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)

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

The reverse transcriptase of HIV-1 (AIDS virus) is characterized by the presence of two highly immunogenic proteins of 66 and 51 kD known to be enzymatically active as a complex p66/51. Using an activity gel procedure that allows identification of catalytic polypeptides in situ after PAGE in denaturing conditions, we visualized two major active bands of 66 and 51 kD of reverse transcriptase from highly purified preparations of HIV-1. We show that both p66 and p51 are enzymatically active. An additional active band was also associated with a 165 kD polypeptide, representing about 2-4% of total activity and possibly corresponding to the putative gag-pol precursor. In H9-infected cells the 66 kD active band became visible 70 hours after infection. These studies show that the two major forms of reverse transcriptase (66 and 51 kD) of HIV-1 are independently active and that a higher Mr form of 165 kD is also enzymatically active.
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PMID:Enzymatically active forms of reverse transcriptase of the human immunodeficiency virus. 246 25

We have used activity gel analysis and immunoblotting to provide evidence linking the hepatitis B virus (HBV) reverse transcriptase with its longest unassigned open reading frame (polymerase [Pol]-ORF). Activity gel analysis demonstrated that infectious HBV particles secreted by the Hep 2.2.15 cell line contain major (approximately 70 kilodaltons [kDa]) and minor (approximately 90 kDa) reverse transcriptase activities. By Western immunoblotting, we detected in both HBV particles and Hep 2.2.15 cell extract a approximately 70-kDa Pol-specific peptide. This approximately 70-kDa peptide reacted with antisera directed against the carboxy terminus of the pol gene product. No such immunoreactivity was observed with antisera against the amino terminus of the Pol peptide. The reverse transcriptase protein which was eluted from the major approximately 70-kDa region detected on an activity gel reacted with Pol-specific antisera. Furthermore, reverse transcriptase activity was immunoprecipitated from dissociated HBV particles by using Pol-specific antisera. On the basis of our results, we suggest that HBV encodes its reverse transcriptase from the Pol-ORF.
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PMID:The hepatitis B virus-associated reverse transcriptase is encoded by the viral pol gene. 246 75

Infectious retrovirus particles consist of a core structure containing RNA and gag-pol polypeptides surrounded by a lipid membrane studded with env proteins. A recombinant vaccinia virus was designed to express the entire gag-pol precursor protein of the human immunodeficiency virus type 1. Synthesis and processing of gag proteins occurred in mammalian cells infected with this live recombinant virus, and reverse transcriptase was detected largely in the medium. Electron micrographs revealed immature retrovirus-like particles budding from the plasma membrane and extracellular particles with morphological characteristics of immature and mature human immunodeficiency virus. The latter contained functional reverse transcriptase as well as processed p24 and p17 gag polypeptides. Thus, assembly and maturation of human immunodeficiency virus-like particles can occur in the absence of either infectious RNA molecules or env proteins. The production of noninfectious virus-like particles by expression vectors should be useful for biochemical studies and could provide a safe source of material for the development of vaccines.
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PMID:Human immunodeficiency virus-like particles produced by a vaccinia virus expression vector. 247 31

The human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT)/ribonuclease H has been expressed to high levels in Escherichia coli from a recombinant plasmid constructed using the polymerase chain reaction (PCR) for in vitro mutagenesis. Translational initiation and termination codons were introduced by the PCR at points corresponding to sites of cleavage of the RT from the gag-pol precursor polyprotein by the HIV-1 protease; the HIV-1 protease is not expressed from this construct. Most of the RT coding sequences derived from PCR were exchanged for a DNA fragment cloned by standard methods to minimize the possibility that an unwanted mutation was introduced during the in vitro amplification. The RT is expressed in bacteria from this plasmid as 66 and 51 kDa proteins, has both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activities, and is indistinguishable from native HIV-1 RT in electrophoretic mobility and immunoreactivity. Peptide sequencing of the amino terminus of the HIV-1 RT purified from bacterial lysates is also presented. A novel activity gel assay was used to confirm that only the 66 kd protein catalyzes the RNase H reaction; this assay will simplify analysis of this catalytic activity. This HIV-1 RT expression plasmid is of interest because of the high level of expression in bacteria and the demonstrated RNase H activity of the enzyme. This plasmid will be distributed for research purposes through the NIH AIDS Repository and will facilitate enzymologic, structural, and immunologic evaluation of reverse transcription and its chemotherapeutic inhibition.
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PMID:HIV-1 reverse transcriptase/ribonuclease H: high level expression in Escherichia coli from a plasmid constructed using the polymerase chain reaction. 247 33

