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)

Viral populations in a human immunodeficiency virus type 1 (HIV-1)-infected individual behave as a quasispecies with a rated distribution of fitness variants. Fitness distributions in naturally occurring viral populations have been difficult to study due to the lack of markers for individual virus clones and complicating inter- and intrahost factors like the presence of multiple cell types with distinct tropisms, differences in route of transmission, and intervening immunity. Here, we quantitated the relative fitness in vivo of three subpopulations of HIV-1 marked by mutations at codons 41 and 215 of reverse transcriptase (RT) directly related to zidovudine resistance in an untreated individual who was infected by a zidovudine-resistant strain transmitted from a donor on therapy. The transmission event did not have a substantial impact on the distribution of mutants within the dominant virus population replicating to high levels in the recipient. The evolution of the RT gene was monitored for 20 months. All 102 clones obtained from the donor and the recipient at the different time points contained the M41L mutation, which is associated with a fourfold reduction in zidovudine sensitivity. The leucine at position 41 was stable, although it was encoded by TTG and CTG triplets that fluctuated in abundance partially due to founder effects of clones with nonsilent mutations at codon 215. Of the three subpopulations in the patient, distinguished by a tyrosine (TAC), aspartic acid (GAC), or serine (TCC) at the 215 position of RT, the relative fitness of the GAC variant was calculated to be 10 to 25% higher than the initial TAC variant, and the relative fitness of the TCC variant was 1% higher than that of the GAC variant. Similar to other RNA viruses, lentivirus populations like HIV-1 in patients with a high virus load apparently consist of a broader spectrum of fitness variants than the 1 to 2% fitness difference sufficient for significant replicative advantage.
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PMID:Broad spectrum of in vivo fitness of human immunodeficiency virus type 1 subpopulations differing at reverse transcriptase codons 41 and 215. 915 39

Genetic polymorphisms occur in many of the drug metabolizing enzymes. However, the effect of polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases is still undescribed, despite the many reported cases of variations in glucuronidation activities. Characterization of the UGT2B15(Y85) cDNA, which was isolated from human prostate and LNCaP cell cDNA libraries, revealed 20 nucleotide differences between UGT2B15(Y85) and the previously characterized UGT2B15 protein UGT2B15(D85). However, only one of the two variations in the coding region leads to an amino acid change from aspartic acid to a tyrosine residue at position 85. The genomic DNA of 27 subjects were analysed by direct sequencing of polymerase chain reaction (PCR) products and demonstrated that UGT2B15(D85) and UGT2B15(Y85) are encoded by variant alleles prevalent in the Caucasian population. Expression of UGT2B15(D85) and UGT2B15(Y85) in HK293 cells demonstrated similar substrate specificities. Of the 65 potential substrates tested for activity, the proteins were active on phenolic compounds, coumarins, flavonoids, drugs and steroid hormones. Both proteins displayed similar Km values of 2.2 and 2.4 microM for androstane-3alpha,17beta-diol and dihydrotestosterone, respectively. However, results suggest that UGT2B15(Y85) has a higher Vmax than UGT2B15(D85). Specific reverse transcriptase (RT)-PCR analysis revealed expression of the UGT2B15 gene in a wide range of extrahepatic tissues including the human liver, kidney, testis, mammary gland, placenta, adipose, skin, uterus, prostate and lung. The wide expression of UGT2B15 in many tissues indicates that it is a major glucuronidation enzyme in humans.
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PMID:Isolation and characterization of UGT2B15(Y85): a UDP-glucuronosyltransferase encoded by a polymorphic gene. 929 60

Two adeno-associated virus (AAV)-derived plasmids were constructed with portions of the N-methyl-d-aspartic acid-R1 (NMDA-R1) receptor subunit downstream from the AAV p40-(pJDT95dlk-aR1) or cytomegalovirus (CMV) promoter (pTRUF3-aR1) in an antisense orientation. Each plasmid drove expression of antisense NMDA-R1 in primary rat neocortical neuronal cultures 4 days after transfection as detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Transfection with pTRUF3-aR1 (2x4 microgram) but not with pJDT95dlk-aR1 decreased neuronal [3H]MK-801 binding in a dose-dependent manner.
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PMID:Reduced MK801 binding in neocortical neurons after AAV-mediated transfections with NMDA-R1 antisense cDNA. 951 73

Sequences of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) domain were determined by direct sequencing of HIV-1 RNA in successive plasma samples from eight seroconverting patients infected with virus bearing the T215Y/F amino acid substitution associated with zidovudine (ZDV) resistance. At baseline, additional mutations associated with ZDV resistance were detected. Three patients had the M41L amino acid change, which persisted. Two patients had both the D67N and the K70R amino acid substitutions; reversion to the wild type was seen at both positions in one of these patients and at codon 70 in the other one. Reversion to the wild type at codon 215 was observed in only one of eight patients. Unusual amino acids, such as aspartic acid (D) and cysteine (C), appeared at position 215 in four patients during follow-up. These variants isolated by coculturing were sensitive to ZDV. Overgrowth of these variants suggests that they have better fitness than the original T215Y variant. Intraindividual nucleoside substitutions over time were 10 times more frequent in codons associated with ZDV resistance (41, 67, 70, 215, and 219) than in other codons of the RT domain. The predominance of nonsynonymous substitutions observed over time suggests that most changes reflect adaptation of the RT function. The variance in sequence evolution observed among patients, in particular at codon 215, supports a role for chance in the evolution of the RT domain.
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PMID:Switch to unusual amino acids at codon 215 of the human immunodeficiency virus type 1 reverse transcriptase gene in seroconvertors infected with zidovudine-resistant variants. 955 30

