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 PAT retroid transposable elements differ from other retroids in that they have a 'split direct repeat' structure, i.e., and internal 300bp sequence is found repeated, about one half at each element extremity. A very abundant transcript of about 900 nt, the start of which maps to the preferentially deleted portion of PAT elements, is detected on total Panagrellus redivius RNA bearing Northern blots. A potentially corresponding ORF encodes a protein of 265 residues having a carboxy terminal Cystein motif, believed to be exclusively characteristic of the GAG protein in retoid elements. A much fainter, 1800nt long transcript, is also detected on Northern blots and maps slightly downstream of the first ORF. The predicted protein sequence of this region bears motifs typical of reverse transcriptase and RNaseH, as found in the Pol genes of retroid elements. Peptide motif similarities are greatest with the DIRS-1 element derived from Dictyostelium discoideum. The possibility of using PAT elements as transposon tagging system for Caenorhabditis elegans is discussed.
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PMID:Unusual features of the retroid element PAT from the nematode Panagrellus redivivus. 131 55

Two distinct site-specific retrotransposon families, named RT1 and RT2, from the sibling mosquito species Anopheles gambiae and A. arabiensis, respectively, were previously identified. Both were shown to occupy identical nucleotide positions in the 28S rRNA gene and to be flanked by identical 17-bp target site duplications. Full-length representatives of each have been isolated from a single species, A. gambiae, and the nucleotide sequences have been analyzed. Beyond insertion specificity, RT1 and RT2 share several structural and sequence features which show them to be members of the LINE-like, or non-long-terminal-repeat retrotransposon, class of reverse transcriptase-encoding mobile elements. These features include two long overlapping open reading frames (ORFs), poly(A) tails, the absence of long terminal repeats, and heterogeneous 5' truncation of most copies. The first ORF of both elements, particularly ORF1 of RT1, is glutamine rich and contains long tracts of polyglutamine reminiscent of the opa repeat. Near the carboxy ends, three cysteine-histidine motifs occur in ORF1 and one occurs in ORF2. In addition, each ORF2 contains a region of sequence similarity to reverse transcriptases and integrases. Alignments of the protein sequences from RT1 and RT2 reveal 36% identity over the length of ORF1 and 60% identity over the length of ORF2, but the elements cannot be aligned in the 5' and 3' noncoding regions. Unlike that of RT2, the 5' noncoding region of RT1 contains 3.5 copies of a 500-bp subrepeat, followed by a poly(T) tract and two imperfect 55-bp subrepeats, the second spanning the beginning of ORF1. The pattern of distribution of these elements among five siblings species in the A. gambiae complex is nonuniform. RT1 is present in laboratory and wild A. gambiae, A. arabiensis, and A. melas but has not been detected in A. quadriannulatus or A. merus. RT2 has been detected in all available members of the A. gambiae complex except A. merus. Copy number fluctuates, even among the offspring of individual wild female A. gambiae mosquitoes. These findings reflect a complex evolutionary history balancing gain and loss of copies against the coexistence of two elements competing for a conserved target site in the same species for perhaps millions of years.
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PMID:Distinct families of site-specific retrotransposons occupy identical positions in the rRNA genes of Anopheles gambiae. 132 71

Several newly discovered potent and selective non-nucleoside inhibitors of human immunodeficiency virus-1 reverse transcriptase (RT) are undergoing evaluation in clinical trials. We studied the potential for development of viral resistance to one of the prototype compounds, BI-RG-587, a dipyridodiazepinone derivative. Human immunodeficiency virus-1 resistant to BI-RG-587 emerged after only one cycle of in vitro infection in the presence of the drug. Resistant virus was cross-resistant to the non-nucleoside tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-thione derivative R82150 but remained susceptible to 2',3'-dideoxynucleosides and phosphonoformate. Both native (virion-associated) and recombinant RT derived from resistant virus were insensitive to BI-RG-587 and R82150. Nucleotide sequence analysis of multiple drug-resistant and -sensitive recombinant RT clones identified a single predicted amino acid change common to all resistant clones (tyrosine-181----cysteine). These studies suggest that the viral resistance to non-nucleoside RT inhibitors may develop in vivo. This possibility should be carefully monitored in clinical trials of these compounds.
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PMID:In vitro selection and molecular characterization of human immunodeficiency virus-1 resistant to non-nucleoside inhibitors of reverse transcriptase. 137 83

