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 are reporting the molecular cloning of gerbil interleukin-18 (IL-18) and caspase-1. The cDNAs encoding the molecules were cloned by cross-species reverse transcriptase-polymerase chain reaction (RT-PCR) and 5'/3' rapid amplification of cDNA ends (RACE). COS-7 cells transfected with plasmids encoding pro-IL-18 and caspase-1 precursor expressed the two proteins intracellularly. When the cells were lysed in the presence of dithiothreitol, caspase-1 precursor became active and converted pro-IL-18 into mature IL-18. A partially purified preparation of gerbil mature IL-18 was bioactive, as it stimulated the proliferation of gerbil spleen cells in a dose-dependent manner.
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PMID:Gerbil interleukin-18 and caspase-1: cloning, expression and characterization. 1270 98

The expression of glutamine synthetase (GS), catalysing the ATP-dependent conversion of glutamate and ammonia into glutamine, is transcriptionally and post-transcriptionally regulated. The genomic structure of dog GS shown in the present study is basically similar to that of other mammals in that it is composed of seven exons and six introns. Using 5'-cRACE (where cRACE stands for circular rapid amplification of cDNA ends) and reverse transcriptase-PCR, we identified an additional exon (120 bp) in the first intron, designated in the present study as exon 1'. By means of alternative splicing, the GS gene produces an altered form of GS transcript with 5'-untranslated region (UTR) containing the exon 1'. This alternative transcript is abundantly expressed in brain, whereas it is found at lower levels in other tissues. In the human and mouse GS genes, extra exons are also found at the corresponding site of the intron 1 but with different sizes. An exon-trapping experiment for the GS gene in COS-7, Madin-Darby canine kidney and SK-N-SH cells revealed that the pattern of alternative splicing is variable in different cell types. The propensity of forming a secondary structure is predicted to be considerably higher in the presence of extra 5'-UTR, suggesting the possibility of a translational effect. To test this, we performed a reporter assay for fusions with different 5'-UTRs, demonstrating that the long form with extra 5'-UTR was translated 20- and 10-fold less than the short one in SK-N-SH and Neuro-2A cells respectively. Similarly, translations of human and mouse transcripts with extra 5'-UTRs were less efficient, showing 6-8-fold reductions in SK-N-SH cells. Furthermore, when we mutated an ATG sequence contained in the exon 1', the suppression of translation was partially relieved, suggesting that the negative regulation by an extra 5'-UTR is, to some extent, due to an abortive translation from the upstream ATG.
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PMID:A splice variant acquiring an extra transcript leader region decreases the translation of glutamine synthetase gene. 1274 66

Inactivating mutations of Phex cause X-linked hypophosphatemia (XLH) by increasing levels of a circulating phosphaturic factor. FGF23 is a candidate for this phosphaturic factor. Elevated serum FGF23 levels correlate with the degree of hypophosphatemia in XLH, suggesting that loss of Phex function in this disorder results in either diminished degradation and/or increased biosynthesis of FGF23. To establish the mechanisms whereby Phex regulates FGF23, we assessed Phex-dependent hydrolysis of recombinant FGF23 in vitro and measured fgf23 message levels in the Hyp mouse homologue of XLH. In COS-7 cells, overexpression of FGF23 resulted in its degradation into N- and C-terminal fragments by an endogenous decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone-sensitive furin-type convertase. Phex-dependent hydrolysis of full-length FGF23 or its N- and C-terminal fragments could not be demonstrated in the presence or absence of decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone in COS-7 cells expressing Phex and FGF23. In a reticulolysate system, apparent cleavage of FGF23 occurred with wild-type Phex, the inactive Phex-3'M mutant, and vector controls, indicating nonspecific metabolism of FGF23 by contaminating enzymes. These findings suggest that FGF23 is not a direct Phex substrate. In contrast, by real-time reverse transcriptase PCR, the levels of fgf23 transcripts were highest in bone, the predominant site of Phex expression. In addition, Hyp mice displayed a bone-restricted increase in fgf23 transcripts in association with inactivating Phex mutations. Increased expression of fgf23 was also observed in Hyp-derived osteoblasts in culture. These findings suggest that Phex, possibly through the actions of unidentified Phex substrates or other downstream effectors, regulates fgf23 expression as part of a potential hormonal axis between bone and kidney that controls systemic phosphate homeostasis and mineralization.
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PMID:Regulation of fibroblastic growth factor 23 expression but not degradation by PHEX. 1287 85

