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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The molecular cloning of a murine receptor-type protein tyrosine phosphatase, termed PTP NU-3, with an extracellular cell-adhesion-molecule-like domain is reported. NU-3 was isolated from 11.5-day total mouse embryonic RNA by reverse-transcriptase PCR using degenerate oligonucleotides flanking the conserved protein tyrosine phosphatase catalytic domain. This produced a 280-bp DNA probe which was subsequently employed to screen a mouse embryonic kidney library. Several overlapping cDNA clones were isolated, collectively forming a cDNA of 6.0 kb that encodes a putative 211-kDa protein. Northern-blot analysis of total RNA from adult and embryonic mouse tissues indicates the existence of two major PTP NU-3 transcripts of approximately 6 kb and 7 kb. Both messages are expressed predominantly in brain tissues and neuronal-derived cell lines, although detectable levels of the 7-kb message were found in other non-neuronal tissues. We have identified a unique 132-bp exon segment that is present in the 7-kb message but is completely absent in the 6-kb transcript, suggesting tissue-specific levels of expression and RNA processing. Analysis of the amino acid sequence encoded by the 132-bp segment reveals that it completes a partial fibronectin type-III element resulting in a protein with a total of nine such elements. Bacterial expression of the two catalytic domains demonstrated that only the first domain possesses enzymic activity towards a tyrosine phosphorylated substrate.
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PMID:Molecular cloning and tissue-specific RNA processing of a murine receptor-type protein tyrosine phosphatase. 752 77

We have identified a T7 RNA polymerase (RNAP) mutant that efficiently utilizes deoxyribonucleoside triphosphates. In vitro this mutant will synthesize RNA, DNA or 'transcripts' of mixed dNMP/rNMP composition depending on the mix of NTPs present in the synthesis reaction. The mutation is conservative, changes Tyr639 within the active site to phenylalanine and does not affect promoter specificity or overall activity. Non-conservative mutations of this tyrosine also reduce discrimination between deoxyribo- and ribonucleoside triphosphates, but these mutations also cause large activity reductions. Of 26 mutations of other residues in and around the active site examined none showed marked effects on rNTP/dNTP discrimination. Mutations of the corresponding tyrosine in DNA polymerase (DNAP) I increase miscoding, though effects on dNTP/rNTP discrimination for the DNAP I mutations have not been reported. This conserved tyrosine may therefore play a similar role in many polymerases by sensing incorrect geometry in the structure of the substrate/template/product due to inappropriate substrate structure or mismatches. T7 RNAP can use RNA templates as well as DNA templates and is capable of both primer extension and de novo initiation. The Y639F mutant retains the ability to use RNA or DNA templates. Thus this mutant can display de novo initiated or primed DNA-directed DNA polymerase, reverse transcriptase, RNA-directed RNA polymerase or DNA-directed RNA polymerase activities depending simply on the templates and substrates presented to it in the synthesis reaction.
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PMID:A mutant T7 RNA polymerase as a DNA polymerase. 755 4

The temperature-sensitive and attenuated phenotypes of the Sabin type 1 vaccine strain of poliovirus result from numerous point mutations which occurred in the virulent Mahoney virus parent. One of these mutations is located in a 3D polymerase (3Dpol) codon (U-6203-->C, Tyr-73-->His) and is involved in attenuation in common mice (M. Tardy-Panit, B. Blondel, A. Martin, F. Tekaia, F. Horaud, and F. Delpeyroux, J. Virol. 67:4630-4638, 1993). This mutation also appears to contribute to temperature sensitivity, in association with at least 1 other of the 10 mutations of the 3'-terminal part of the genome including the 3Dpol coding and 3' noncoding regions. To map the other mutation(s), we constructed poliovirus mutants by mutagenesis and recombination of Mahoney and Sabin 1 cDNAs. Characterization of these poliovirus mutants showed that a second mutation in a 3Dpol codon (C-7071-->U, Thr-362-->Ile) contributes to temperature sensitivity. A mutation in the 3' noncoding region of the genome (A-7441-->G), alone or linked to another mutation (U-7410-->C), also appeared to be involved in this phenotype. The temperature-sensitive effect associated with the 3'-terminal part of the Sabin 1 genome results from the cumulative and/or synergistic effects of at least three genetic determinants, i.e., the His-73 and Ile-362 codons of 3Dpol and nucleotide G-7441. Sequence analysis of strains isolated from patients with vaccine-associated paralytic poliomyelitis showed that these genetic determinants are selected against in vivo, although the Ile-362 codon appeared to be more stable than either the His-73 codon or G-7441. These genetic determinants may contribute to the safety of Sabin 1 in vaccines.
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PMID:Mapping of mutations contributing to the temperature sensitivity of the Sabin 1 vaccine strain of poliovirus. 763 70

