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)

Replacement of the putative active site Asp residue of cloned HIV-1 protease with Ala yields a molecule incapable of autocatalytic processing. Similarly, protease/reverse transcriptase and protease/reverse transcriptase/endonuclease polyproteins containing the same mutation accumulate as enzymatically inert polyproteins. Introduction of a second, wild-type, copy of protease in trans alleviates this defect, leading in the case of individually cloned protease to cleavage of the mutant protein, and with the polyprotein mutants to release of the reverse transcriptase and endonuclease polypeptides, the former of which recover enzymatic activity. In related experiments, a similar inhibition and trans-complementation of a genetically engineered gag--protease fusion protein was observed.
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PMID:Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation. 246 Dec 97

Elimination of the protease domain from the polymerase open reading frame (pol) of the human immunodeficiency virus type 1 (HIV-1) leads, in Escherichia coli, to synthesis and accumulation of a non-processed 98-kDa reverse transcriptase/endonuclease (RT/ENDO) polyprotein. A partially purified preparation of this reverse RT/ENDO polyprotein displays little or no RT activity. Introduction of the pol protease domain as a separate transcriptional unit on the same plasmid restores the processing program, generating correctly sized RT and ENDO polypeptides. Concomitant with restoration of processing is the reappearance of RT activity. These results suggest that for HIV-1 RT to be active, it must be matured from the pol polyprotein.
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PMID:Biosynthesis and analysis of a genetically engineered HIV-1 reverse transcriptase/endonuclease polyprotein in Escherichia coli. 246 29

Molecular evolution and phylogeny of different human immunodeficiency virus type 1 (HIV1) strains, of a type 2 (HIV2) strain, and of two simian immunodeficiency viruses (SIVAGM and SIVMAC) have been studied by comparing the nucleotide sequences of the two regions of their pol genes which encode the reverse transcriptase (RT) and endonuclease/integrase (EN). The analyses show that the different HIV 1s form one cluster (HIV1 group) and that the SIVs and HIV2 form another (HIV2 group). When the entire genomes of a HIV1, a HIV2, and the two SIVs were compared, the SIVAGM showed a unique pattern of mutation accumulations; that is, the SIVAGM has accumulated more nonsynonymous changes than synonymous changes in the RT and EN regions after its recent divergence from SIVMAC-142, and, furthermore, it has a deletion of approximately 350 bp in the region between the pol and env genes. The SIVAGM was apparently derived from cell cultures infected with a macaque isolate, SIVMAC-251. The contamination provides an opportunity to measure the maximum rate of evolution in the SIVAGM by comparing its DNA sequence to those of SIVMAC-251 and SIVMAC-142. The analysis shows that the rates are given approximately by (1.95 +/- 1.37) x 10(-3)/site/year for one SIVAGM sequence and (5.18 +/- 2.25) x 10(-3)/site/year for another.
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PMID:Molecular evolution of the human and simian immunodeficiency viruses. 246 34

A series of test substrates have been synthesized to establish the effect of termini on the putative exoribonuclease H activity of reverse transcriptase. Recombinant reverse transcriptase from human immunodeficiency virus, natural enzyme from avian myeloblastosis virus, and a known endonuclease, Escherichia coli ribonuclease H, cleaved relaxed, circular, covalently closed plasmids in which 770 consecutive residues of one strand were ribonucleotides. The avian enzyme also deadenylated capped globin mRNA with a covalently attached oligo(dT) tail at the 3' end. These results resolve a long-standing controversy--that the viral enzymes are obligatory exonucleases in vitro, based on their failure to cleave certain substrates for E. coli ribonuclease H, including circular poly(A).linear poly(T) and ribonucleotide-substituted supercoiled plasmids, but resemble endonucleases in vivo, based on their ability to degrade RNA in complex DNA.RNA hybrids. The data strongly suggest that the viral enzymes are endonucleases with exquisite sensitivity to the conformation of heteroduplexes. Inhibition of viral, but not cellular, ribonuclease H with ribonucleoside-vanadyl complexes further distinguishes these enzymes.
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PMID:Ribonuclease H activities associated with viral reverse transcriptases are endonucleases. 247 Nov 88

