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)

A collection of influenza virus PB2 mutant genes was prepared, including N-terminal deletions, C-terminal deletions, and single-amino-acid insertions. These mutant genes, driven by a T7 promoter, were expressed by transfection into COS-1 cells infected with a vaccinia virus encoding T7 RNA polymerase. Mutant proteins accumulated to levels similar to that of wild-type PB2. Immunofluorescence analyses showed that the C-terminal region of the protein is essential for nuclear transport and that internal sequences affect nuclear localization, confirming previous results (J. Mukaijawa and D. P. Nayak, J. Virol. 65:245-253, 1991). The biological activity of these mutants was tested by determining their capacity to (i) reconstitute RNA polymerase activity in vivo by cotransfection with proteins NP, PB1, and PA and a virion-like RNA encoding the cat gene into vaccinia virus T7-infected COS-1 cells and (ii) complete with the wild-type PB2 activity. In addition, when tested at different temperatures in vivo, two mutant PB2 proteins showed a temperature-sensitive phenotype. The lack of interference shown by some N-terminal deletion mutants and the complete interference obtained with a C-terminal deletion mutant encoding only 124 amino acids indicated that this protein domain is responsible for interaction with another component of the polymerase, probably PB1. To further characterize the mutants, their ability to induce in vitro synthesis of viral cRNA or mRNA was tested by using ApG or beta-globin mRNA as a primer. One of the mutants, 1299, containing an isoleucine insertion at position 299, was able to induce cRNA and mRNA synthesis in ApG-primed reactions but required a higher beta-globin mRNA concentration than wild-type PB2 for detection of in vitro synthesis. This result suggested that mutant I299 has diminished cap-binding activity.
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PMID:Mutational analysis identifies functional domains in the influenza A virus PB2 polymerase subunit. 862 88

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

By transfection of COS cells with an expression vector containing CD70 cDNA we demonstrate that two previously described MoAbs (ED6 and LD6) recognize CD70. By means of these MoAbs, we show that the surface expression of CD70 inversely correlates with the expression of its receptor, CD27, on activated T and NK cell populations and clones, although a subpopulation of cells expressing low density of both molecules exists. In addition, culture in the presence of IL-4 significantly enhances CD27 and reduces CD70 surface expression in phytohaemagglutinin (PHA)-activated peripheral blood lymphocytes (PBL), while tumour necrosis factor-alpha (TNF-alpha) displays opposite effects, indicating that receptor and ligand are reciprocally regulated by these cytokines. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of CD27 and CD70 mRNA suggests a transcriptional control of CD27 antigen expression in T cell clones. In addition, we show by the use of a re-directed killing assay that in cytotoxic T cell receptor (TCR) alpha/beta+ T cell clones, CD27 molecule may be involved in the regulation of cytolytic functions and may act synergistically with CD2. Finally, CD70 also acts as a signal-transducing molecule in some activated CD70+ TCR gamma/delta+ T or NK cell clones. In conclusion, our data indicate that CD27 and CD70 molecules are differentially expressed and regulated on long term-activated T and NK cells and are involved in the control of cellular functions.
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PMID:Reciprocal expression of CD70 and of its receptor, CD27, in human long term-activated T and natural killer (NK) cells: inverse regulation by cytokines and role in induction of cytotoxicity. 906 41

Interest in extra-adrenal corticosteroid synthesis has been revived by technological advances and the quest for answers to clinical problems. The cytochrome P450 21-hydroxylase converts progesterone to deoxycorticosterone, the obligatory substrate for the production of the main adrenal steroids aldosterone, cortisol and corticosterone. The rat P450 21-hydroxylase was cloned and two constructs, 21OH-5 and 21OH-6, sequenced. The constructs are similar, except that 21OH-6 has three additional major insertions of 64, 70 and 84 bp, a 3 bp deletion, and four extra base pairs immediately before the poly-A sequence. The entire coding region of 21OH-5 has 87 and 71% homology with the mouse and human 21-hydroxylase cDNA, respectively, whereas the encoded protein has 84 and 65% homology. Reverse transcriptase-polymerase chain reaction (RT-PCR) combined with Southern blot demonstrated expression of both transcripts in the kidney, aorta, liver, cerebellum, hypothalamus and brain stem, heart and cerebrum, but not the hippocampus, in addition to the adrenal. The entire coding region of 21OH-5 and the corresponding region of 21OH-6 including the three introns were cloned into pCR3 and the plasmids transiently transfected into COS-7 cells. Only 21OH-5 was translated into active protein, converting approximately 64% of 3H-progesterone to deoxycorticosterone in 2 h.
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PMID:Cloning of two alternatively spliced 21-hydroxylase CDNAs from rat adrenal. 940 81