Variations in glucuronidation activities among different individuals have been reported; however, genetic polymorphisms in the genes encoding phase II drug metabolizing UDP-glucuronosyltransferases have not been studied extensively. A novel UGT2B cDNA clone UGT2B4(E458) was isolated from human prostate and LNCaP cell cDNA libraries. The cDNA encoding UGT2B4(E458) is 2097 bp in length and has an open reading frame of 1584 nucleotides encoding a protein of 528 amino acids. Characterization of the UGT2B4(E458) cDNA revealed nucleotide differences with the previously published UGT2B4 and UGT2B11 cDNAs. These variations in the UGT2B4 sequence lead to an amino acid change from aspartic acid to glutamic acid at position 458. In the previous UGT2B11 cDNA (which has subsequently been renamed UGT2B4 (L109,396, D458)), leucine residues are found at positions 109 and 396, whereas phenylalanines are present at these positions in the UGT2B4(D458) and UGT2B4(E458) enzymes. Analysing the genomic DNA of 26 unrelated Caucasian individuals demonstrated the presence of variant alleles encoding UGT2B4(D458) and UGT2B4(E458). Stable expression of UGT2B4(E458) cDNA in HK293 cells demonstrates the presence of a 52 kDa protein, which is in agreement with other characterized (UGT2B proteins. UGT2B4(E458) conjugates hyodeoxycholic acid (HDCA) as well as 4-hydroxyestrone (4-OH-E1), androstane-3alpha,17beta-diol (3alpha-diol) and androsterone (ADT). Specific reverse transcriptase-polymerase chain reaction analysis revealed expression of UGT2B4(D458) and UGT2B4(E458) transcripts in a wide range of extrahepatic tissues, including the liver, kidney, testis, mammary gland, prostate, placenta, adipose, adrenal, skin and lung. Our results suggest that UGT2B4(E458) and UGT2B(E458) are two widely expressed isoenzymes, and that polymorphism in the UGT2B4 gene might be responsible for differences in UGT2B4 enzymatic properties.
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PMID:Characterization and substrate specificity of UGT2B4 (E458): a UDP-glucuronosyltransferase encoded by a polymorphic gene. 1037 68

The beta-glucosidase from Aspergillus niger (CMI CC 324262) was purified, and an N-terminal sequence and two internal sequences were determined. BglI genomic gene and the cDNA were cloned from a genomic library and by reverse transcriptase-polymerase chain reaction, respectively. The cDNA was successfully expressed in Saccharomyces cerevisiae and Pichia pastoris. Sequence analysis revealed that the gene encodes a 92-kDa enzyme that is a member of glycosidase family 3. (1)H-NMR analysis of the reaction catalyzed by this enzyme confirmed that, in common with other family 3 glycosidases, this enzyme hydrolyzes with net retention of anomeric configuration. Accordingly, the enzyme was inactivated by 2-deoxy-2-fluoro beta-glucosyl fluoride, with kinetic parameters of k(i) = 4.5 min(-1), K(I) = 35.4 mM, through the trapping of a covalent glycosyl enzyme intermediate. The catalytic competence of this intermediate was demonstrated by the fact that incubation with linamarin resulted in reactivation, presumably via a transglycosylation mechanism. Peptic digestion of the 2-deoxy-2-fluoroglucosyl enzyme and subsequent analysis of high pressure liquid chromatography eluates by electrospray ionization triple quadrupole mass spectrometry in the neutral loss mode allowed the localization of a 2-deoxy-2-fluoroglucosyl-peptide. Sequence determination of this labeled peptide by tandem mass spectrometry in the daughter ion scan mode permitted the identification of Asp-261 as the catalytic nucleophile within the sequence VMSDW. Asp-261 is fully conserved within this family, consistent with its key role, and aligns with the aspartic acid residue previously identified in the Aspergillus wentii enzyme by labeling with conduritol B epoxide (Bause, E., and Legler, G. (1974) Hoppe-Seyler's Z. Physiol. Chem. 355, 438-442).
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PMID:Cloning, expression, characterization, and nucleophile identification of family 3, Aspergillus niger beta-glucosidase. 1067 36

