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)

1. The sodium-dependent amino acid transport systems responsible for proline, glycine and glutamine transport, together with the sodium-independent systems for leucine and tryptophan, have been investigated in isolated bovine chondrocytes by inhibition studies and ion replacement. Each system was characterized kinetically. 2. Transport via system A was identified using the system-specific analogue alpha-methylaminoisobutyric acid (MeAIB) as an inhibitor of proline, glycine and glutamine transport. 3. Uptake of proline, glycine and glutamine via system ASC was identified by inhibition with alanine or serine. 4. System Gly was identified by the inhibition of glycine transport with excess sarcosine (a substrate for system Gly) whilst systems A and ASC were inhibited. This system, having a very limited substrate specificity and tissue distribution, was also shown to be Na+ and Cl- dependent. Evidence for expression of the system Gly component GLYT-1 was obtained using the reverse transcriptase-polymerase chain reaction (RT-PCR). 5. System N, also of narrow substrate specificity and tissue distribution, was shown to be present in chondrocytes. Na+-dependent glutamine uptake was inhibited by high concentrations of histidine (a substrate of system N) in the presence of excess MeAIB and serine. 6. System L was identified using the system specific analogue 2-aminobicyclo(2,2, 1)heptane-2-carboxylic acid (BCH) and D-leucine as inhibitors of leucine and tryptophan transport. 7. The presence of system T was tested by using leucine, tryptophan and tyrosine inhibition. It was concluded that this system was absent in the chondrocyte. 8. Kinetic analysis showed the Na+-independent chondrocyte L system to have apparent affinities for leucine and tryptophan of 125 +/- 27 and 36 +/- 11 microM, respectively. 9. Transport of the essential amino acids leucine and tryptophan into bovine chondrocytes occurs only by the Na+-independent system L, but with a higher affinity than the conventional L system.
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PMID:Neutral amino acid transport in bovine articular chondrocytes. 988 51

The Tth DNA polymerase gene from the thermophilic Thermus thermophilus (strain HB8) was amplified, cloned and expressed in Escherichia coli. The recombinant DNA polymerase containing a polyhistidine tag at the N-terminus was isolated in a single step by Ni2+ affinity chromatography. The purified recombinant enzyme, showing high polymerase activity contained 43 additional amino-acid residues (including a cluster of six histidine residues inserted for purification of the recombinant protein by metal-affinity chromatography) at N-terminus. The applied overexpression system was very efficient giving 700,000 u of DNA polymerase activity from 1 liter of induced culture. The enzyme was characterized and displayed high DNA polymerase and reverse transcriptase activities and high thermostability as compared to the native Tth DNA polymerase.
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PMID:Recombinant His-tagged DNA polymerase. I. Cloning, purification and partial characterization of Thermus thermophilus recombinant DNA polymerase. 991 91

We have selected a human immunodeficiency virus type 1 (HIV1) using the technique of in vitro selection to generate variants that are resistant to didanosine and/or stavudine. After serial passages of the Lai strain of HIV1 in MT-2 cells in increased concentrations of didanosine-stavudine association, 2 novel mutations in reverse transcriptase at codon 57 (Asp-->His) and at codon 98 (AIa-->Val) were observed. These mutations were associated with an 11.5-fold increase in the didanosine and a 4.5-fold increase in the stavudine 50% inhibitory concentration.
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PMID:Mutations in the human immunodeficiency virus type 1 reverse transcriptase gene observed in stavudine and didanosine strains obtained by in vitro passages. 992 11

Seven types of zinc finger protein (ZFP) genes based on the combinations of cysteine and histidine residues were found in a human heart cDNA database. Here we report the isolation of 360 cDNA clones encoding putative ZFPs. Of these, 154 (42.8%) represent C2H2-type ZFPs, 101 (28.1%) represent C2C2-type, five (1.4%) represent C2HC-type, 71 (19.7%) represent C2HC4C(HD)-type, three (0.8%) represent C3H-type, eight (2.2%) represent C3HC4-type and 18 (0.5%) represent combination type (genes containing more than one type of zinc finger). Among these 360 ZFPs, a novel ZFP cDNA named HFHZ (human fetal heart ZFP) with sequence homology to a Kruppel-associated box (KRAB) was identified. Sequencing the full-length of this cDNA clone identified an open reading frame of 711 bp that encodes a 237 amino acid protein with a predicted molecular weight of 27.7 kDa. Sequence analysis indicated that HFHZ contained a truncated KRAB box at the N-terminus and two C2H2 zinc fingers at the C-terminus. The transcript of HFHZ is highly expressed in fetal heart and moderately expressed in fetal brain but not expressed in fetal liver as revealed by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis suggesting that HFHZ is not expressed ubiquitously. The 3.3-fold higher expression in the fetal heart than in the adult heart suggests that HFHZ mRNA is downregulated in the process of development. In addition, the relatively high expression (1.9-fold) of HFHZ observed in the hypertrophic as compared to the normal adult heart suggests that this fetal gene is reactivated in response to hypertrophic stimuli. Chromosomal localization by in situ hybridization revealed that this gene is in 19q13.1, a region containing genes involved in both cell cycle and developmental regulation.
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PMID:Characterization of a novel gene encoding zinc finger domains identified from expressed sequence tags (ESTs) of a human heart cDNA database. 992 72