The HSPDE4A gene spans 50 kb, consists of at least 17 exons and is orientated 5'-3', telomere to centromere. It is located at chromosome 19p13.2, being 350 kb proximal to the gene encoding TYK2 and 850 kb distal to the gene encoding the low-density lipoprotein receptor. Its structure is consistent with the production of active 'long' and 'short' isoenzymes as the result of alternative mRNA splicing at two splice junctions. Identified is the single alternatively spliced 5' exon encoding the unique N-terminal region of the long isoenzyme HSPDE4A4B (pde46). The upstream conserved regions, UCR1 and UCR2, which form characteristic domains of PDE4 long forms are each encoded by three exons. The PDE4A-subfamily-specific linker region LR1, which joins UCR1 and UCR2, is encoded by two exons, whereas LR2, which joins UCR2 to the catalytic unit, is encoded by a single exon. Identification of exons encoding an enzymically inactive product of this gene, HSPDE4A8A (2el), indicates that this is an authentic gene product. The 5' exon encoding the unique N-terminal region of the human homologue of the rodent isoform RNPDE4A1A (RD1) was located, and the splice junction used to produce this short PDE4A isoform shown to occur at a different position from that seen in both the rat PDE4B and PDE4D genes. Reverse transcriptase PCR analysis indicates that RD1 homologues are conserved across species, having a conserved membrane-targeting region and a hypervariable LR2 region. Human RD1 was expressed transiently in COS-7 cells and detected as an 83 kDa species primarily associated with the high-speed membrane fraction. Human RD1 exhibited a Km for cAMP of about 3 microM, an IC50 value for inhibition by the PDE4-selective inhibitor rolipram of about 0.3 microM and was considerably more thermostable than rat RD1. Human RD1 was generated as a mature 80 kDa species in an in vitro transcription-translation system and shown to be capable of binding to membranes. Knowledge of the gene structure and the associated sequence information should facilitate analysis of the involvement of PDE4A in hereditary disorders that may result from alterations in enzyme expression, activity, regulation and intracellular targeting and serve as a resource for determining authenticity of cloned PDE4A species.
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PMID:Identification and characterization of the human homologue of the short PDE4A cAMP-specific phosphodiesterase RD1 (PDE4A1) by analysis of the human HSPDE4A gene locus located at chromosome 19p13.2. 967 30

The cholesterol side-chain cleavage enzyme, cytochrome P450scc, initiates the biosynthesis of all steroid hormones. Adrenal and gonadal strategies for P450scc gene transcription are essentially identical and depend on the orphan nuclear receptor steroidogenic factor-1, but the placental strategy for transcription of P450scc employs cis-acting elements different from those used in the adrenal strategy and is independent of steroidogenic factor-1. Because placental expression of P450scc is required for human pregnancy, we sought factors that bind to the -155/-131 region of the human P450scc promoter, which participates in its placental but not adrenal or gonadal transcription. A yeast one-hybrid screen of 2.4 x 10(6) cDNA clones from human placental JEG-3 cells yielded two unique clones; one is the previously described transcription factor LBP-1b, which is induced by HIV, type I infection of lymphocytes, and the other is a new factor, termed LBP-9, that shares 83% amino acid sequence identity with LBP-1b. When expressed in transfected yeast, both factors bound specifically to the -155/-131 DNA; antisera to LBP proteins supershifted the LBP-9.DNA complex and inhibited formation of the LBP-1b.DNA complex. Reverse transcriptase-polymerase chain reaction detected LBP-1b in human placental JEG-3, adrenal NCI-H295A, liver HepG2, cervical HeLa, and monkey kidney COS-1 cells, but LBP-9 was detected only in JEG-3 cells. When the -155/-131 fragment was linked to a minimal promoter, co-expression of LBP-1b increased transcription 21-fold in a dose-dependent fashion, but addition of LBP-9 suppressed the stimulatory effect of LBP-1b. The roles of LBP transcription factors in normal human physiology have been unclear. Their modulation of placental but not adrenal P450scc transcription underscores the distinctiveness of placental strategies for steroidogenic enzyme gene transcription.
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PMID:Cloning of factors related to HIV-inducible LBP proteins that regulate steroidogenic factor-1-independent human placental transcription of the cholesterol side-chain cleavage enzyme, P450scc. 1064 52

