined by reverse transcriptase polymerase chain reaction (RT-PCR) and Northern blot analysis. The SVV UDG is encoded on a 2.0 kb transcript which also appears to encode the SVV glycoprotein L (gL) and the VZV gene 58 homolog. The SVV UDG is enzymatically active as determined by the ability of a SVV UDG-maltose binding protein fusion construct to remove [(3)H]-uracil incorporated into DNA.
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PMID:Identification and characterization of the simian varicella virus uracil DNA glycosylase. 1060 70


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