We describe a new human immunodeficiency virus type 1 (HIV-1) mutational pattern associated with phenotypic resistance to lamivudine (3TC) in the absence of the characteristic replacement of methionine by valine at position 184 (M184V) of reverse transcriptase. Combined genotypic and phenotypic analyses of clinical isolates revealed the presence of moderate levels of phenotypic resistance (between 4- and 50-fold) to 3TC in a subset of isolates that did not harbor the M184V mutation. Mutational cluster analysis and comparison with the phenotypic data revealed a significant correlation between moderate phenotypic 3TC resistance and an increased incidence of replacement of glutamic acid by aspartic acid or alanine and of valine by isoleucine at residues 44 and 118 of reverse transcriptase, respectively. This occurred predominantly in those isolates harboring zidovudine resistance-associated mutations (41L, 215Y). The requirement of the combination of mutations 41L and 215Y with mutations 44D and 44A and/or 118I for phenotypic 3TC resistance was confirmed by site-directed mutagenesis experiments. These data support the assumption that HIV-1 may have access to several different genetic pathways to escape drug pressure or that the increase in the frequency of particular mutations may affect susceptibility to drugs that have never been part of a particular regimen.
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PMID:A novel human immunodeficiency virus type 1 reverse transcriptase mutational pattern confers phenotypic lamivudine resistance in the absence of mutation 184V. 1068 19

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP; phosphophoryn) are two principal dentin-specific non-collagenous proteins. DPP is extremely acidic and is rich in aspartic acid and serine. By virtue of this structure, DPP may bind large amounts of calcium and may facilitate initial mineralization of dentin matrix collagen as well as regulate the size and shape of the crystals. The function of DSP is not known. DSP and DPP are encoded by a single gene in both rat and mouse, and are uniquely expressed in odontoblasts and transiently in pre-ameloblasts. Because DSP and DPP are isolated from dentin as distinct proteins and appear to be present in different amounts, the nascent dentin sialophosphoprotein (DSPP) is likely cleaved to yield DSP and DPP. However, when, where and how the DSPP is cleaved into DSP and DPP is not clear. To further elucidate the structure and function of human DSP and DPP, we have cloned DPP and DSP cDNA by reverse transcriptase-polymerase chain reaction (RT-PCR) strategies, and then cloned and initiated characterization of a human dentin sialophosphoprotein gene. The genomic organization of human DSPP is very similar to that of mouse, containing five exons and four introns, suggesting it is a homologue of mouse dentin sialophosphoprotein (DSPP). Exons 1-4 encode for DSP, while exon 5 encodes for the C-terminus of DSP and the whole DPP. A 4.6-kb RNA transcript was detected on Northern blot analyses of total RNA extracted from immature (open root apices) human teeth using either a human DPP or DSP probe.
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PMID:Molecular cloning of a human dentin sialophosphoprotein gene. 1070 75

The genes encoding the alpha (63-kDa) and beta (95-kDa) subunits of Rous sarcoma virus (RSV) reverse transcriptase (RT) or the entire Pol polypeptide (99 kDa) were mutated in the conserved aspartic acid residue Asp 181 of the polymerase active site (YMDD) or in the conserved Asp 505 residue of the RNase H active site. We have analyzed heterodimeric recombinant RSV alphabeta and alphaPol RTs within which one subunit was selectively mutated. When alphabeta heterodimers contained the Asp 181-->Asn mutation in their beta subunits, about 42% of the wild-type polymerase activity was detected, whereas when the heterodimers contained the same mutation in their alpha subunits, only 7.5% of the wild-type polymerase activity was detected. Similar results were obtained when the conserved Asp 505 residue of the RNase H active site was mutated to Asn. RNase H activity was clearly detectable in alphabeta heterodimers mutated in the beta subunit but was lost when the mutation was present in the alpha subunit. In summary, our data imply that the polymerase and RNase H active sites are located in the alpha subunit of the heterodimeric RSV RT alphabeta.
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PMID:Asymmetric subunit organization of heterodimeric Rous sarcoma virus reverse transcriptase alphabeta: localization of the polymerase and RNase H active sites in the alpha subunit. 1070 41

We constructed human immunodeficiency virus (HIV) mutants by replacing the matrix domain with sequences encoding the viral protease or p6* and protease. The chimeras retaining matrix myristylation and processing signals underwent efficient autoprocessing with severely defective particle budding. The budding defects of the chimeras were rescued by suppressing the chimera protease activity either through addition of an HIV protease inhibitor or through inactivating the chimera protease via a substitution mutation of the catalytic aspartic acid residue. This resulted in the release of chimeric virus-like particles with the density of a wild-type retrovirus particle. In addition, the assembly-competent but processing-defective chimeras produced proteolytically processed particles with significant reverse transcriptase activity when a downstream native pol gene was present. These results suggest that HIV has the potential to adapt heterologous sequences in place of the matrix sequence without major effects on virus-like particle budding. In addition, the positions of the protease and substrate accessibility may contribute significantly toward avoiding a premature Gag or Gag-Pol process, which leads to severe defects in both particle budding and incorporation.
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PMID:Assembly and processing of human immunodeficiency virus Gag mutants containing a partial replacement of the matrix domain by the viral protease domain. 1070 61


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