We have isolated from the rat cerebellum cDNA library a complementary DNA encoding a new member of the tandem pore K(+) channel family. Its amino acid sequence shares 54% identity with that of TASK-1, but less than 30% with those of TASK-2 and other tandem pore K(+) channels (TWIK, TREK, TRAAK). Therefore, the new clone was named TASK-3. Reverse transcriptase-polymerase chain reaction analysis showed that TASK-3 mRNA is expressed in many rat tissues including brain, kidney, liver, lung, colon, stomach, spleen, testis, and skeletal muscle, and at very low levels in the heart and small intestine. When expressed in COS-7 cells, TASK-3 exhibited a time-independent, noninactivating K(+)-selective current. Single-channel conductance was 27 pS at -60 mV and 17 pS at 60 mV in symmetrical 140 mM KCl. TASK-3 current was highly sensitive to changes in extracellular pH (pH(o)), a hallmark of the TASK family of K(+) channels. Thus, a change in pH(o) from 7.2 to 6.4 and 6.0 decreased TASK-3 current by 74 and 96%, respectively. Mutation of histidine at position 98 to aspartate abolished pH(o) sensitivity. TASK-3 was blocked by barium (57%, 3 mM), quinidine (37%, 100 microM), and lidocaine (62%, 1 mM). Thus, TASK-3 is a new member of the acid-sensing K(+) channel subfamily (TASK).
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PMID:TASK-3, a new member of the tandem pore K(+) channel family. 1073 76

We raised a polyclonal antibody against maltose binding protein fusion human cGMP-binding, cGMP-specific phosphodiesterase (PDE5) produced in E. coli. This antibody immunoreacted specifically with recombinant human and rat PDE5 proteins expressed in transfected COS-7 cells and with a native form of PDE5 in extracts of rat platelets, lung, and cerebellum. Immunohistochemical analysis showed that the anti-PDE5 antibody detected immunoactive materials in Purkinje cell layers of the cerebellum, proximal renal tubules, collecting renal ducts, and epithelial cells of pancreatic ducts in rats. Reverse transcriptase-polymerase chain reaction analysis demonstrated that PDE5 transcripts are also present in rat cerebellum, kidney, and pancreas. Here we described a cell-specific localization of PDE5 in various rat tissues, suggesting the possibility of the presence of a cGMP/PDE5 pathway in these tissues.
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PMID:Immunohistochemical localization of cGMP-binding cGMP-specific phosphodiesterase (PDE5) in rat tissues. 1076 52

In order to determine how efficiently the polymerase proteins derived from human and avian influenza A viruses can interact with each other in the context of a mammalian cell, a genetic system that allows the in vivo reconstitution of active ribonucleoproteins was used. The ability to achieve replication of a viral-like reporter RNA in COS-1 cells was examined with heterospecific mixtures of the core proteins (PB1, PB2, PA and NP) from two strains of human viruses (A/Puerto Rico/8/34 and A/Victoria/3/75), two strains of avian viruses (A/Mallard/NY/6750/78 and A/FPV/-Rostock/34), and a strain of avian origin (A/Hong Kong/156/97) that was isolated from the first human case of H5N1 influenza in Hong Kong in 1997. In accordance with published observations on reassortant viruses, PB2 amino acid 627 was identified as a major determinant of the replication efficiency of heterospecific complexes in COS-1 cells. Moreover, the results showed that replication of the viral-like reporter RNA was more efficient when PB2 and NP were both derived from the same avian or human virus or when PB1 was derived from an avian virus, whatever the origin of the other proteins. Furthermore, the PB1 and PB2 proteins from the A/Hong- Kong/156/97 virus exhibited intermediate properties with respect to the corresponding proteins from avian or human influenza viruses, suggesting that some molecular characteristics of PB1 and PB2 proteins might at least partially account for the ability of the A/Hong Kong/156/97 virus to replicate in humans.
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PMID:Genetic analysis of the compatibility between polymerase proteins from human and avian strains of influenza A viruses. 1076 71

We have isolated from a human genomic library the human 5-hydroxytryptamine 5-HT(5A) and 5-HT(5B) genes. The human 5-HT(5A) gene encodes a protein with similar characteristics to its mouse homologue. When expressed in monkey COS-7 cells, the human 5-HT(5A) receptor displayed a high affinity for tritiated 5-carbamidotryptamine ([3H]5-CT; K(D)=2.8 nM) and iodinated lysergic acid diethylamide ([125I]LSD; K(D)=187 pM). These binding sites displayed the following displacement profile: Ergotamine>Methiothepin>5-CT, Ritanserin>5-HT. Reverse transcriptase polymerase chain reaction (RT-PCR) experiments revealed the presence of human 5-HT(5A) mRNA in the central nervous system but not in peripheral organs. When expressed in Xenopus oocytes, the 5-HT(5A) receptor was able to couple to the inwardly rectifying K(+) channel, GIRK(1). In contrast to the human 5-HT(5A) gene and the mouse 5-HT(5B) gene, the human 5-HT(5B) gene does not encode a functional protein because its coding sequence is interrupted by stop codons. Our results suggest, therefore, that the 5-HT(5B) receptor has been lost during evolution after the divergence between rodents and primates. The 5-HT(5B) receptor is the first example of a brain-specific protein that is absent in human.
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PMID:Human 5-HT(5) receptors: the 5-HT(5A) receptor is functional but the 5-HT(5B) receptor was lost during mammalian evolution. 1134 85


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