The intrinsic properties of reverse transcriptase in reverse transcription were studied using a synthetic, partial ovalbumin mRNA with a synthetic DNA oligonucleotide annealed to the 3'-end of the RNA as a model substrate. With or without concomitant cDNA synthesis, the RNase H activity of avian myeloblastosis virus (AMV)-reverse transcriptase cleaved the substrate at a site which would leave a hybrid of between 7 and 14 base pairs between the 3' termini of the RNA and DNA oligonucleotide. Variability in the exact size of the hybrid probably reflects some weak base preference for cleavage by the enzyme. These short hybrids can be recognized as substrates by Escherichia coli RNase H and can be utilized by reverse transcriptase as sites for continuation of cDNA synthesis. Substrates with 5'-triphosphorylated termini, 3'-OH, 3'-phosphate, 3'-end hairpin structures and 20 base pair hybrids on the middle region of long RNA more than 300 bases or on circular RNA were all cleaved by AMV-reverse transcriptase-associated RNase H, indicating that the RNase H activity is essentially regarded as an endonuclease degrading RNA moiety in RNA-DNA hybrid. The modes of action of reverse transcriptase from murine leukemia virus and Rous-associated virus 2 were the same as that of AMV-reverse transcriptase, except that the size of the remaining hybrid and the specificity for cleavage depended on the reverse transcriptase. We propose a possible model to explain the mode of action of RNase H and RNA-dependent DNA polymerase activities in reverse transcription.
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PMID:Intrinsic properties of reverse transcriptase in reverse transcription. Associated RNase H is essentially regarded as an endonuclease. 247 53

A highly purified thyroglobulin (Tg) mRNA was isolated from human nodal euthyroid goiter. Ultracentrifugation in a 5-20% sucrose density gradient revealed that the protein has a sedimentation coefficient of 33S. cDNA was synthesized from the 33S RNA, using reverse transcriptase in the presence of a human placental ribonuclease inhibitor. In order to detect the polymorphism of restriction fragment length, screening of high molecular weight DNA from normal human thyroid gland and from nodal euthyroid and diffuse toxic goiters was carried out, using Tg cDNA probes and plasmid DNA containing a Tg cDNA fragment. After restriction with endonuclease Hind III, three Tg-containing fragments were identified both in the normal and nodal euthyroid goiters, whereas restriction with endonuclease Bam HI revealed two Tg-containing fragments in nodal euthyroid and diffuse toxic goiters.
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PMID:[Mapping of the thyroglobulin gene in high molecular weight DNA from the human thyroid in normal conditions and in thyroid pathology]. 256 30

The rapid identification of anti-HIV compounds in the laboratory following the isolation of the causative virus in 1983 and their subsequent use in the clinic was not unexpected. Three decades of previous work had established a scientific basis for the evaluation of antiviral compounds. However, no antiviral yet discovered can cause total blockade of a virus replicating in a cell. The combination of properties of HIV including latency, antigenic and biochemical variation is unusual and the virus represents a daunting challenge for chemotherapy. But at least 90 antiviral compounds have been discovered, many inhibiting the virus reverse transcriptase. Other targets for inhibition are possible including viral regulatory gene products, viral protease and endonuclease enzymes but compounds for initial study will have to be found by random searching. X-ray crystallography of HIV proteins will shortly be possible, enabling the commencement of a more molecular specific search for inhibitors. Meanwhile, advantage can be taken of comparative nucleotide sequences of the HIV-1 and -2 genomes to test short oligonucleotides as potential inhibitors of mRNA transcription. The pol gene also has a zinc finger amino acid sequence suggesting that chelation chemotherapy may have a potential role. In the absence of HIV vaccines, and associated theoretical problems in their development, antiviral chemotherapy is expected to occupy a central role in combating the AIDS epidemic.
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PMID:Potential target sites for antiviral inhibitors of human immunodeficiency virus (HIV). 265 20

