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)

Tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step in the biosynthesis of catecholamines. Among the various mechanisms implicated in the regulation of TH activity, alternative splicing of TH primary transcript has been described as a characteristic of higher primates and Drosophila. We investigated whether there is such a regulatory mechanism in the rat. Reverse transcriptase-PCR experiments were performed with RNA from PC12 cells. A new TH mRNA species was evidenced, resulting from the use of an alternative donor site in exon 2. RNase protection assays and in situ hybridization experiments detected this mRNA species in the adrenal medulla but not in the main catecholaminergic nuclei of the CNS. The corresponding putative protein lacks 33 amino acids in the N-terminal regulatory domain. A recombinant protein was produced in E. coli. Its in vitro specific activity was similar to that of the previously identified TH protein.
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PMID:A novel rat tyrosine hydroxylase mRNA species generated by alternative splicing. 878 6

The use of conventional PCR can amplify target DNA from both viable and nonviable cells of Vibrio cholera. Detection of only viable microbial pathogens in biological samples, especially clinical and food samples, is usually desired to ensure positive test results are associated with active agents, and not the remains of dead cells. Positive identifications caused by nonliving causative agents may lead to misguided decisions concerning the effectiveness of treatment, and whether patient treatment should be continued or whether the food should be discarded. Consequently, this work was directed toward development of a reverse-transcriptase polymerase chain reaction (RT-PCR)-based in vitro DNA amplification method, which specifically detects only viable cells. Total RNA from both viable and nonviable cells was purified by using a FastPrep Cell Disrupter ([symbol: see text]Bio 101/Savant) and FastRNA extraction reagents ([symbol: see text]Bio 101). The purified RNA was treated with DNase I (RNase-free) to avoid any amplification from the contaminating target DNA. An RT-PCR approach using this rapid and effective method for RNA purification showed amplification of the target mRNA only from the viable cells. The sensitivity of detection of viable cells of V. cholerae was > or = 10(3), which is well within the minimum number of cells (10(5)-10(6)) required for infection. The use of a reliable prokaryotic RNA extraction method followed by RT-PCR amplification of the target mRNA can be used for specific detection of viable microbial pathogen, such as V. cholerae.
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PMID:Detection of viable Vibrio cholerae by reverse-transcriptase polymerase chain reaction (RT-PCR). 885 11

POU domain transcription factors are required for neuropeptide expression in selected subsets of hypothalamic neuroendocrine neurons. We now report that expression of the gonadotropin-releasing hormone (GnRH) gene, which controls sexual development, is regulated by the POU protein SCIP/Oct-6/Tst-1. Reverse transcriptase PCR cloning and RNase protection assays demonstrated the presence of SCIP/Oct-6/Tst-1 mRNA in the GnRH-producing neuronal cell line GT1-7. The physiological relevance of this regulatory activity was suggested by the detection of SCIP/Oct-6/Tst-1 mRNA in a subset of GnRH neurons in the hypothalamus of prepubertal female rats. Coexpression of SCIP/Oct-6/Tst-1 in neuronal cells inhibited rat GnRH (rGnRH) promoter activity via three regions of the proximal rGnRH promoter containing SCIP/Oct-6/Tst-1 binding sites. DNase I footprinting, gel shift assays, and DNA and protein mutagenesis studies indicated that both direct DNA binding and protein-protein interactions are required for SCIP/Oct-6/Tst-1 modulation of GnRH gene expression. Activation of SCIP/Oct-6/Tst-1 expression in terminally differentiated GnRH neurons may be a factor determining the ratio of phenotypically "inactive" versus "active" GnRH neurons during postnatal life.
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PMID:Repression of gonadotropin-releasing hormone promoter activity by the POU homeodomain transcription factor SCIP/Oct-6/Tst-1: a regulatory mechanism of phenotype expression? 903 92

Purified preparations of La France isometric virus (LIV), an unclassified, double-stranded RNA (dsRNA) virus of Agaricus bisporus, were associated with an RNA-dependent RNA polymerase (RDRP) activity. RDRP activity cosedimented with the 36-nm isometric particles and genomic dsRNAs of LIV during rate-zonal centrifugation in sucrose density gradients, suggesting that the enzyme is a constituent of the virion. Enzyme activity was maximal in the presence of all four nucleotides, a reducing agent (dithiothreitol or beta-mercaptoethanol), and Mg2+ and was resistant to inhibitors of DNA-dependent RNA polymerases (actinomycin D, alpha-amanitin, and rifampin). The radiolabeled enzyme reaction products were predominantly (95%) single-stranded RNA (ssRNA) as determined by cellulose column chromatography and ionic-strength-dependent sensitivity to hydrolysis by RNase A. Three major size classes of ssRNA transcripts of 0.95, 1.3, and 1.8 kb were detected by agarose gel electrophoresis, although the transcripts hybridized to all nine of the virion-associated dsRNAs. The RNA products synthesized in vitro appeared to be of a single polarity, as they hybridized to an ssDNA corresponding to one strand of a genomic dsRNA and not to the complementary strand. Similarly, reverse transcription-PCR with total cellular ssRNA as a template and strand-specific primers targeting a genomic dsRNA during synthesis of cDNA suggested that only the coding strand was transcribed in vivo. Our data indicate that the RDRP activity associated with virions of LIV is probably a transcriptase engaged in the synthesis of ssRNA transcripts corresponding to each of the virion-associated dsRNAs.
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PMID:Characterization of an RNA-dependent RNA polymerase activity associated with La France isometric virus. 903 61

