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)

We isolated a cDNA encoding a novel glucuronyltransferase from human placenta cDNA with the use of the degenerate reverse transcriptase-polymerase chain reaction method. Degenerate primers were designed based upon the amino acid sequence alignment of rat glucuronyltransferase (GlcAT-P) involved in the biosynthesis of the carbohydrate epitope HNK-1 with putative proteins in Caenorhabditis elegans and Schistosoma mansoni. The new cDNA sequence revealed an open reading frame coding for a protein of 335 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 43% identity to the rat GlcAT-P, and the highest sequence identity was found in the COOH-terminal catalytic domain. The expression of a soluble recombinant form of the protein in COS-1 cells produced an active glucuronyltransferase with marked specificity for a glycoserine Galbeta1-3Galbeta1-4Xylbeta1-O-Ser. In contrast, asialoorosomucoid, which contains the Galbeta1-4GlcNAc sequence and is a good acceptor substrate for the GlcAT-P, did not serve as an acceptor. The reaction product was sensitive to beta-glucuronidase digestion and co-chromatographed with authentic GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser in high-performance liquid chromatography, suggesting that the enzyme is a beta1, 3-glucuronyltransferase. These results indicate that this new member of the glucuronyltransferase gene family is the enzyme previously described as glucuronyltransferase I that forms the glycosaminoglycan-protein linkage region, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser, of proteoglycans.
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PMID:Molecular cloning and expression of glucuronyltransferase I involved in the biosynthesis of the glycosaminoglycan-protein linkage region of proteoglycans. 950 57

Tat is a virally expressed regulatory protein involved in the replication of HIV-1, the etiological agent of AIDS. To investigate the effect of tat inhibition on HIV replication, we constructed a retroviral vector to express an anti-tat hammerhead ribozyme as part of the 3' untranslated region of beta-galactosidase transcripts. Initial testing of this vector in tat-expressing COS-7 cells reduced tat activity by 85-95% as measured by tat-dependent CAT assays. Amphotropic and HIV-pseudotyped retroviral particles generated with this vector were used in HIV challenge experiments to determine the ability of this reagent to control HIV replication. CD4(+) peripheral blood lymphocytes (PBLs) stably transduced with this vector were subsequently challenged with HIV. These cells were able to resist HIV infection for up to 20 days as measured by cell death and reverse transcriptase activity. These data yield proof of principle that a pseudotyped retroviral vector can target and deliver a protective ribozyme to CD4(+) cells.
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PMID:Inhibition of HIV-1 replication by an anti-tat hammerhead ribozyme. 953 87

Heparan-sulfate 6-sulfotransferase (HS6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-sulfoglucosamine residue of heparan sulfate. The enzyme was purified to apparent homogeneity from the serum-free culture medium of Chinese hamster ovary (CHO) cells (Habuchi, H., Habuchi, O., and Kimata, K. (1995) J. Biol Chem. 270, 4172-4179). From the amino acid sequence data of the purified enzyme, degenerate oligonucleotides were designed and used as primers for the reverse transcriptase-polymerase chain reaction using poly(A)+ RNA from CHO cells as a template. The amplified cDNA fragment was then used as a probe to screen a cDNA library of CHO cells. The cDNA clone thus obtained encoded a partial peptide sequence composed of 236 amino acid residues that included the sequences of six peptides obtained after endoproteinase digestion of the purified enzyme. This cDNA clone was applied to the screening of a human fetal brain cDNA library by cross-hybridization. The isolated cDNA clones contained a whole open reading frame that predicts a type II transmembrane protein composed of 401 amino acid residues. No significant amino acid sequence identity to any other proteins, including heparan-sulfate 2-sulfotransferases, was observed. When the cDNA for the entire coding sequence of the protein was inserted into a eukaryotic expression vector and transfected into COS-7 cells, the HS6ST activity increased 7-fold over the control. The FLAG fusion protein purified by anti-FLAG affinity chromatography showed the HS6ST activity alone. Northern blot analysis revealed the occurrence of a single transcript of 3.9 kilobases in both human fetal brain and CHO cells. The results, together with the ones from our recent cDNA analysis of heparan-sulfate 2-sulfotransferase (Kobayashi, M., Habuchi, H., Yoneda, M., Habuchi, O., and Kimata, K. (1997) J. Biol. Chem. 272, 13980-13985), suggest that at least two different gene products are responsible for 6- and 2-O-sulfations of heparan sulfate.
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PMID:Molecular characterization and expression of heparan-sulfate 6-sulfotransferase. Complete cDNA cloning in human and partial cloning in Chinese hamster ovary cells. 953 12