Protein tyrosine phosphorylation is thought to play an important role in the regulation of neural function. To elucidate the role that protein tyrosine phosphatases (PTPs) may play in the postischemic brain, PTPs expressed in regions of the rat brain vulnerable to transient forebrain ischemia were examined. With the reverse-transcriptase polymerase chain reaction using degenerate primers, three PTPs, STEP, PTP delta, and SH-PTP2, were identified. They were expressed in the hippocampus 12 h after transient ischemia for 20 min. During the reperfusion period, the mRNA levels of these PTPs were not different from those in sham-operated rats. In contrast, a fourfold increase in the mRNA level of CL100 (3CH134), a PTP that is inducible by oxidative stress, was detected by Northern blotting in the hippocampus and cerebral cortex 1 h after the onset of reperfusion. In situ hybridization histochemistry showed a slight increase in the level of CL100 mRNA in neuronal cells in the hippocampus and cortex of postischemic rats compared to control rats. These findings suggest that PTPs play a role in the normal function of the hippocampus and cerebral cortex and demonstrate that ischemia induced CL100 expression.
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PMID:Induction of CL100 protein tyrosine phosphatase following transient forebrain ischemia in the rat brain. 779 38

We have investigated the mRNA expression of 2 human protein tyrosine phosphatases with sequence homology to cytoskeletal proteins, PTPH1 and PTPMEG. Northern-blot analysis of PTPH1 using poly (A)+ RNA from normal human colon tissue showed a low-abundance message of 4.3 kb. Reverse-transcriptase/polymerase-chain reaction (RT-PCR) was therefore used to detect it in a wide variety of cell lines including 9 colorectal, 5 gastric, 5 hepatic and 6 hematopoietic tumor cells. PTPH1 mRNA was not detected only in Colo 320 cells over-expressing c-myc mRNA, among the colorectal cancer cell lines examined. When Colo 320 cells were incubated with 5 mM sodium butyrate for 5 days, PTPH1 mRNA became detectable, concomitant with the marked decrease in the expression level of c-myc mRNA. Moreover, the chromosomal localization of PTPH1 gene was investigated by fluorescence in situ hybridization. Interestingly, PTPH1 gene was mapped to 9q31 where the gene for Gorlin syndrome, a putative tumor suppressor gene, exists.
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PMID:Expression and chromosomal assignment of PTPH1 gene encoding a cytosolic protein tyrosine phosphatase homologous to cytoskeletal-associated proteins. 825 32

The highly conserved Wnt genes belong to a widely distributed family of presumptive signaling molecules that have been implicated not only in the regulation of normal pattern formation during embryogenesis and differentiation of cell lineages, but also in oncogenic events. All of the known vertebrate Wnt genes encode for 38- to 43-kDa cysteine-rich putative glycoproteins, which have features typical of secreted growth factors: a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence, and 22 conserved cysteine residues whose relative spacing is maintained. In this study, we report the cloning and sequencing of several overlapping cDNAs encoding approximately 4.1 kb of the human homologue of Wnt-5A. The mature protein contained 343 residues (M(r) approximately 38,000 excluding any post-translational modifications) with a > 93% homology to the reported sequences of other Wnt-5A proteins (> 99% homologous to mouse Wnt-5A). This protein maintained certain features--a hydrophobic signal sequence, the Wnt-1 family "signature sequence" (CKCHGvSGSC), and a number of other conserved amino acid residues: 24 cysteine residues, 4 asparagine-linked oligosaccharide consensus sequences, and a tyrosine sulfation site--that have been found in all other Wnt-5A proteins. Reverse transcriptase PCR analysis of RNA from a variety of human embryonic, neonatal, and adult cells and/or tissues showed that human Wnt-5A expression was detected only in neonatal heart and lung. It may be relevant, however, that the 3'-untranslated region contained numerous AT-rich motifs that could be involved in the rapid degradation of mRNA. Finally, using a combination of Southern blotting, PCR amplification, and in situ hybridization, the human Wnt-5A (WNT5A) gene was mapped to chromosome 3p14-p21.
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PMID:Molecular cloning of the human proto-oncogene Wnt-5A and mapping of the gene (WNT5A) to chromosome 3p14-p21. 828 27