We have identified a novel membrane form of guanylate cyclase (GC) from a mouse testis cDNA library and termed it mGC-G (mouse GC-G) based on its high sequence homology to rat GC-G. It encodes a potential type I transmembrane receptor, with the characteristic domain structure common to all members of the family of membrane GCs, including an extracellular, putative ligand-binding domain, a single membrane-spanning segment and cytoplasmic protein kinase-like and cyclase catalytic domains. Real-time quantitative reverse transcriptase--PCR and Northern-blot analyses showed that mGC-G is highly and selectively expressed in mouse testis. Phylogenetic analysis based on the extracellular protein sequence revealed that mGC-G is closely related to members of the subfamily of natriuretic peptide receptor GCs. When overexpressed in HEK-293T cells (human embryonic kidney 293T cells) or COS-7 cells, mGC-G manifests as a membrane-bound glycoprotein, which can form either homomeric or heteromeric complexes with the natriuretic peptide receptor GC-A. It exhibits marked cGMP-generating GC activity; however, notably, all ligands known to activate other receptor GCs failed to stimulate enzymic activity. The unique testis-enriched expression of mGC-G, which is completely different from the broader tissue distribution of rat GC-G, suggests the existence of as-yet-unidentified ligands and unappreciated species-specific physiological functions mediated through mGC-G/cGMP signalling in the testis.
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PMID:Identification of an orphan guanylate cyclase receptor selectively expressed in mouse testis. 1471 86

By employing the reverse transcriptase-polymerase chain reaction technique in conjunction with 3' rapid amplification of cDNA ends, a full-length cDNA encoding a zebrafish (Danio rerio) tyrosylprotein sulfotransferase (TPST) was cloned and sequenced. Sequence analysis revealed that this zebrafish TPST is, at the amino acid sequence level, 66% and 60% identical to the human and mouse TPST-1 and TPST-2, respectively. The recombinant form of the zebrafish TPST, expressed in COS-7 cells, exhibited a pH optimum at 5.75. Manganese appeared to exert a stimulatory effect on the zebrafish TPST. The activity of the enzyme determined in the presence of 20 mM MnCl2 was more than 2.5 times that determined in the absence of MnCl2. Of the other nine divalent metal cations tested at a 10 mM concentration, Co2+ also showed a considerable stimulatory effect, while Ca2+, Pb2+, and Cd2+ exerted some inhibitory effects. The other four divalent cations, Fe2+, Cu2+, Zn2+, and Hg2+, inhibited completely the sulfating activity of the zebrafish TPST. Using the wild-type and mutated P-selectin glycoprotein ligand-1 N-terminal peptides as substrates, the zebrafish TPST was shown to exhibit a high degree of substrate specificity for the tyrosine residue on the C-terminal side of the peptide. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic TPST.
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PMID:Zebrafish tyrosylprotein sulfotransferase: molecular cloning, expression, and functional characterization. 1506 Jun 24