A mouse liver homogenate was shown to contain enzymatic activities catalyzing the sulfation of 3,4-dihydroxyphenylalanine (Dopa) and tyrosine isomers with a pH optimum of 8.25. Western blot analysis revealed a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against rat liver SULT1B1 sulfotransferase. By employing the reverse transcriptase-polymerase chain reaction (RT-PCR) technique, a 910-base pair product encoding the putative mouse liver SULT1B1 sulfotransferase was obtained. Using this PCR product as a probe, a cDNA containing the entire open reading frame of the mouse liver SULT1B1 sulfotransferase was cloned from a mouse liver Lambda ZAP cDNA library. The nucleotide sequence indicated it is a new enzyme. The deduced amino acid sequence exhibited 87.6, 72.3, 55.9, 54.2, 52.8, 51.1, and 49.4% identity to the amino acid sequences of the rat liver SULT1B1 sulfotransferase, human thyroid hormone sulfotransferase, mouse phenol sulfotransferase, rat liver phenol sulfotransferase, rat liver hydroxyarylamine sulfotransferase, mouse estrogen sulfotransferase, and rat estrogen sulfotransferase. Upon transfection of COS-7 cells with an expression vector (pcDNA3) harboring the cDNA encoding this new enzyme, a 34 kDa protein exhibiting immunologic cross-reactivity to antiserum against the rat liver SULT1B1 sulfotransferase was expressed. The recombinant sulfotransferase exhibited enzymatic activities toward Dopa and tyrosine isomers, as well as dopamine and 3,3',5-triiodo-L-thyronine. Northern blot analyses indicated the SULT1B1 sulfotransferase was predominantly expressed in liver, but not in the other ten mouse organs examined. Furthermore, the enzyme was found to be expressed in a developmental stage-dependent manner, being at a very low level in liver samples from 1-day-old mice and then gradually increasing to the maximum level in liver samples from 4-week-old mice.
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PMID:Molecular cloning, expression, and characterization of a novel mouse liver SULT1B1 sulfotransferase. 964 46

We have found a novel isoform of the mouse type 2 Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] mRNA by reverse transcriptase-mediated PCR analysis. The novel isoform, which was expressed specifically in skeletal muscle and heart, was generated by the inclusion of a novel exon. As this exon contains a stop codon, the isoform encodes a putative protein (designated TIPR) consisting of 175 acid residues of the type 2 Ins(1,4,5)P3R and the following six residues derived from this exon. We transfected the cDNA of this isoform into COS-7 cells; these cells expressed a 24 kDa protein that was recognized by an antibody against TIPR produced in Escherichia coli. The isoform encoding TIPR was also found in human skeletal muscle and heart. The N-terminal region of Ins(1,4,5)P3R is suggested to have a role in ligand binding and to interact with the C-terminal channel domain of Ins(1,4,5)P3R itself. TIPR might regulate the Ins(1,4,5)P3 signal pathway in both muscles.
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PMID:Muscle-specific mRNA isoform encodes a protein composed mainly of the N-terminal 175 residues of type 2 Ins(1,4,5)P3 receptor. 972 62

We have selectively mutagenized specific residues at the junction between the protease (PR) and reverse transcriptase (RT) genes of human immunodeficiency virus type 1 (HIV-1) to study the effects of PR-RT fusion proteins in the context of a full-length, infectious proviral construct. Mutant viruses derived from COS-7 cells transfected with this construct were analyzed in regard to each of viral replication, maturation, and infectivity. Immunoblot analysis revealed that the mutation prevented cleavage between the PR and RT proteins and that both existed as a PR-RT fusion protein in each of cellular and viral lysates. Interestingly, intracellular PR that existed within the PR-RT fusion protein remained functionally active, whereby HIV-1 precursor proteins were processed efficiently. Furthermore, the RT component of the fusion protein also retained its enzymatic activity as shown in RT assays. Electron microscopy revealed that the mutant viruses containing the PR-RT fusion protein possessed wild-type morphology. These viruses also displayed wild-type sensitivities to inhibitors of each of the HIV-1 PR and RT activities. However, viruses containing the PR-RT fusion protein were 20 times less infectious than wild-type viruses. This defect was further pronounced when mutated Gag-Pol proteins were overexpressed as a consequence of an additional mutation that interfered with frameshifting. Thus, unlike cleavage site mutations at the N terminus of PR, a cleavage site mutation between PR and RT did not affect the enzymatic activities of either PR or RT and viruses containing PR-RT fusion proteins were viable.
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PMID:Characterization of human immunodeficiency virus type-1 (HIV-1) particles that express protease-reverse transcriptase fusion proteins. 981 41