Ty3, a retrotransposon of Saccharomyces cerevisiae, is found within 20 base pairs (bp) of the 5' ends of different tRNA genes. Determination of the complete nucleotide sequence of one Ty3 retrotransposon (Ty3-2) shows that the element is composed of an internal domain 4,748 bp long flanked by long terminal repeats of the 340-bp sigma element. Three open reading frames (ORFs) longer than 100 codons are present in the sense strand. The first ORF, TYA3, encodes a protein with a motif found in the nucleic acid-binding protein of retroviruses. The second ORF, TYB3, has homology to retroviral pol genes. The deduced amino acid sequence of the reverse transcriptase domain shows the greatest similarity to Drosophila retrotransposon 17.6, with 43% identical residues. The inferred order of functional domains within TYB3--protease, reverse transcriptase, and endonuclease--resembles the order in Drosophila element 17.6 and in animal retroviruses but is different from that found in yeast elements Ty1 and Ty2. A second Ty3 element (Ty3-1) from a standard laboratory strain was overexpressed and shown to transpose.
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PMID:Ty3, a yeast retrotransposon associated with tRNA genes, has homology to animal retroviruses. 285 94

The DNA sequence of the gag and pol regions of a provirus cloned from a bovine tumor is presented. In order to confirm these results the sequence of portions of a second clone, derived from a virus-producing cell line, was also determined. The gag gene was found to consist of 1179 nucleotides, which probably encode only three proteins: an N-terminal protein of 109 amino acids, a major core protein (p24) of 215 amino acids, and a nucleic acid binding protein (p12) of 69 residues. An open reading frame, whose translated product showed clear homology to the avian and murine proteases, was found beginning immediately upstream of the 3' end of gag. Following this protease region, a third long open reading frame, encoding 852 amino acids, showed clear homology to both avian and murine pol genes. The mechanism of translation of the protease and pol gene products cannot be predicted with certainty. Like Moloney murine leukemia virus (M-MuLV), BLV has a termination signal at the 3' end of gag, but unlike M-MuLV the protease is in a different reading frame. Like Rous sarcoma virus (RSV), BLV has a termination signal at the 3' end of the protease region and the reverse transcriptase is in a different (i.e., the third) reading frame. Possible translation mechanisms are discussed. Finally, the BLV gag and pol gene products are highly related to those of the human T-cell leukemia virus (HTLV); relatedness varied from 37% amino acid identities within the N terminal gag protein to 54% within the nucleic acid binding protein. Highly significant homology with both murine and avian type-C proteins was found within p24, p12, and the putative protease, reverse transcriptase, and endonuclease. Based on this homology, the BLV-HTLV family of viruses appears about equally distantly related to murine and avian type-C viruses.
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PMID:The gag and pol genes of bovine leukemia virus: nucleotide sequence and analysis. 299 90

DNA from human T-lymphoid (Molt-4) and hamster kidney (BHK-21) cells infected with the T-lymphotropic simian foamy virus LK-3 was shown to be infectious, when assayed by transfection of BHK-21 cells. The proviral genome was further characterized by blot hybridization to a specific cDNA probe, which had been prepared by reverse transcription in vitro using viral RNA and RNA-dependent DNA polymerase present in cytoplasmic extracts of infected BHK-21 cells. This probe hybridized to a DNA species of 14 kbp in extracts from LK-3-infected diploid human fibroblasts, Molt-4 and BHK-21 cells, whereas no hybridization occurred with DNA from the respective uninfected controls. No integrated proviral DNA could be demonstrated, and the 14 kbp DNA was shown not to represent circular DNA. The patterns of restriction endonuclease and S1 nuclease fragments indicated a unique configuration of linear double-stranded DNA containing a single-stranded section separating two subunits one of which may be sufficient to transmit LK-3 by transfection with DNA.
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PMID:Detection and characterization of infectious DNA intermediates of a primary foamy virus. 301 32


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