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)

Two conserved sequence motifs, occurring in HIV-1 reverse transcriptase at residues 110-116 and 183-190, have been studied using site-directed mutagenesis of the cloned gene. In particular, aspartates at positions 185 and 186 have each been mutated to either asparagine or glutamate. The resulting mutant proteins were catalytically inactive but still able to bind the template-primer complex, poly rA-oligo dT. Other mutations in these regions resulted in reduced reverse trascriptase activity but the mutation of tyrosine-183 to serine caused a significant increase in the Km for dTTP and the Ki for inhibition by 3'-azidothymidine-triphosphate, 2',3'-dideoxythymidine-triphosphate and phosphonoformic acid.
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PMID:Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase. 170 76

To extend our previous studies of the function of the Cys-His box of Rous sarcoma virus NC protein, we have constructed a series of point mutations of the conserved or nonconserved amino acids of the proximal Cys-His box and a one-amino-acid deletion. All mutants were characterized for production of viral proteins and particles, for packaging and maturation of viral RNA, for reverse transcriptase activity, and for infectivity. Our results indicated the following. (i) Mutations affecting the strictly conserved amino acids cysteine 21, cysteine 24, and histidine 29 were lethal; only the mutant His-29----Pro was still able to package viral RNA, most of it in an immature form. (ii) Mutation of the highly conserved glycine 28 to valine reduced viral RNA packaging by 90% and infectivity 30-fold, whereas mutant Gly-28----Ala was fully infectious. This suggests a steric hindrance limit at this position. (iii) Shortening the distance between cysteine 24 and histidine 29 by deleting one amino acid abolished the maturation of viral RNA and yielded noninfectious particles. (iv) Substitution of tyrosine 22 by serine lowered viral RNA packaging efficiency and yielded particles that were 400-fold less infectious; double mutant Tyr-22Thr-23----SerSer had the same infectivity as Tyr-22----Ser, whereas mutant Thr-23----Ser was fully infectious. (v) Changing glutamine 33 to a charged glutamate residue did not affect virus infectivity. Similarities and differences between our avian mutants and those in murine retroviruses are discussed.
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PMID:Point mutations in the proximal Cys-His box of Rous sarcoma virus nucleocapsid protein. 216 81

A number of cDNA clones coding for 417 amino acid residues of the central part of the rat liver elongation factor 2 (EF-2) have been isolated. The oligonucleotides complementary to EF-2 mRNA were used as primers for synthesis of the first strand of cDNA cloned. Structures of these oligonucleotides were determined in course of 3'----5' sequencing coding strand of EF-2 cDNA. This method of synthesis of specific cDNA enabled one to reduce essentially the number of recombinant clones to be screened for EF-2 cDNA. Comparative studies of deduced protein sequences of rat liver EF-2 and hamster EF-2 [1] revealed the only substitution of aspartate residue for glutamate residue (hamster EF-2). The homology between nucleotide sequences of rat and hamster EF-2 cDNA was 89%. Northern-blot analysis of rat liver poly(A)+ mRNA revealed the only species of mRNA 3000 nucleotides long. A strong stop-signal for reverse transcriptase in the 5'-region of rat liver EF-2 mRNA is discovered: probability of dissociation of the enzyme from mRNA is at least 97.5%.
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PMID:[Cloning and determination of the primary structure of cDNA, coding the central part of elongation factor 2 from the rat liver]. 248 Jan 35

A process is described in which the baboon endogenous virus (BaEV) is produced under optimum conditions in cell culture, and concentrated by hollow-fiber ultrafiltration technology under conditions of large-scale production. This system has advantages over conventional systems in that the flow rate is increased 2.5-fold during concentration. Thermal inactivation of BaEV was retarded by the addition of lactose glutamate to the harvested tissue culture fluid. After concentration, at least 91% of the virus RNA-directed DNA polymerase is recovered with a concomitant increase in infectious virus. Materials needed for modifying described systems may be obtained from commercial sources.
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PMID:Concentration of baboon endogenous virus in large-scale production by use of hollow-fiber ultrafiltration technology. 676 70

S-2720 and other members of the quinoline/quinoxaline class of HIV-1-specific nonnucleoside reverse transcriptase inhibitors (NNRTIs) select for a glycine to glutamate substitution at residue 190 (Gly 190 Glu) of the reverse transcriptase (RT), when drug-resistant viruses are generated in cell culture. This mutation has not been described to appear upon selection for resistant viral variants using derivatives of any other class of NNRTIs. Notably, the RNA-dependent DNA polymerase activity of the Gly 190 Glu mutant enzyme is drastically diminished with respect to the wild-type RT. We describe here the effects of other amino acid substitutions at position 190 of the RT that were introduced by using site-directed mutagenesis. Polymerase activities and sensitivities to inhibition by a number of NNRTIs were determined for the different RT mutants. In general, an inverse correlation was found between the enzymatic activity and increasing length of the side chain, whereas the size of the residue and the level of resistance to NNRTIs appeared to be positively related. Double mutants, which contain the Gly 190 Glu mutation together with substitutions that confer resistance to other RT inhibitors, were all shown to possess severely diminished polymerase activity.
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PMID:Mutational analysis of residue 190 of human immunodeficiency virus type 1 reverse transcriptase. 751 21