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent protein kinase A-activated chloride channel that resides on the apical surface of epithelial cells. One unusual feature of this protein is that during biogenesis, approximately 75% of wild type CFTR is degraded by the endoplasmic reticulum (ER)-associated degradative (ERAD) pathway. Examining the biogenesis and structural instability of the molecule has been technically challenging due to the limited amount of CFTR expressed in epithelia. Consequently, investigators have employed heterologous overexpression systems. Based on recent results that epithelial specific factors regulate both CFTR biogenesis and function, we hypothesized that CFTR biogenesis in endogenous CFTR expressing epithelial cells may be more efficient. To test this, we compared CFTR biogenesis in two epithelial cell lines endogenously expressing CFTR (Calu-3 and T84) with two heterologous expression systems (COS-7 and HeLa). Consistent with previous reports, 20 and 35% of the newly synthesized CFTR were converted to maturely glycosylated CFTR in COS-7 and HeLa cells, respectively. In contrast, CFTR maturation was virtually 100% efficient in Calu-3 and T84 cells. Furthermore, inhibition of the proteasome had no effect on CFTR biogenesis in Calu-3 cells, whereas it stabilized the immature form of CFTR in HeLa cells. Quantitative reverse transcriptase-PCR indicated that CFTR message levels are approximately 4-fold lower in Calu-3 than HeLa cells, yet steady-state protein levels are comparable. Our results question the structural instability model of wild type CFTR and indicate that epithelial cells endogenously expressing CFTR efficiently process this protein to post-Golgi compartments.
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PMID:Efficient intracellular processing of the endogenous cystic fibrosis transmembrane conductance regulator in epithelial cell lines. 1506 92

In this paper, we have characterized the structure, evolutionary origin, and function of rat and human carcinoembryonic antigen-related cell adhesion molecule1 (CEACAM1) multifunctional Ig-like cell adhesion proteins that are expressed by many epithelial tissues. Restriction enzyme digestion reverse transcriptase-PCR analysis identified three cDNAs encoding novel CEACAM1 N-domains. Comparative sequence analysis showed that human and rat CEACAM1 N-domains segregated into two groups differing in similarity to rat CEACAM1(a)-4L and human CEACAM1. Sequence variability analysis indicated that both human and rat N-domains possessed two variable regions, and one contained a major adhesive epitope. Recombination analysis showed that the group of rat but not human N-domains with high sequence similarity was derived at least in part by recombination. Binding assays revealed that three monoclonal antibodies with strong reactivity for the CEACAM1(a)-4L N-domain showed no reactivity with CEACAM1(b)-4S, an allele with a different N-domain sequence. CEACAM1(b)-4S displayed adhesive activity efficiently blocked by a synthetic peptide corresponding to the adhesive epitope in CEACAM1(a)-4L. Blocking analysis also showed that the adhesive epitope for rat CEACAM1 was located downstream from the equivalent human and mouse epitopes. Glycosylation analysis demonstrated O-linked sugars on rat CEACAM1(b)-4S from COS-1 cells. However, this was not the alteration responsible for the lack of monoclonal antibody reactivity. When considered together with previous studies, our findings suggest an inverse relationship between functionality and amino acid sequence similarity to CEACAM1. Like IgG, the N-domain of CEACAM1 appears to tolerate 10-15% sequence diversification without loss of function but begins to show either altered specificity or diminished functionality at higher levels.
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PMID:Two variable regions in carcinoembryonic antigen-related cell adhesion molecule1 N-terminal domains located in or next to monoclonal antibody and adhesion epitopes show evidence of recombination in rat but not in human. 1518 66

The two COX (cyclo-oxygenase) isoenzymes COX-1 and -2 catalyse the initial step in the conversion of arachidonic acid into PG (prostaglandin) hormones. The identification of an mRNA transcript encoding a splice variant of human COX-1 was reported more than a decade ago [Diaz, Reginato and Jimenez (1992) J. Biol. Chem. 267, 10816-10822], yet catalytic activity and tissue expression of the corresponding spliced protein remained uncharacterized. The splice variant lacks amino acids 396-432, corresponding to the last 37 amino acids of exon 9 of the gene encoding COX-1. These amino acids form a loop at one side of the peroxidase active site of the protein. We expressed the full-length and spliced COX-1 cDNAs in COS-7 and Sf9 insect cells, and determined the PG-forming activity using incubations with radiolabelled arachidonic acid and HPLC analyses. When expressed in either system, abundant PG formation was observed with the full-length COX-1, whereas the spliced protein did not form any detectable product. Peroxidase activity was readily detected in microsomes prepared from COS-7 cells transfected with COX-1 but not with the splice variant. In reverse transcriptase-PCR experiments, we detected the mRNA for the alternatively spliced and full-length COX-1 in human brain, tonsil and colon tissue, yet we were unable to detect expression of the spliced protein in the same tissues using immunoprecipitation and Western-blot analyses. We conclude that, whereas the mRNA transcript for the spliced COX-1 is present in various human tissues, the corresponding protein is either not formed or subject to rapid proteolytic degradation.
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PMID:Human cyclo-oxygenase-1 and an alternative splice variant: contrasts in expression of mRNA, protein and catalytic activities. 1536 Oct 66