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) has only 2 cysteine residues at positions 38 and 280. In order to investigate the role of these cysteines in the structure and function of the enzyme, we have previously modified each of the cysteines to serines employing site-directed mutagenesis. Two of the mutant forms of HIV-1 RT, the single mutant of cysteine 280 and a double mutant with both cysteines modified, were purified. In the present study we have compared the catalytic properties of the DNA-polymerizing and the ribonuclease H (RNase H) functions of the two mutant RTs to those of the native enzyme. The results indicate that the single mutant RT closely resembles the wild type enzyme in almost all the catalytic functions tested. The double cysteine mutant RT, on the other hand, exhibits several unique features. First, the specific activities of the RNA- and DNA-directed DNA synthesis are significantly lower than the corresponding activities of the other two enzymes. This probably results from the lower Vmax values exhibited by the double mutant RT, since the Km values calculated for all enzymes were similar. Second, the most outstanding differences are associated with the RNase H activity of the double mutant RT. The specific activity of RNase H is about 4-fold higher than the wild type and the single mutant RTs. Furthermore, the heat stability of the RNase H function of the double mutated RT is at least 15-fold higher than that of the other two RTs. The substantial resistance to heat denaturation is apparent only for the RNase H activity, since the DNA polymerizing function of the double mutant RT is as sensitive to heat denaturation as the other two proteins.
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PMID:The effects of cysteine mutations on the catalytic activities of the reverse transcriptase of human immunodeficiency virus type-1. 137 33

The active site of human immunodeficiency virus reverse transcriptase (HIV1-RT) was probed using three group-specific reagents: phenylglyoxal (PG), N-ethylmaleimide (NEM), and pyridoxal 5'-phosphate (PLP). The inactivation of HIV1-RT by arginine-specific PG was found to be completely protected against by adding primer-template. The potential active site arginine was localized to position 277 in the primary structure, suggesting that the polymerase domain of the enzyme should be considered as extending at least this far from the N terminus. The sulfhydryl-modifying reagent NEM completely inhibits NY5-HIV1-RT, which contains a cysteine at position 162, and such inhibition is protected against by primer-template. However, it does not strongly inhibit LAV-HIV1-RT, in which C162 is replaced by S162, indicating that while C162 may be at or near the active site or interact allosterically with primer-template, it is not essential for activity. The lysine-specific reagent PLP was found to be a noncompetitive inhibitor with respect to both primer-template [poly(rA).oligo(dT)] and dTTP. The latter result differentiates HIV1-RT from other RTs, for which PLP has been shown to be a competitive inhibitor with respect to dTTP.
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PMID:Active site studies of human immunodeficiency virus reverse transcriptase. 138 Aug 26

We have generated by site-directed mutagenesis plasmids that induce the synthesis of specific mutants of the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). These recombinant mutants of HIV-1 RT, designed on the basis of our previous studies of HIV-1 and HIV-2 RTs, were analyzed for structure-function relationship by assessing their RNA-dependent and DNA-dependent DNA polymerase as well as the ribonuclease H activities. Three groups of mutants were studied. 1) We have investigated the importance of the only two sets of highly conserved double prolines found in the sequence of HIV-1 RT. The results indicate that the conversion of either one or both prolines (at positions 225 and 226) to threonines have no significant effect on all catalytic activities of the enzyme. The mutants in which prolines 419 and 420 were individually modified to threonines exhibit full activities, whereas the double proline 419/420 mutant lost most of its RNase H activity (although the DNA polymerase function was fully retained). 2) We have deleted phenylalanine 346 from HIV-1 RT, which is absent in wild type HIV-2 RT. This mutant of HIV-1 RT lost practically all catalytic activities. 3) A mutant of HIV-1 RT in which a cysteine residue substituted for alanine 446, was found to be slightly hyperactive for both DNA polymerase and RNase H activities.
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PMID:Functional analysis of novel selective mutants of the reverse transcriptase of human immunodeficiency virus type 1. 138 52

Lamprey liver mRNA sequences were amplified by reverse transcriptase-polymerase chain reaction using primers synthesized according to the amino acid sequences at the thioester region common to the mammalian C3, C4, and alpha 2-macroglobulin (alpha 2M). Two different cDNA species were identified that showed a close similarity to the mammalian C3 or alpha 2M sequences, respectively. Using the C3-like sequence as a probe, two overlapping cDNA clones were isolated from the lambda ZAP library, which together covered the entire region encoding the putative lamprey pro-C3. The deduced amino acid sequence of the putative lamprey pro-C3 contained 1660 amino acids and showed 31%, 22%, 23%, and 16% amino acid sequence identity with mouse C3, C4, C5, and human alpha 2M, respectively. The distributions of cysteine residues were completely identical between the mouse C3 and the putative lamprey C3 except that the lamprey sequence had two additional cysteine residues in the alpha-chain. The possible beta-alpha and alpha-gamma processing sites were found at exactly the same positions as in mammalian C4. These results suggest that the putative lamprey C3 retains a close similarity to the common ancestor of the mammalian C3 and C4, which appeared to have had a three-subunit chain structure.
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PMID:Complete complementary DNA sequence of the third component of complement of lamprey. Implication for the evolution of thioester containing proteins. 157 50

