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 present study describes the separation and purification of a reverse transcriptase and cellular DNA polymerases from the human spleen of a patient with myelofibrotic syndrome. The specific requirements with respect to bivalent cations and template-primers for DNA polymerase-alpha, DNA polymerase-beta and DNA polymerase-gamma, as well as for the reverse transcriptase, are reported. Sedimentation-velocity measurements of the purified enzymes gave values of 150000, 40000, 100000 and 70000 daltons for DNA polymerase-alpha DNA polymerase-beta, DNA polymerase-gamma and the reverse transcriptase respectively. Serological studies have shown that the reverse transcriptase from human spleen is not antigenically related to cellular DNA polymerase-alpha, -beta or -gamma, but is antigenically related to reverse transcriptase from simian sarcoma virus and gibbon-ape leukaemia virus.
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PMID:Purification, biochemical characterization and serological analysis of cellular deoxyribonucleic acid polymerases and a reverse transcriptase from spleen of a patient with myelofibrotic syndrome. 7 8

The present study describes the separation and purification of a reverse transcriptase and cellular DNA polymerases from the human spleen of a patient with myelofibrotic syndrome. The specific requirements with respect to bivalent cations and templateprimers for DNA polymerase-alpha, DNA polymerase-beta and DNA polymerase-gamma, as well as for the reverse transcriptase, are reported. Sedimentation velocity measurements of the purified enzymes gave values of 150 000, 40 000, 100 000 and 70 000 daltons for DNA polymerase-alpha, DNA polymerase-beta, and DNA polymerase-gamma and the reverse transcriptase respectively. The purified reverse transcriptase was specifically inhibited by antisera to the reverse transcriptases of the two primate viruses, SiSV and GaLV. Antisera raised against the myelofibrotic spleen reverse transcriptase inhibited the homologous enzyme and also the reverse transcriptase from SiSV and GaLV. DNA polymerases alpha, beta and gamma from the same spleen were not inhibited by the antisera. These results constitute the first indication of a possible retroviral etiology for myelofibrotic syndrome. Since SiSV and GaLV are exogenous to all primates the results indicate that this polymerase was acquired and the results are most simply interpreted as indicating that virus related to the SiSV-GaLV group is present in man.
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PMID:[Experiments towards the viral etiology of a preleukemic syndrome: osteomyelofibrosis (author's transl)]. 8 39

A synthetic procedure has been developed by which stable abasic sites are introduced into oligodeoxynucleotides at any desired position in the sequence. A modified tetrahydrofuran moiety, isosteric with 2'-deoxyribofuranose, serves as a structural analog of the natural apurinic/apyrimidinic site. We have also prepared oligodeoxynucleotides that lack cyclic structure at the abasic site but retain the carbon atoms of the phosphodiester backbone. These synthetic oligodeoxynucleotides are cleaved on the 5' side of the abasic site by endonuclease IV and by exonuclease III; they serve also as templates for avian myeloblastosis virus reverse transcriptase, Escherichia coli DNA polymerase I (Klenow fragment), and calf thymus DNA polymerase-alpha. Extension of primed templates by these DNA polymerases is blocked initially at the position immediately 3' to the abasic site; nucleoside monophosphates are subsequently incorporated opposite the lesion. The nucleotide most frequently incorporated opposite all abasic sites, regardless of structure, is dAMP. Significant "readthrough" at the abasic site was observed in experiments using avian myeloblastosis virus reverse transcriptase and DNA polymerase-alpha and, to a much lesser degree, with DNA polymerase I. We conclude that a modified tetrahydrofuran group can serve as a stable structural analog of 2'-deoxyribose in the apurinic/apyrimidinic site. These modified oligodeoxynucleotides should prove useful for studies of chemical mutagenesis.
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PMID:Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/apyrimidinic endonucleases. 244 Aug 61

In the present study, effects of various 2- and 2'-substituted polyadenylic acid analogs on eukaryotic, bacterial and viral DNA polymerases were investigated. The polymer containing 2'-deoxy-2'fluoroadenosine, (dAfl)n, showed a concentration dependent stimulation of (rA)n . (dT)12-catalyzed reverse transcriptase reaction from Rauscher Leukemia Virus (RLV). A similar stimulation of the (rA)n . (dT)12-catalyzed DNA polymerase-gamma reaction was also observed. However, the (rC)n . (dG)12-dependent reverse transcriptase activity was inhibited by (dAfl). The DNA polymerase-beta activity catalyzed by (dA)n . (dT)12 was also inhibited by (dAfl)n. The reported data indicate that (dAfl)n closely resembles (rA)n as a functional template. In contrast, the 2-substituted derivatives, poly(2-methylthioadenylic acid) and poly(2-ethylthioadenylic acid), are not able to discriminate between the reactions catalyzed by different templates. For example, both derivatives inhibit (rA)n . (dT)12- and (rC)n . (dG)12-catalyzed reverse transcriptase reaction to the same extent; though the methylthio derivative is a much better inhibitor than the ethylthio analog. The DNA polymerase-alpha was less sensitive to these inhibitors; whereas the bacterial DNA polymerase (Kornberg enzyme; DNA polymerase I) was completely resistant to the action of all the derivatives used in this study.
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PMID:A study of antitemplate inhibition of mammalian, bacterial and viral DNA polymerases by 2- and 2'-substituted derivatives of polyadenylic acid. 697 88