We report on the establishment of one transgenic and two endogenous reporter gene assays to determine androgenic/antiandrogenic activity. A transient transactivation assay was developed in COS-7 African green monkey kidney cells. Three plasmids were co-transfected by electroporation: the human androgen receptor expression vector pSG5AR, the reporter gene vector pMamneoLuc, expressing luciferase under the control of the mouse mammary tumor virus (MMTV) promotor containing 4 hormone responsive elements (HREs), and the control plasmid pSVbeta. Transcriptional activation was measured by luciferase-mediated chemoluminescence. In T47D human breast cancer cells two endogenous reporter gene systems were established: one based on the androgen-induced expression of prostate-specific antigen (PSA), the other based on the androgen-repressed expression of testosterone repressed message 2 (TRPM-2). PSA protein was measured by enzyme-linked immunosorbent assay (ELISA), TRPM-2 m-RNA by reverse transcriptase polymerase chain reaction (RT-PCR). All three test systems were validated using the physiological androgen dihydrotestosterone (DHT) as agonist and the established antiandrogens hydroxyflutamide and p,p'-dichlorodiphenylethene (p,p'-DDE) as antagonists. The PSA assay was the most sensitive test system with an EC50 of 0.7 nM for DHT-induced response. The transient transactivation assay in COS-7 cells was less sensitive with an EC50 of 9.7 nM DHT. In the PSA assay hydroxyflutamide was a more potent antagonist (IC30 = 0.02 microM) than p,p'-DDE (IC30 = 0.9 microM). In the transient transactivation assay in COS-7 cells, both compounds elicited about 30% of the agonistic response induced by 100 nM DHT, thus showing partial agonistic properties. In summary, three highly sensitive and complementary in vitro test systems, together achieving enhanced specificity for detection and assessment of androgenic/antiandrogenic activity have been established and validated.
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PMID:Sensitive in vitro test systems to determine androgenic/antiandrogenic activity. 1549 79

The serotonin 5-HT4 receptor (where 5-HT stands for 5-hydroxy-tryptamine) is a member of the seven transmembrane-spanning G-protein-coupled family of receptors and mediates many cellular functions both in the central nervous system and at the periphery. In the present study, we isolated and characterized the 5'-flanking region of the h5-HT4 (human 5-HT4) receptor. We demonstrate the existence of a novel exon that corresponds to the 5'-untranslated region of the h5-HT4 receptor gene. RNase protection analysis and reverse transcriptase-PCR experiments performed on human atrial RNA demonstrated that the major transcription start site of the h5-HT4 receptor gene is located at -3185 bp relative to the first ATG codon. In addition, a 1.2 kb promoter fragment which drives the transcription of the 5-HT4 receptor was characterized. The promoter region lacks TATA and CAAT canonical motifs in the appropriate location, but contains putative binding sites for several transcription factors. Transient transfection assays revealed that the (-3299/-3050) gene fragment possesses the ability to promote the expression of the luciferase reporter gene in human cell lines. In contrast, the promoter was silent in monkey COS-7 cells, indicating the requirement of specific factors to initiate transcription in human cells. In addition to the promoter element, enhancer activity was found in a region (-220/-61) located in the long 5'-untranslated region. Mutational analysis, gel shift and transfection assays identified an Nkx2.5 (NK2-transcription-factor-related 5)-like binding site as a regulatory sequence of this enhancer. Our results suggest a complex regulation of the h5-HT4 receptor gene expression involving distinct promoters and non-coding exons.
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PMID:Functional studies of the 5'-untranslated region of human 5-HT4 receptor mRNA. 1557 21


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