Several single-cell clones were isolated from a fetal lamb kidney cell line (FLK) persistently infected with bovine leukemia virus (BLV). The majority of isolated cell clones were virus productive, several were nonproductive based on the determination of the activity of reverse transcriptase and production of 3H-uridine labeled virus particles. Two nonproductive clones, NP-1 and NP-2, were further characterized in comparison with the virus productive cells. All clones contained three integrated BLV proviruses in the nonproducer cells. The virus specific mRNAs were expressed both in the virus productive and nonproductive cells. The virus-specific protein products were found different in the nonproducing cells. The gag pol precursor Pr145 was missing in NP-1 cells, in NP-2 its Mr was 120 only. In NP-2 cells the precursors Pr70gag and Pr45gag were absent. The env precursor gPr72 and both of the two glycoproteins were detected. The nonproductive cells NP-2 produced mainly env gene products, therefore they were tested as a potential material for anti-BLV vaccine. The NP-2 cells after inoculation to rats and cattle were able to induce formation of neutralizing antibodies directed against the env gene products.
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PMID:Bovine leukemia virus: isolation and characterization of nonproducer cell clones. 282 41

The gag-pol precursor protein of the avian sarcoma-leukosis virus is processed into three known pol-encoded mature polypeptides; the 95- and 63-kilodalton (kDa) beta and alpha subunits, respectively, of reverse transcriptase and the 32-kDa pp32 protein. The pp32 protein possesses DNA endonuclease activity and is produced from the precursor by two proteolytic cleavage events, one of which removes 4.1 kDa of protein from the C terminus. A 36-kDa protein (p36pol) which retains this C-terminal segment is detectable in small quantities in virions. We have constructed Escherichia coli plasmid clones that express the C-terminal domains of pol corresponding to pp32 and p36. These proteins have been purified by column chromatographic methods to near homogeneity. No significant differences could be detected in the enzymatic properties of the bacterially produced p32pol and p36pol proteins. Both possess DNA endonuclease activity and, like the pp32 protein isolated from virions, can cleave near the junction of two tandem avian sarcoma-leukosis virus long terminal repeats in double-stranded supercoiled DNA substrates. In the presence of Mg2+, both p32pol and viral pp32 cleave either strand of DNA 2 nucleotides 5' to the junction.
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PMID:Properties of avian sarcoma-leukosis virus pp32-related pol-endonucleases produced in Escherichia coli. 283 18

The ability of dihydrothymidine (DHdTTP) and thymidine glycol (dTTP-GLY) 5'-triphosphates to serve as substrates for different DNA polymerases was investigated. DHdTTP but not dTTP-GLY was used as a substrate by E. coli DNA polymerase I (Pol I). Within the detection limit of the assay used, neither T4 DNA polymerase nor avian myeloblastosis virus (AMV) reverse transcriptase used DHdTTP or dTTP-GLY as substrates. The ability of DHdTTP and dTTP-GLY to undergo enzyme-catalyzed turnover to the monophosphate paralleled their ability to serve as substrates for polymerization. These results, along with kinetic parameters for the incorporation of DHdTTP with Pol I, strongly suggest that the saturation of thymine C5-C6 bond and the substituent groups at C5 and C6 differentially exert effects on binding to DNA polymerases. DNA sequencing gel analysis of the polymerization products revealed that most single adenine sites were capable of templating DHdTTP, however, DNA synthesis was partially arrested at multiple adenine sites, suggesting that sequential incorporation of DHdTTP produced significant disorder in the primer terminus.
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PMID:Dihydrothymidine and thymidine glycol triphosphates as substrates for DNA polymerases: differential recognition of thymine C5-C6 bond saturation and sequence specificity of incorporation. 306 Aug 57

We have purified a 10,774-dalton protein from human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame (ORF). Radiochemical amino acid microsequencing identified 12 amino acids from the stretch of 39 N-terminal residues of this protein, beginning with a PQITLW sequence at position 69 of the pol ORF. Radiosequencing of selected tryptic peptides of the protein identified 11 additional residues (Leu-9 and Val-2) in six peptides encompassing the entire molecule of 99 residues. A protein of similar size and identical N-terminal sequence (determined through the first 39 residues) was present among the processed HIV pol gene products in Escherichia coli which expressed the entire HIV pol ORF. The C terminus of both the viral and E. coli-expressed proteins was inferred to be contiguous with the N terminus of the p64-p51 reverse transcriptase on the basis of tryptic mapping and specific immunoreactivity with an antiserum against a dodecapeptide located upstream of the reverse transcriptase. Thus, the initial processing of the pol precursor that generates the native protease is apparently preserved across phylogenetic barriers. Although the purified viral protease lacked measurable proteolytic activity, the bacterial extracts were capable of processing an HIV gag precursor protein synthesized in E. coli.
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PMID:Purification and structural characterization of the putative gag-pol protease of human immunodeficiency virus. 329 93

Monoclonal antibodies were prepared against the avian myeloblastosis virus reverse transcriptase. These monoclonal antibodies specifically immunoprecipitated the alpha and beta subunits of the reverse transcriptase molecule, as well as the Pr180gag-pol precursor protein present in virus-infected cells. In addition, these monoclonal antibodies inhibited the DNA polymerase activity associated with the reverse transcriptase molecule but not the RNase H activity. The monoclonal antibody preparations were specific for the amino-terminal portion of the protein, as determined by the immunoprecipitation of a reverse transcriptase-beta-galactosidase fusion protein produced in Escherichia coli by molecular cloning procedures.
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PMID:Production and characterization of monoclonal antibodies against avian retrovirus reverse transcriptase. 618 37


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