NK cells migrate in response to C-C chemokines, including monocyte chemotactic protein-1 (MCP-1) and MCP-3. Increased migration was observed in IL-2-activated NK cells. It was therefore of interest to define the expression in resting and activated NK cells of the MCP-1 receptor (CCR2) for which two cDNAs (A and B) have been described. Specific oligonucleotides and reverse-transcriptase PCR revealed the presence in activated NK cells and mononuclear phagocytes of the fragments expected on the basis of the reported cDNAs. In addition, amplification with a common A/B- and an A-specific oligonucleotide yielded an unexpected, abundant, 1649-bp fragment. Sequence analysis as well as Northern blotting and RNase protection with different probes revealed that the CCR2 gene is expressed in activated NK cells and mononuclear phagocytes as a predominant long transcript (3.4 kb) consisting of CCR2B, followed by a novel sequence (X), corresponding to an intron in the genome, and by a CCR2A-specific portion. The predominant long transcript is polyadenylated and present in the cytoplasm. The augmented migratory capacity of IL-2 activated vs resting NK cells was associated with increased CCR2 transcript levels.
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PMID:IL-2-regulated expression of the monocyte chemotactic protein-1 receptor (CCR2) in human NK cells: characterization of a predominant 3.4-kilobase transcript containing CCR2B and CCR2A sequences. 905 2

Two basic processes are involved in protein evolution: One is amino acid replacement and another is reorganization of structural or functional units of proteins. Multidomain or multifunctional proteins are thought to have evolved by fusion of smaller structural units such as modules or domains. Reverse transcriptase (RT) is one of such fused proteins. The N-terminal part forms of globular domain with polymerase activity and the C-terminal part forms another globular domain with ribonuclease H activity (RNase H domain). There are single-domain enzymes which are homologous with the RNase H domain. The group of enzymes is called type I ribonuclease H (RNase HI). It is most likely that the ancestors of RNase HI and the polymerase domain were fused and became contemporary RT. At fusion, amino acid replacements presumably occurred at the interface of the domains to reinforce the interdomain interactions. Such replaced amino acid residues are conserved during evolution of the fused enzyme. We analyzed the pattern of amino acid replacement at each residue site in the free form, RNase HI group, and the integrated form, RNase H domain group. Then we compared the patterns between the two forms. Drastic fitting replacements of amino acid residues occurred at four of 29 residue sites involved in interdomain contact. Hydrophilic amino acid residues of the free form were substituted with hydrophobic or ambivalent ones in the integrated form. These substitutions aid in stabilizing the fused conformation by hydrophobic interactions at the interface of the domains. These observations imply that domain fusion could have occurred with only a relatively small number of adaptive amino acid substitutions.
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PMID:Adaptive amino acid replacements accompanied by domain fusion in reverse transcriptase. 907 Oct 24

Human T cell leukemia/lymphotropic virus (HTLV) is a complex 9 kb human retrovirus with at least eight alternatively spliced mRNAs expressed from the 3' or pX region of the genome. These mRNAs allow for the expression of novel proteins from the previously recognized pX open reading frames I and II in addition to Tax, Rex and p21rex encoded from orf III and IV. These alternatively spliced messages have been detected using reverse-transcriptase polymerase chain reaction (RT/PCR) amplification in HTLV-I-transformed T cell lines as well as in peripheral blood mononuclear cells (PBMC) from infected patients with and without disease. To gain insight into the role of these alternatively spliced mRNAs in pathogenesis, we developed a semi-quantitative non-PCR-based RNase protection assay to detect and quantitate their presence in HTLV-I-infected cells. Analysis of RNA from HTLV-I-infected cells established from patients with adult T cell leukemia (ATL) as well as tropical spastic paraparesis/HTLV-I-associated myelopathy (TSP/HAM) and both IL-2-dependent and IL-2-independent HTLV-I-infected cell lines by RNase protection has confirmed the existence of all of the alternatively spliced messages in each cell line analyzed. However, the relative quantity of each message was significantly different among these lines suggesting that splice site utilization is an important viral regulatory pathway.
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PMID:Differential expression of alternatively spliced pX mRNAs in HTLV-I-infected cell lines. 917 42