A cDNA coding for the beta 4 subunit of murine integrin (m beta 4) has been cloned and sequenced using mRNA from a murine lung carcinoma as the template. The 5' sequence contains two AUG codons, the second of which initiates synthesis of the mature protein. The cDNA sequence has an open reading frame coding for 1748 amino acids (aa), including a signal peptide, cysteine-rich region, serine- and threonine-rich region, transmembrane domain, and a cytoplasmic domain of over 1000 aa. Overall, the deduced m beta 4 aa sequence has 88% identity with the human beta 4 subunit (h beta 4) sequence deduced from the sequence of placental mRNA. Reverse transcriptase-polymerase chain reaction using primers flanking splice sites for two variant forms of h beta 4 transcripts provided evidence for alternate splicing of RNA in the murine spleen and to a lesser extent in the skin, uterus, and thymus but was found at only one of the two alternative sites. Five potential glycosylation sites present in the extracellular domain of h beta 4 are conserved in m beta 4. One tyrosine in the terminal region of the cytoplasmic domain (position 1600) is conserved between m beta 4 and h beta 4 and has the consensus sequence for tyrosine phosphorylation. Finally, a genomic restriction map of m beta 4 shows that the gene is about 40 kb in length. No restriction-fragment length polymorphisms were detected between BALB/c liver and BALB/c lung carcinoma DNA.
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PMID:Sequence of a cDNA encoding the beta 4 subunit of murine integrin. 835 87

The poliovirus RNA-dependent RNA polymerase (3Dpol) shares a region of homology with all RNA polymerases, centered around the amino acid motif YGDD, which has been postulated to be involved in the catalytic activity of the enzyme. Using oligonucleotide site-directed mutagenesis, we substituted the tyrosine at this motif of the poliovirus RNA-dependent RNA polymerase with cysteine, histidine, isoleucine, methionine, phenylalanine, or serine. The enzymes were expressed in Escherichia coli, and in vitro enzyme activity was tested. The phenylalanine and methionine substitutions resulted in enzymes with activity equal to that of the wild-type enzyme. The cysteine substitution resulted in an enzyme with approximately 50% of the wild-type activity, while the serine substitution resulted in an enzyme with approximately 10% of the wild-type activity; the isoleucine and histidine substitutions resulted in background levels of enzyme activity. To assess the effects of the mutants in viral replication, the mutant polymerase genes were subcloned into the infectious cDNA clone of poliovirus. Transfection of poliovirus cDNA containing the phenylalanine mutation in 3Dpol gave rise to virus in all of the transfection trials, while cDNA containing the methionine mutation resulted in virus in only 3 of 40 transfections. Transfection of cDNAs containing the other substitutions at the tyrosine residue did not result in infectious virus. The recovered viruses demonstrated kinetics of replication similar to those of the wild-type virus, as measured by [3H]uridine incorporation at either 37 or 39 degrees C. RNA sequence analysis of the 3Dpol gene of both viruses demonstrated that the tyrosine-to-phenylalanine or tyrosine-to-methionine mutation was still present. No other differences in the 3Dpol gene between the wild-type and phenylalanine-containing virus were found. The virus containing the methionine mutation also contained two other nucleotide changes from the wild-type 3Dpol sequence; one resulted in a glutamic acid-to-aspartic acid change at amino acid 108 of the polymerase, and the other resulted in a C-to-T base change at nucleotide 6724, which did not result in an amino acid change. To confirm that the second amino acid mutation found in the 3Dpol gene of the methionine-substituted virus allowed for replication ability, a mutation corresponding to the glutamic acid-to-aspartic acid change was made in the polymerase containing the methionine substitution, and this double-mutant polymerase was expressed in E. coli. The double-mutant enzyme was as active as the wild-type enzyme under in vitro assay conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Enzymatic activity of poliovirus RNA polymerases with mutations at the tyrosine residue of the conserved YGDD motif: isolation and characterization of polioviruses containing RNA polymerases with FGDD and MGDD sequences. 838 83

