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)

Three new cell lines of human glioblastoma have been established. These cells co-expressed hepatocyte growth factor (HGF) and its receptor, c-Met, genes in vitro. Reverse-transcriptase/polymerase-chain reaction study revealed that the cells also expressed gene for HGF activator, a recently cloned serine proteinase, suggesting that HGF might have a role in glioma cells in vitro as an autocrine factor. The activator mRNA was also detected in other well-established glioma cell lines, glioma tissues and normal brain. The concomitant expression of HGF, HGF activator and c-met was also detected in one glioblastoma case in vivo out of five tested.
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PMID:Concomitant expression of hepatocyte growth factor (HGF), HGF activator and c-met genes in human glioma cells in vitro. 755 48

Dengue virus type 2, a member of the family Flaviviridae, encodes a single polyprotein precursor consisting of 3391 amino acids residues that is processed to at least 10 mature proteins by host and viral proteases. The NS3 protein contains a domain commonly found in cellular serine proteinases that in cooperation with NS2B is involved in polyprotein processing. In addition, NS3 and NS5 proteins contain conserved motifs found in several RNA helicases and RNA-dependent RNA polymerases, respectively. Both enzymatic activities have been suggested to be involved in viral RNA replication. In this report, we demonstrate that the NS3 and NS5 proteins interact in vivo in dengue virus type 2-infected monkey kidney (CV-1) cells and in HeLa cells coinfected with recombinant vaccinia viruses encoding these proteins as shown by coimmunoprecipitations and immunoblotting methods. We also show by immunofluorescence, metabolic labeling, and two-dimensional peptide mapping that NS5 is a nuclear phosphoprotein and that phosphorylation occurs on serine residues at multiple sites. Furthermore, NS5 exists in differentially phosphorylated states in the nuclear and the cytoplasmic fractions, and only the cytoplasmic form of NS5 is found to coimmunoprecipitate with NS3, suggesting that differential phosphorylation may control the interaction between these proteins and its function in the viral RNA replicase.
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PMID:Association between NS3 and NS5 proteins of dengue virus type 2 in the putative RNA replicase is linked to differential phosphorylation of NS5. 764 75

Reverse transcriptase and polymerase chain reaction methods were used to amplify and clone actin cDNAs from the chlorophylls a + C-containing unicellular alga, Emiliania huxleyi (Prymnesiophyta). Actins in E. huxleyi are defined by a gene family containing at least six distinct coding regions that were derived from relatively recent gene duplications. Five of the coding regions (types 1, 2, and 4-6) varied only among synonymous codons. A nonsynonomous change in a sixth coding region (type 3 actin) produced a serine-to-phenylalanine replacement. The G + C composition of third positions in E. huxleyi actin genes is 98%, which contrasts with the mean value of 50% G + C content for first and second positions. Distance-matrix and parsimony analyses of actin genes identified the prymnesiophytes as a photosynthetic lineage that is not already related to other eukaryotic algal groups.
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PMID:Isolation and molecular phylogenetic analysis of actin-coding regions from Emiliania huxleyi, a Prymnesiophyte alga, by reverse transcriptase and PCR methods. 768 35

We have isolated the murine cDNA homologue of the human protein tyrosine phosphatase PTP-PEST (MPTP-PEST) from an 18.5-day mouse embryonic kidney library. The cDNA isolated has a single open reading frame predicting a protein of 775 amino acids. When expressed in vitro as a glutathione S-transferase fusion protein, the catalytic domain (residues 1-453) shows intrinsic phosphatase activity. Reverse transcriptase PCR and Northern-blot analysis show that MPTP-PEST mRNA is expressed throughout murine development. Indirect immunofluorescence in COS-1 cells against a heterologous epitope tag attached to the N-terminus of MPTP-PEST, together with cellular fractionation and Western-blot experiments from different murine cell lines, indicate that MPTP-PEST is a free cytosolic protein of 112 kDa. Finally, sequence analysis indicates that the C-terminal portion of the protein contains four regions rich in proline, glutamate, serine and threonine, otherwise known as PEST sequences. These are characteristic of proteins that display very short intracellular half-lives. Despite the presence of these motifs, pulse-chase labelling experiments demonstrate that MPTP-PEST has a half-life of more than 4 h.
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PMID:Murine protein tyrosine phosphatase-PEST, a stable cytosolic protein tyrosine phosphatase. 777 23

The class II IL-8 receptor (IL-8R) binds both melanoma growth stimulatory activity (MGSA) and IL-8 with high affinity. Reverse transcriptase polymerase chain reaction demonstrates that the class II IL-8R mRNA, which has previously been detected only in cells of hematopoietic lineage, is also expressed in non-hematopoietic cell types shown to respond to MGSA or IL-8. To study the signaling mechanism by MGSA through the class II IL-8R in non-hematopoietic cells, this receptor was overexpressed in the 3ASubE human placental and the 293 human kidney cell lines. Membrane preparations of the class II IL-8R expressing 3ASubE transfectants exhibited a 2.3 +/- 0.2-fold increase in GTP gamma 35S binding, which was sensitive to pertussis toxin, in response to MGSA treatment (0.2 microM). This MGSA response was not observed in cells transfected with the parental expression vector. In vivo phosphorylation studies demonstrated that the class II IL-8R was basally phosphorylated in the untreated transfectants, and MGSA (5 nM) treatment markedly enhanced the phosphorylation of this receptor. The MGSA-induced receptor phosphorylation was both time and concentration dependent and could be mimicked by treatment with the calcium ionophore A23187. Phosphoamino acid analysis indicated that the MGSA-induced receptor phosphorylation was on serine residue(s), suggesting that a serine kinase is activated in response to MGSA binding to the class II IL-8R in non-hematopoietic cells.
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PMID:Melanoma growth stimulatory activity enhances the phosphorylation of the class II interleukin-8 receptor in non-hematopoietic cells. 829 49