In order to study salmon thyroid-stimulating hormone (TSH), we designed a highly specific, sensitive, and rapid RNase protection assay (RPA) for quantification of steady-state levels of salmon TSH beta-subunit mRNA expression. The cDNA encoding the beta-subunit of TSH was isolated from coho salmon pituitary total RNA by reverse-transcriptase PCR, partially sequenced, and used as template for synthesizing a radioactively labeled, sequence-specific, antisense probe, and sense standard for the RPA. This assay, along with a similar RPA previously designed for coho salmon total alpha-subunit mRNA, was used to examine the effects of feeding T3 (0, 10, 100 micrograms/g) and methimazole (a thyroid inhibitor) (2.5 mg/g) on TSH subunit gene expression after 2 and 4 weeks. The low dose of T3 (10 micrograms/g) caused no change in TSH beta mRNA after 2 and 4 weeks and a transient increase in alpha mRNA after 2 weeks, followed by no significant effect after 4 weeks. The high dose of T3 (100 micrograms/g) caused a decrease in TSH beta mRNA after 4 weeks and no change in total alpha mRNA after 2 and 4 weeks. In contrast, methimazole treatment caused significant increases in both TSH beta mRNA (250%) and alpha mRNA (50%) levels after 4 weeks. These findings confirm that, as in mammals, TSH alpha- and beta-subunit expression in teleosts may be differentially regulated by negative feedback from the thyroid hormones.
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PMID:Quantification of salmon alpha- and thyrotropin (TSH) beta-subunit messenger RNA by an RNase protection assay: regulation by thyroid hormones. 920 9

Human eosinophils contain a number of granule proteins for which specific physiological roles remain unclear. The combined ribonucleolytic and membrane disruptive properties of the eosinophil-derived neurotoxin and eosinophil cationic protein, respectively, suggest the possibility that eosinophils might participate in host defense against enveloped single-stranded RNA viruses. To test this hypothesis, stocks of a replication-defective retrovirus encoding the reporter gene beta-galactosidase were pretreated with isolated human eosinophils, then used to transduce human erythroleukemia (K-562) target cells. Histochemical staining for beta-galactosidase activity was used to detect and quantitate the transduced cells. Co-incubation of retrovirus with eosinophils (0.4 x 10[6]/mL) before target cell transduction resulted in a marked decrease in transduction efficiency corresponding to an approximately 20-fold dilution of viral stock (P < 0.01), an effect that was directly proportional to the concentration of eosinophils, and that was reversed in the presence of ribonuclease inhibitor. Reverse transcriptase-polymerase chain reaction analysis demonstrated loss of the retroviral RNA genome as a result of eosinophil pretreatment, indicating that eosinophils are capable of mediating direct ribonucleolytic destruction of the isolated retroviral particles. Our results demonstrate that eosinophils function as effective anti-retroviral agents in vitro via the actions of their secreted ribonucleases, and suggest that eosinophils may represent an unrecognized arm of host defense against enveloped single-stranded RNA viral pathogens.
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PMID:Eosinophils inhibit retroviral transduction of human target cells by a ribonuclease-dependent mechanism. 930 75

The genome of infectious salmon anemia virus (ISAV), which infects farmed Atlantic salmon (Salmo salar L.), is characterized here. The virus has an RNA genome, as shown by using specific DNA virus metabolic inhibitors and radioactive in vivo labeling of ISAV nucleic acid. Electrophoresis of [14C]uridine-labeled ISAV RNA revealed that the ISAV genome is segmented. The genome consists of eight segments that range from 1.0 to 2.3 kb, with a total molecular size of approximately 14.5 kb. One ISAV-specific molecular clone, corresponding to the smallest genome segment, was obtained by cDNA cloning of mRNA from an ISAV-infected cell culture. This clone gave a positive hybridization signal on Northern blots of pelleted ISAV. Pretreatment of the ISAV pellet with RNase A resulted in the disappearance of the positive hybridization signal, demonstrating that the genome is single stranded. Reverse transcriptase PCR with primers corresponding to sequences from the molecular clone and target RNA from ISAV-infected and noninfected fish tissues gave specific positive reactions. Alignments of the nucleotide sequence of the molecular clone did not reveal significant homology with any other available sequence in databases. However, the data presented here, together with morphological and replicational properties previously described, indicate that ISAV has a strong resemblance to members of the Orthomyxoviridae family. This is the first thoroughly characterized orthomyxo-like virus isolated from a teleost.
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PMID:Genomic characterization of the virus causing infectious salmon anemia in Atlantic salmon (Salmo salar L.): an orthomyxo-like virus in a teleost. 931 51


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