The attenuated Sabin strain of poliovirus type 1 (PV-1) differs from the neurovirulent PV-1 Mahoney strain by 55 nucleotide mutations. Only one of these mutations (A-480-->G, in the 5' noncoding (5' NC) region of the genome, is well characterized, and it confers a strong attenuating effect. We attempted to identify genetic attenuation determinants in the 3'-terminal part of the Sabin 1 genome including the 3D polymerase (3Dpol) gene and the 3' NC region. Previous studies suggested that some of the 11 mutations in this region of the Sabin 1 genome, and in particular a mutation in the polymerase gene (U-6203-->C, Tyr-73-->His), are involved to some extent in the attenuation of PV-1. We analyzed the attenuating effect in the mouse model by using the mouse-adapted PV-1/PV-2 chimeric strain v510 (a Mahoney strain carrying nine amino acids of the VP1 capsid protein from the Lansing strain of PV-2). Mutagenesis of locus 6203 was performed on the original v510 (U-6203-->C) and also on a hybrid v510/Sabin 1 (C-6203-->U) carrying the downstream 1,840 nucleotides of the Sabin 1 genome including the 3Dpol and 3' NC regions. Statistical analysis of disease incidence and time to disease onset in numerous mice inoculated with these strains strongly suggested that nucleotide C-6203 is involved in the attenuation of the Sabin 1 strain. Results also suggested that, among the mutations located in the 3Dpol and 3' NC regions, nucleotide C-6203 may be the principal or the only one to be involved in attenuation in this mouse model. We also found that the effect of C-6203 was weaker than that of nucleotide G-480; the two nucleotides acted independently and may have a cumulative effect on attenuation. The U-6203-->C substitution also appeared to contribute to the thermosensitivity of the Sabin 1 strain.
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PMID:A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice. 839 4

In neutrophils, binding and phagocytosis facilitate subsequent intracellular killing of microorganisms. Activity of Na+/H+ exchangers (NHEs) participates in these events, especially in regulation of intracellular pH (pHi) by compensating for the H+ load generated by the respiratory burst. Despite the importance of these functions, comparatively little is known regarding the nature and regulation of NHE(s) in neutrophils. The purpose of this study was to identify which NHE(s) are expressed in neutrophils and to elucidate the mechanisms regulating their activity during phagocytosis. Exposure of cells to the phagocytic stimulus opsonized zymosan (OpZ) induced a transient cytosolic acidification followed by a prolonged alkalinization. The latter was inhibited in Na+-free medium and by amiloride analogues and therefore was due to activation of Na+/H+ exchange. Reverse transcriptase PCR and cDNA sequencing demonstrated that mRNA for the NHE-1 but not for NHE-2, 3, or 4 isoforms of the exchanger was expressed. Immunoblotting of purified plasma membranes with isoform-specific antibodies confirmed the presence of NHE-1 protein in neutrophils. Since phagocytosis involves Fcgamma (FcgammaR) and complement receptors such as CR3 (a beta2 integrin) which are linked to pathways involving alterations in intracellular [Ca2+]i and tyrosine phosphorylation, we studied these pathways in relation to activation of NHE-1. Cross-linking of surface bound antibodies (mAb) directed against FcgammaRs (FcgammaRII > FcgammaRIII) but not beta2 integrins induced an amiloride-sensitive cytosolic alkalinization. However, anti-beta2 integrin mAb diminished OpZ-induced alkalinization suggesting that NHE-1 activation involved cooperation between integrins and FcgammaRs. The tyrosine kinase inhibitors genistein and herbimycin blocked cytosolic alkalinization after OpZ or FcgammaR cross-linking suggesting that tyrosine phosphorylation was involved in NHE-I activation. An increase in [Ca2+]i was not required for NHE-1 activation because neither removal of extracellular Ca2+ nor buffering of changes in [Ca2+]i inhibited alkalinization after OpZ or Fc-gammaR cross-linking. In summary, Fc-gammaRs and beta2 integrins cooperate in activation of NHE-1 in neutrophils during phagocytosis by a signaling pathway involving tyrosine phosphorylation.
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PMID:Na+/H+ exchange activity during phagocytosis in human neutrophils: role of Fcgamma receptors and tyrosine kinases. 860 83


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