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

We previously isolated cDNA clones, MLL-a and MLL-b, derived from the 11q23 breakpoint region and detected gene rearrangements with MLL-b cDNA in infantile leukemia cell lines with 11q23 abnormalities. We also showed chimeric mRNAs between MLL and genes on partner chromosomes such as 4q21 and 19p13. In the present study, we isolated overlapping MLL cDNA clones of 11 kb and demonstrated that MLL-a and MLL-b were derived from the same gene, MLL/ALL-1/HRX. Northern analysis with an MLL cDNA probe detected different signals in t(11;19) cell lines, one being sized 10 kb in two cell lines, KOCL-33 and KOCL-44, and the other being 9.2 kb in the cell line, KOPN-1. To elucidate the molecular basis for the heterogeneity, we isolated cDNA clones of a translocation-associated gene on chromosome 19, LTG19, as well as chimeric cDNAs from KOPN-1. Northern analysis with LTG19 cDNA demonstrated the identical gene, encoding serine/proline rich 559 amino acid polypeptide, to be involved in all three cell lines. Sequence comparison revealed that the LTG19 portion of the predicted chimeric protein of KOPN-1 was fused in frame and contained the C-terminal 189 amino acids. This was shorter by 366 amino acids than those of KOCL-33 and KOCL-44, also fused in frame. Reverse transcriptase-PCR analysis demonstrated complex chimeric mRNAs in cell lines and leukemia samples. Although a chimeric mRNA of KOPN-1 type was rare, its presence suggested that the shared C-terminal portion of 189 amino acids of LTG19 contains important signal(s) for malignant transformation.
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PMID:Two distinct portions of LTG19/ENL at 19p13 are involved in t(11;19) leukemia. 837 76

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

Met-ase-1 is a 30 000 Mr serine protease (granzyme) that was first isolated in the cytolytic granules of rat CD3(-) large granular lymphocytes. We screened a mouse genomic library with rat Met-ase-1 cDNA, and obtained bacteriophage clones that contained the mouse Met-ase-1 gene. The mouse Met-ase-1 gene comprises five exons spanning approximately 5.2 kilobases (kb) and exhibits a similar structural organization to its rat homologue and a family of neutrophil elastase-like serine proteases. Mouse Met-ase-1 mRNA was only detected in total cellular and poly A mRNA of mouse CD3(-) GM1(+) large granular lymphocytes derived from splenocytes stimulated with IL-2 and the mouse NK1.1(+) cell line 4 - 16. Spleen T-cell populations generated by Concanavalin A stimulation and a number of mouse pre-NK and T cell lines did not express mouse Met-ase-1 mRNA. The 5' flanking region of the mouse Met-ase-1 gene also shares considerable regions of identity with the 5' flanking region of the rat Met-ase-1 gene. A 3.3 kb segment of 5' sequence flanking the mouse Met-ase-1 gene was inserted upstream of the chloramphenicol acetyltransferase reporter gene and this construct transiently transfected into a variety of mouse and rat large granular lymphocyte leukemia and T-cell lines. The transcriptional activity of the mouse Met-ase-1 5' flanking region was significant in the RNK-16 large granular lymphocyte leukemia, strongest in the 4 - 16 mouse NK1.1(+) cell line, and weak in several mouse pre-NK cell lines. Reverse transcriptase polymerase chain reaction of mouse large granular lymphocyte mRNA was used to derive the full-length coding sequence for mouse Met-ase-1. The predicted hexapropeptide of mouse Met-ase-1 (Asn-6 to Gln-1), was deleted by polymerase chain reaction mutagenesis to enable expression of active mouse Met-ase-1 in mammalian COS-7 cells. Northern blot analysis and protease assays of transfected COS cell lysates against a panel of thiobenzyl ester substrates formally demonstrated that the mouse Met-ase-1 gene encodes a serine proteinase that hydrolyzes substrates containing a long narrow hydrophobic amino acids like methionine, norleucine, and leucine in the P1.
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PMID:Cloning and expression of the recombinant mouse natural killer cell granzyme Met-ase-1. 878 Nov 19

In this study, we isolated two cDNA molecules encoding putative glutamate receptor subunits, fGluR1 alpha and fGluR1 beta, from an Oreochromis sp. brain cDNA library by hybridizing with the glutamate receptor cDNA, fGluR2 beta, of the same fish. The deduced amino acid sequence of the fGluR1 alpha consists of 908 residues with an 18-residue signal peptide and displays a sequence identity of 74% to the amino acid sequence of rat GluR1 subunit. Northern blotting indicates that the expression level of fGluR1 alpha in telencephalon is higher than that in optic tectum and cerebellum in adult fish brain. Reverse-transcriptase polymerase chain reaction and genomic analyses reveal the presence of variants created by alternative splicing at the flip-flop module and the carboxyl terminus of fGluR1 alpha transcripts. The amino acid sequence of fGluR1 alpha is unique in that it contains a glutamine-rich sequence inserted at the loop 1 (L1) between transmembrane domains 1 and 2. A second incomplete cDNA clone, designated fGluR1 beta, coding for a polypeptide showing sequence identity to the rat GluR1 and fGluR1 alpha was isolated from the same library. Insertion of a serine- and glutamine-rich sequence at the L1 was also detected in the translated sequence of fGluR1 beta.
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PMID:Molecular analysis of cDNA molecules encoding glutamate receptor subunits, fGluR1 alpha and fGluR1 beta, of Oreochromis sp. 883 30


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