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

The 1,839-base pair complementary DNA (cDNA) for rat lung carboxylesterase was cloned by reverse transcriptase polymerase chain reaction from total rat lung RNA using specific primers derived from the 5' and 3' untranslated regions of rat hepatic cholesteryl ester hydrolase (CEH). The unique cDNA was sequenced and found to be similar to hepatic CEH, pI 6.1 esterase, and hydrolase A. In Northern blot analysis, the cDNA hybridized with a single band from lung messenger RNA (mRNA). The 1.7-kb coding sequence, predicting a 62-kD protein, was transfected into COS-7 cells and Chinese hamster ovary (CHO) cells. Expression in COS-7 and CHO cells was accompanied by 4- and 3.2-fold increases in carboxylesterase activity (hydrolysis of p-nitrophenyl acetate), respectively. Unlike the hepatic CEH, the expressed lung carboxylesterase described here did not hydrolyze cholesterol esters. In situ hybridization experiments localized the lung carboxylesterase mRNA to the airway epithelium. The organophosphorus compound phosphoric acid diethyl 4-nitrophenyl ester, paraoxon, completely inhibited this lung carboxylesterase, placing it in the family of B esterases by this criterion.
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PMID:Molecular cloning and expression of rat lung carboxylesterase and its potential role in the detoxification of organophosphorus compounds. 1034 Sep 39

1. A degenerate polymerase chain reaction (PCR) homology screening procedure was applied to rat brain cDNA in order to identify novel genes belonging to the amiloride-sensitive Na+ channel and degenerin (NaC/DEG) family of ion channels. A single gene was identified that encodes a protein related to but clearly different from the already cloned members of the family (18-30 % amino acid sequence identity). Phylogenetic analysis linked this protein to the group of ligand-gated channels that includes the mammalian acid-sensing ion channels and the Phe-Met-Arg-Phe-amide (FMRFamide)-activated Na+ channel. 2. Expression of gain-of-function mutants after cRNA injection into Xenopus laevis oocytes or transient transfection of COS cells induced large constitutive currents. The activated channel was amiloride sensitive (IC50, 1.31 microM) and displayed a low conductance (9-10 pS) and a high selectivity for Na+ over K+ (ratio of the respective permeabilities, PNa+/PK+ >= 10), all of which are characteristic of NaC/DEG channel behaviour. 3. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed a predominant expression of its mRNA in the small intestine, the liver (including hepatocytes) and the brain. This channel has been called the brain-liver-intestine amiloride-sensitive Na+ channel (BLINaC). 4. Corresponding gain-of-function mutations in Caenorhabditis elegans degenerins are responsible for inherited neurodegeneration in the nematode. Besides the BLINaC physiological function that remains to be established, mutations in this novel mammalian degenerin-like channel might be of pathophysiological importance in inherited neurodegeneration and liver or intestinal pathologies.
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PMID:Cloning and functional expression of a novel degenerin-like Na+ channel gene in mammals. 1045 52

Thrombocytosis is occasionally seen in patients with carcinomas and has been assumed to be attributable to interleukin-6 or granulocyte-macrophage colony-stimulating factor produced by carcinoma cells. In this study, we clarified whether thrombopoietin (TPO) is involved in carcinoma-associated thrombocytosis. Expression of TPO mRNA was observed in the majority of 27 carcinoma cell lines as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). There were 6 PCR products differing in size; sequence analysis showed the full-length TPO mRNA (TPO-1), 12- and 116-bp deleted variants (TPO-2 and TPO-3, respectively), and 3 novel isoforms (197- and 128-bp deleted forms and a 60-bp insert form of TPO-3; named TPO-4, TPO-5, and TPO-6, respectively). Of 27 lines, 24 expressed TPO-1 mRNA with various other isoforms. Culture supernatants of COS-1 cells transfected with TPO-5 or TPO-6 cDNA did not promote the proliferation of TPO-responsive cells, whereas Western blot analysis on the cell lysates demonstrated TPO-5 but not TPO-6 protein, suggesting poor extracellular secretion (TPO-5) or poor protein synthesis (TPO-6). TPO protein was detected in 10-fold concentrated culture supernatants of cells of these carcinoma lines, with a median concentration of 0.38 fmol/mL as evaluated by enzyme-linked immunosorbent assay. High blood TPO levels were observed with a median value of 3.46 fmol/mL (range, 0.34 to 8.67 fmol/mL) in patients with advanced carcinomas associated with thrombocytosis. These results indicate that thrombocytosis in patients with carcinomas might be caused, at least in part, by TPO produced by carcinoma cells.
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PMID:Production of thrombopoietin by human carcinomas and its novel isoforms. 1047 24


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