Approximately 50% of the ribosomal DNA (rDNA) units of Drosophila melanogaster are inactivated by two different 28 S RNA ribosomal gene insertions (type I and type II). We present here the nucleotide sequence of complete type I and type II elements. Conceptual translation of these sequences revealed open reading frames (ORFs) encoding amino acid residues conserved in all retrotransposable elements. Full-length type I elements are 5.35 x 10(3) base-pairs in length and contain two overlapping ORFs. The smaller ORF (471 amino acid residues) has similarity to gag genes, while the larger ORF (1021 residues) has similarity to pol genes. Full-length type II elements are 3.6 x 10(3) base-pairs and contain one large ORF (1056 residues) that appears to represent a gag-pol fusion. Type I and type II elements are similar in structure, in the proteins they encode, and in insertion specificity to the R1Bm and R2Bm retrotransposable elements of Bombyx mori. We suggest that the D. melanogaster elements be called R1Dm and R2Dm, to reflect their structure as retrotransposons. Comparison of the R1 and R2 elements from these two widely different species revealed regions of the ORF that are likely to play an important role in the propagation of the elements. Four distinct regions of sequence conservation separated by regions of little or no sequence similarity were detected for both the R1 and R2 elements: (1) cysteine motifs of the gag gene, with three such motifs for R1 and one motif for R2; (2) a reverse transcriptase domain; (3) an integrase domain located carboxyl terminal to the reverse transcriptase region; and (4) a small region amino terminal to the reverse transcriptase domain, whose function is not known. The level of identity of the amino acid residues for these segments is 28 to 34% between the R1 elements, and 34 to 39% for the R2 elements. Finally, it may be predicted that the mechanism of unequal crossover might eventually eliminate R1 and R2 from the rDNA locus. The long history of selection at the protein level exhibited by these elements indicates that it is their active transposition that maintains them in the locus. The high level of sequence homogeneity between copies of each element within the same species is consistent with the high turnover rate expected to result from these processes.
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PMID:Type I (R1) and type II (R2) ribosomal DNA insertions of Drosophila melanogaster are retrotransposable elements closely related to those of Bombyx mori. 169 Aug 12

The enzyme reverse transcriptase (RT) is crucial in the early steps of the life cycle of retroviruses. We have expressed in bacteria the RTs from human immunodeficiency viruses (HIV) types 1 and 2 in order to study the structural-functional relationships of these two multifunctional enzymes that share a relatively high degree of amino acid sequence homology. For comparison purposes, we have analyzed several catalytic functions of both enzymes. The two HIV RTs show a high similarity in many aspects studied but exhibit profound differences in several other properties. For instance, the specific RNase H activity of HIV-2 RT is about 10 times lower than the corresponding activity of HIV-1 RT. There are also significant dissimilarities between some of the apparent Km values calculated for the DNA polymerizing functions of both enzymes. Furthermore, the heat stability of the DNA polymerizing activity of HIV-2 RT is about 15-fold higher than that of HIV-1 RT. On the other hand, the susceptibility of the RNase H activities of the two enzymes to heat inactivation was found to be similar. Other treatments also enable discrimination between the RNase H and DNA polymerizing catalytic properties of the two enzymes (although both reverse transcriptases respond similarily). Thus, the RNase H activity was inactivated by N-ethylmaleimide, suggesting the possible involvement of cysteine residues in performing this activity, whereas the DNA polymerizing functions of the two enzymes were fully resistant to this chemical modification. The zinc chelator 1,10-phenanthroline affected the DNA polymerase activities of both enzymes to a significantly higher extent than the RNase H activity. In all, the two HIV RTs were shown to be substantially different one from the other in several of their properties and also distinct from other RTs thus far studied.
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PMID:Catalytic properties of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. 170 12

Various combinations of inhibitors of HIV reverse transcriptase were tested for inhibition of HIV replication in order to reveal any potential synergism or antagonism. PFA, a pyrophosphate analogue, gave synergistic inhibition of HIV replication in combination with both of the thymidine analogues AZT and FLT. The combination of PFA and AZT-TP gave only additive or weakly synergistic inhibition in a reverse transcriptase enzyme assay. The combination of AZT and FLT also gave synergistic inhibition of HIV replication, whilst the combination of AZT-TP and FLT-TP gave only additive or weakly synergistic inhibition of reverse transcriptase. Thus, the synergy does not arise from effects on reverse transcriptase alone but must be owing to other, cellular factors, such as effects on nucleoside metabolism or metabolism of the analogues. The results are consistent with the hypothesis that AZT may have an alternative mechanism of inhibition other than inhibition of reverse transcriptase. The diminished cytotoxicity observed in addition to the synergistic inhibition makes these combinations attractive from the point of view of combination chemotherapy. The inhibition of HIV replication by peptides from various parts of the V3 region of gp120 whose sequences were homologous with the tryptase inhibitor trypstatin was tested. Inhibitory activity was displayed by two peptides containing cysteine in their sequence. Antibodies to two peptides containing the two conserved cysteine residues from opposite sides of the neutralizing loop of gp120 were previously associated with protection from vertical transmission of HIV. The V3 region thus seems to be important for the function of gp120 and the transmission of HIV.
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PMID:Synergistic combinations and peptides in the inhibition of human immunodeficiency virus. 171 18


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