The active form of HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C-terminal proteolytic cleavage of p66. A well-known problem of p66 recombinant expression is partial cleavage of a 15-kDa peptide from the C-terminus by host proteases that can not be completely suppressed. In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions. We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N-terminal fusion protein of glutathione S-transferase (GST) with p51 and a C-terminally His-tagged p66, respectively. The two-plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis. The combination of glutathione-agarose, phenyl-superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination. Truncated forms of p51 are efficiently removed. Mobility-shift assay revealed that the preparations are free of p66 homodimer. In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility-shift assays. This indicates the p51 subunit has an active role in DNA binding
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PMID:Mixed reconstitution of mutated subunits of HIV-1 reverse transcriptase coexpressed in Escherichia coli - two tags tie it up. 1010 27

During human immunodeficiency virus type 1 (HIV-1) assembly, the primer tRNA for the reverse transcriptase-catalyzed synthesis of minus-strand strong-stop cDNA, tRNA3Lys, is selectively packaged into the virus and annealed onto the primer binding site on the RNA genome. Annealing of tRNA3Lys in HIV-1 is independent of polyprotein processing and is facilitated in vitro by p7 nucleocapsid (NCp7). We have previously shown that mutations in clusters of basic amino acids flanking the first Cys-His box in NC sequence inhibit annealing of tRNA3Lys in vivo by 70 to 80%. In this report, we have investigated whether these NC mutations act through Pr55(gag) or Pr160(gag-pol). In vivo placement of tRNA3Lys is measured with total viral RNA as the source of primer tRNA-template in an in vitro reverse transcription assay. Cotransfection of COS cells with a plasmid coding for either mutant Pr55(gag) or mutant Pr160(gag-pol), and with a plasmid containing HIV-1 proviral DNA, shows that only the NC mutations in Pr55(gag) inhibit tRNA3Lys placement. The NC mutations in Pr55(gag) reduce viral infectivity by 95% and are trans-dominant-negative, i.e., they inhibit genomic placement of tRNA3Lys even in the presence of wild-type Pr55(gag). This dominant phenotype may indicate that the mutant Pr55(gag) is disrupting an ordered Pr55(gag) structure responsible for the annealing of tRNA3Lys to genomic RNA.
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PMID:The role of Pr55(gag) in the annealing of tRNA3Lys to human immunodeficiency virus type 1 genomic RNA. 1019 52

We have expressed the recombinant reverse transcriptase (RT) of bovine leukemia virus (BLV) in bacteria. The gene encoding the RT was designed to start at its 5' end next to the last codon of the mature viral protease, namely the amino terminus of the RT matches the last 26 codons of the pro gene and is coded for by the pro reading frame. The RT sequence extends into the pol gene, utilizing the pol reading frame after overcoming the stop codon by adding an extra nucleotide (thus imitating the naturally occurring frameshift event). Hence we have generated a transframe polypeptide that is a 584-residues-long protein (see Rice, Stephens, Burny, and Gilden (1985) Virology 142, 357-377). This protein was partially purified after adding a six-histidine tag and studied biochemically testing a variety of parameters. The enzyme exhibits all activities typical of RTs, i.e., both RNA- and DNA-dependent DNA polymerase as well as a ribonuclease H (RNase H) activity. Unlike most RTs, the BLV RT is enzymatically active as a monomer even after binding a DNA substrate. The enzyme shows a preference for Mg2+ over Mn2+ in both its DNA polymerase and RNase H activities. BLV RT is relatively resistant to nucleoside triphosphate analogues, which are known to be potent inhibitors of other RTs such as that of HIV.
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PMID:Catalytic features of the recombinant reverse transcriptase of bovine leukemia virus expressed in bacteria. 1036 2