The unusually high frequency of misincorporation by HIV-1 reverse transcriptase (HIV RT) is likely to be the major factor in the rapid accumulation of viral mutations in AIDS, especially in the env gene. To investigate the ability of HIV RT to copy the env gene, we subcloned an HIV env gene fragment into a single-stranded DNA vector and measured the progression of synthesis by HIV RT. We observed that HIV RT, but not RT from avian myeloblastosis virus, DNA polymerase-alpha or T7 DNA polymerase, pauses specifically at poly-deoxyadenosine stretches within the env gene. The frequency of bypassing the polyadenosine stretches by HIV RT is enhanced by increasing the ratio of enzyme to template. We measured the fidelity of DNA synthesis within a segment of the hypervariable region 1 of the env gene (V-1) containing a poly-deoxyadenosine sequence by repetitively copying the DNA by HIV RT, and then cloning and sequencing the copied fragments. We found that 27% of the errors identified in V-1 sequence were frameshift mutations opposite the poly-adenosine tract, a site where strong pausing was observed. Pausing of HIV RT at the polyadenosine tract could be enhanced by either distamycin A or netropsin, (A-T)-rich minor groove binding peptides. Moreover, netropsin increases the frequency of frameshift mutations in experiments in which HIV RT catalyzes gap filling synthesis within the lacZ gene in double-stranded circular M13mp2 DNA. These combined results suggest that the enhanced mutation frequency may be due to increased pausing at netropsin-modified polyadenosine tracts. Therefore, netropsin and related A-T binding chemicals may selectively enhance frameshift mutagenesis induced by HIV RT and yield predominantly non-viable virus.
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PMID:Mutagenicity and pausing of HIV reverse transcriptase during HIV plus-strand DNA synthesis. 751 Mar 88

To study the mechanism of azidothymidine (AZT) cytotoxicity, human DNA was transfected to a variant of Chinese hamster V79 fibroblasts, the tr5 line. This cell line was used for this study for its elevated sensitivity to 5 microM AZT. Primary and secondary transfectants of tr5 cells using total human DNA and pSV2neo plasmid were selected by sequential incubations in AZT (20-50 microM), G418 (400 micrograms/ml active dose), and medium containing hypoxanthine, aminopterin, and thymidine (HAT). One DNA Alu fragment was detected in transfectants using primer TC-65, specific for human Alu sequences in the polymerase chain reaction (PCR). Moreover, cDNA of Chinese hamster alpha-type DNA polymerases was detected in transfectants by reverse transcriptase PCR (RT-PCR) using specific oligo-primer from a DNA polymerase-alpha cDNA sequence and in elevated annealing temperatures. In untransfected tr5 cells, neither of these sequences was detected. The data suggested that the genetic basis for AZT sensitivity may be related to the expression of alpha-type DNA polymerase, and the result indicated that AZT cytotoxicity could be reversed by transfection of appropriate human DNA into tr5 cells. This animal cell model has applications for studies of AZT metabolism and the isolation of the human gene that modulates AZT cytotoxicity.
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PMID:Enhanced expression of alpha-type DNA polymerase genes reduces AZT cytotoxicity in hamster tr5 cells. 833 31

Two lignans, phyllamycin B and retrojusticidin B isolated from Phyllanthus myrtifolius Moon have been demonstrated to have a strong inhibitory effect on human immunodeficiency virus-1 reverse transcriptase activity (HIV-1 RT), but much less inhibitory effect on human DNA polymerase-alpha (HDNAP-alpha) activity. Fifty percent inhibitory concentrations of phyllamycin B and retrojusticidin B were determined to be 3.5 and 5.5 microM for HIV-1 RT, and 289 and 989 microM for HDNAP-alpha, respectively. The mode of inhibition was found to be non-competitive inhibition with respect to template-primer and triphosphate substrate. Several tannins such as caffeoylquinates (CQs) isolated from Lonicera japonica Thunb, galloylquinates (GQs) and galloylshikimates (GSs) purified from Castanopsis hystrix were shown to have a much less selective inhibitory effect on HIV-1 RT.
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PMID:Differential inhibition of reverse transcriptase and cellular DNA polymerase-alpha activities by lignans isolated from Chinese herbs, Phyllanthus myrtifolius Moon, and tannins from Lonicera japonica Thunb and Castanopsis hystrix. 854 Jul 56

The human fragile-X syndrome, a major cause of inherited mental retardation, is associated with expansion of the trinucleotide repeat GGC:GCC. Repetitive sequences in DNA are subject to slippage during catalysis by DNA polymerases. We characterized the extent of slippage of synthetic GGC:GCC repeats by various DNA polymerases: Taq DNA polymerase, Klenow fragment of DNA polymerase I, DNA Sequence, DNA polymerase-alpha and polymerase-beta, as well as HIV reverse transcriptase. All of these enzymes were found to expand GGC:GCC repeats, with the most extensive expansion exhibited by Taq DNA polymerase. Starting with a template and primer, each 15 nucleotides (nt) in length, the product of one round of synthesis by Taq polymerase is as long as 250 nt. Sequence analysis of cloned DNA fragments expanded by Taq polymerase indicates that expansion involves multiple triplet additions and that it is asymmetric. The asymmetric distribution of terminal nucleotides in the expanded product is consistent with active expansion of the GCC strand and passive additions onto the GGC strand. The preferential elongation and expansion of the GCC strand was confirmed in studies utilizing longer repeats within a single-stranded M-13 template.
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PMID:In vitro expansion of GGC:GCC repeats: identification of the preferred strand of expansion. 875 19

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