We demonstrate by reverse transcriptase-polymerase chain reaction and Southern blotting that an immortalized rat oligodendroglial cell line (CG-4) expresses the non-N-methyl-D-aspartate (non-NMDA) glutamate receptor (GluR) genes GluR2-7, KA-1, and KA-2 and that nonimmortalized cells of the rat oligodendroglial lineage express the GluR1-3, GluR5-7, KA-1, and KA-2 genes. Lactic dehydrogenase release assays show that both immortalized and nonimmortalized cells of the oligodendroglial lineage are damaged by a 24-h exposure to 500 microM kainate or 5 mM L-glutamate, but not by a 24-h exposure to up to 10 mM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA). Damage is prevented by the non-NMDA GluR channel inhibitor 6-cyano-7-nitroquinoxaline-2,3-dione and is also averted if Ca2+ is removed from the culture medium. Cyclothiazide, which blocks desensitization of AMPA-preferring GluRs, increases cytotoxicity of kainate as well as inducing toxicity of AMPA. We conclude that cells of the oligodendroglial lineage express a population of AMPA-preferring and possibly also kainate-preferring GluR channels that are capable of mediating Ca(2+)-dependent excitotoxicity and that AMPA-induced cytotoxicity is blocked by desensitization of AMPA-preferring GluRs.
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PMID:Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors mediate excitotoxicity in the oligodendroglial lineage. 753 52

Although the excitatory amino acid glutamate and its receptors play crucial roles in many functions of the central nervous system (CNS), their presence in the peripheral tissues has remained unclear. In the present study, we have identified kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) receptor subtype mRNAs in pancreatic islets, using reverse transcriptase polymerase chain reaction (RT-PCR). Intracellular calcium ([Ca2+]i) measurements and electrophysiological recordings indicate that kainate, AMPA, and NMDA all elicit increases of [Ca2+]i in single pancreatic beta-cells and depolarize them. In addition, kainate and AMPA stimulate insulin secretion from isolated pancreatic islets, whereas NMDA does not. Also, immunocytochemical study shows the presence of intense glutaminase immunoreactivity in pancreatic alpha-cells and intrapancreatic ganglia, a finding compatible with the possibility that glutamate is released from alpha-cells as well as from neurons. Because the inhibitory amino acid gamma-amino butyric acid (GABA) is present in beta-cells as well as in neurons and inhibits glucagon secretion from alpha-cells, the present study suggests that glutamate and GABA are coordinated in the regulation of hormone secretion in pancreatic islets.
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PMID:Expression and role of ionotropic glutamate receptors in pancreatic islet cells. 776 62

Expression of mRNAs for glutamate transporter (GLT-1) and glutamate aspartate transporter (GLAST) was investigated in three different types of purified glial cells by the reverse transcriptase-polymerase chain reaction (RT-PCR). Cultured astrocytes, oligodendrocytes, and microglia expressed mRNAs for GLAST and GLT-1; mRNA for GLAST was expressed more prominently than that for GLT-1 in astrocytes. Oligodendrocytes and microglia expressed mRNAs for both GLT-1 and GLAST equally, but the expression in microglia was not prominent, suggesting glutamate uptake is not essential in microglia. In astrocytes cultured from different brain regions, GLAST mRNA was equally expressed. GLT-1 mRNA was also detected in these astrocytes, but the expression level was lower than that of GLAST.
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PMID:Expression of glutamate transporters in cultured glial cells. 779 59

We report that a non-neuronal cell line, MIN6, derived from insulin-secreting pancreatic beta-cells, naturally expresses functional ionotropic glutamate receptors. Electrophysiological recordings show that kainate, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), and N-methyl-D-aspartate (NMDA) depolarize single MIN6 cells and evoke inward ionic currents. These agents also increase the intracellular calcium concentration in MIN6 cells. Furthermore, insulin secretion from MIN6 cells is stimulated by kainate, AMPA, and NMDA. The presence of AMPA/kainate and NMDA receptor subtypes is confirmed by reverse transcriptase-polymerase chain reaction. These results demonstrate that ionotropic glutamate receptors with properties similar to those in neuronal cells are expressed in a non-neuronal cell line, MIN6. Thus, MIN6 provides a useful and valuable model system for biochemical, pharmacological, and physiological studies of ionotropic glutamate receptors.
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PMID:Functional neuronal ionotropic glutamate receptors are expressed in the non-neuronal cell line MIN6. 800 3

Congenital methemoglobinemia caused by an erythrocytic deficiency of cytochrome b5 reductase (b5R; type I) in African-American individuals was first reported by this laboratory. The rarity of this observation is possibly due to the difficulty detecting cyanosis that is masked by naturally occurring dark skin pigment. Since previous biochemical studies on the African-American family with variant enzyme b5R-Shreveport showed enzyme instability, we focused on molecular analysis of its transcript. The transcript size was the same as that of a normal control. The nucleotide sequence of both normal and variant transcripts were examined by directly sequencing reverse transcriptase-polymerase chain reaction (RT-PCR) product. The propositus was found to be homozygous for a G to A transition at codon 212 in exon 8, changing a glutamate to a lysine (E212K). In addition, a C to G transversion was found at codon 116 in exon 5, changing a threonine to a serine (T116S). Using allele-specific PCR, we determined that E212K was found only in the propositus and her heterozygous mother. Furthermore, E212K is predicted to disrupt an alpha-helix peptide structure of b5R, suggesting that this is likely the disease-causing mutation. In contrast, T116S was found to be a high-frequency polymorphism specific for the African-American population. The E212K mutation is uniquely present in the 3' end of the b5R gene (exon 8), which differs from those b5R mutations found among Japanese subjects (exons 3 and 5) and in an Italian subject (exon 4) and, thus, further contributes to our understanding of the structure/function relationship of this housekeeping enzyme.
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PMID:A novel mutation found in the 3' domain of NADH-cytochrome B5 reductase in an African-American family with type I congenital methemoglobinemia. 863 21


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