We studied the phenotype and the nucleotide sequence for the cDNAs of the Aldh3a1a and Aldh3a1c allelic forms of the dioxin-inducible cytosolic aldehyde dehydrogenase (ALDH3A1) present in inbred mouse strains. This gene is constitutively expressed in cornea, stomach, skin, urinary bladder and lungs. The Aldh3a1a allele is found in most inbred mouse strains and codes for a 'high-activity' corneal enzyme compared to the 'low-activity' encoded by the Aldh3a1c allele in SWR/J strain. The 'low-activity' variant is associated with extensive corneal clouding after a single exposure to ultraviolet light. The ALDH3A1 phenotype was examined in tissues from inbred mouse strains carrying the Aldh3a1a allele including, SJL/J, C57BL/6 J/Ibg, DBA/2 J/Ibg, C3H/Ibg and the Aldh3a1c allele (SWR/J). Only trace levels of ALDH3A1 activity were found in all SWR/J tissues. All other strains had significant levels of ALDH3A1 activity in eye, stomach, skin, less in urinary bladder and lungs and only trace amounts in liver. However, no differences were found in corneal and stomach ALDH3A1 mRNA levels between the 'low-' and 'high-activity' variants. A 1556-bp ALDH3A1 cDNA fragment, containing the entire coding region plus 5' and 3' untranslated regions, was amplified by reverse transcriptase-polymerase chain reaction from SWR/J and DBA/2 J/Ibg mouse strains. Sequence analysis revealed 13 nucleotide changes in the Aldh3a1c allele. Four of these changes result in G88R, I154N, H305R and I352V substitutions, whereas nine changes are silent. The I154N disrupts a potential alpha helix, which belongs to the Rossmann fold. Replacement of Arg with the more ionizable His at position 305 of a beta strand might directly affect catalytic activity of the enzyme. It is likely that structural changes associated with these amino acid changes are responsible for the loss of ALDH3A1 enzymatic activity in SWR/J mice.
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PMID:Four amino acid changes are associated with the Aldh3a1 locus polymorphism in mice which may be responsible for corneal sensitivity to ultraviolet light. 1037 61

By using degenerate primers designed from glutamate decarboxylase (GAD) sequences of mammals, Xenopus and Drosophila, a 270-bp cDNA fragment was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR) from cerebellum total RNA of rainbow trout. This partial cDNA shows 90% identity with mammalian GAD 65 and presents the Asn-Pro-His-Lys (NPHK) sequence corresponding to the pyridoxal-binding region of porcine DOPA decarboxylase or mammalian GAD. The distribution of GAD 65 mRNA-expressing neurons in the forebrain of the trout was studied by in situ hybridization using either digoxigenin- or 35S-labeled probes. The results demonstrate that gamma-amino butyric acid (GABA) neurons are widely distributed throughout the forebrain, with a high density in the periventricular regions. In this study, we report their precise distribution in the telencephalon and diencephalon. GAD mRNA-expressing cells were particularly abundant in the preoptic region and the mediobasal hypothalamus, two major neuroendocrine and estrogen-sensitive regions in fish. The presence of GAD mRNA-expressing neurons was observed in visually related structures such as the suprachiasmatic nucleus, the pretectal region, and the thalamus. Immunohistochemistry with antibodies directed against mouse GAD failed to demonstrate the presence of immunoreactive cell bodies, but showed a very high concentration of GAD-immunoreactive fibers in many brain regions, notably in the preoptic area, hypothalamus, and neurohypophyseal digitations of the pituitary, in particular in the proximal pars distalis. These results indicate that GABA neurons are ideally placed to modulate neuroendocrine activities at the hypothalamic and pituitary levels and to participate in the processing of sensorial information.
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PMID:Distribution of glutamic acid decarboxylase mRNA in the forebrain of the rainbow trout as studied by in situ hybridization. 1041 33

Hepatitis B virus (HBV), although a DNA virus, replicates using reverse transcriptase encoded by the HBV polymerase (pol) gene. The biochemical dissection of HBV pol has been hampered by failure to liberate enzymatically active protein from nucleocapsids. Here, we have employed a yeast-based genetic approach to express the HBV reverse transcriptase. In this strategy, the reverse transcriptase of yeast retrotransposon Ty1 element is replaced with the HBV pol gene to produce the hybrid Ty1/HBV element. Additionally, the indicator gene his3AI is combined in an antisense orientation to the transcripts of the hybrid Ty1/HBVRT element. The splicing of his3AI, cDNA synthesis of the Ty1/HBVRT RNA and subsequent integration relies on the reverse transcriptase activity. The production of histidine prototrophs results from the successful reverse transcription of Ty1/HBVRThis3AI transcripts followed by either homologous recombination or integrase-mediated insertion and subsequent expression of HIS3 gene. Using this approach we successfully detected the reverse transcriptase activity of HBV in yeast strains defective in endogenous Ty1 expression. Consistent with the unique priming activity associated with HBV pol, the minus strand DNA synthesis was protein-primed. Deletion of HBV reverse transcriptase (RT) or RNase H domains resulted in a dramatic drop in histidine prototrophs. The addition of HBV encoded HBx protein in virus-like particles during in vitro RT reaction stimulated the RT reaction by severalfold. Furthermore, in the presence of 3TC, a known inhibitor of HBV reverse transcriptase, yeast His(+) growth of His protrophs was not observed. Thus, this approach, which is based on genetic selection in yeast, is safe, economic, and a reliable strategy with a potential for large scale screening of cofactors and inhibitors of HBV polymerase functions.
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PMID:Expression of hepatitis B virus polymerase in Ty1-his3AI retroelement of Saccharomyces cerevisiae. 1053 36


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