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)

Rabies virus M protein was expressed in Escherichia coli in the form of a fusion protein with maltose binding protein (MBP) and purified by amylose affinity column chromatography after extraction. In order to investigate the possible regulatory role of M protein in viral transcription, an assay system for rabies virion-associated transcriptase activity was established by using the ribonucleoprotein (RNP) cores prepared from purified virions. Analysis of the products of the transcription assay system showed that the products are sensitive to RNase and are positive-strand RNA. Addition of the fusion protein to the system after cleavage with a proteinase Factor Xa (FXa), which cleaves the fusion protein into the M protein and MBP, resulted in an efficient and dose-dependent inhibition of the transcription. Furthermore, addition to the system of anti-M protein monoclonal antibody significantly restored the transcription. Control experiments with the same transcription assaying system using rabies virus nucleoprotein expressed as a fusion protein with MBP and cleaved with FXa did not result in an inhibition of the transcription. These results suggest that the M protein of rabies virus has the property to down-regulate virion-associated transcription.
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PMID:Rabies virus M protein expressed in Escherichia coli and its regulatory role in virion-associated transcriptase activity. 864 3

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

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

Cucumber mosaic virus (CMV) is an icosahedrion plant virus and contains three different single-stranded positive sense genomic RNAs. The very 3' ends of each of the genomic RNAs can fold into a tRNA-like structure. Based on the structural analysis of the 3' tRNA-like structure of the brome mosaic virus (BMV), we superimposed and redrew the 3' tRNA-like structure of CMV. We homogenized virus infected or healthy tobacco leaves with polytron and carried out low speed centrifugation twice and ultra-centrifugation three times to get detergent solubilized membrane bound fractions. We accidentally found that these fractions were enriched with a host-encoded RNA-dependent RNA polymerase (RdRp) activity. Similar activity could also be found in other plants tested. Alternately, the membrane bound fraction could be simply precipitated by low speed centrifugation (3,000 g) and high speed ultra-centrifugation (40,000 g). The pellet was then suspended in a detergent-containing buffer, after which 25%-55% glycerol gradient fractionation was performed. Activity was tested through the incorporation of [alpha-32P]UTP using endogenous CMV RNAs as templates on each fraction collected. It was found that most of the fractions contained the viral-encoded RNA-dependent RNA polymerase. The products of RdRp reaction were found to have a double-stranded from through further analysis of the RNase protection assay.
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PMID:The preparation of RNA-dependent RNA polymerase complex from virus infected plants. 961 71

Some foreign genes introduced into plants are poorly expressed, even when transcription is controlled by a strong promoter. Perhaps the best examples of this problem are the cry genes of Bacillus thuringiensis (B.t.), which encode the insecticidal proteins commonly referred to as B.t. toxins. As a step toward overcoming such problems most effectively, we sought to elucidate the mechanisms limiting the expression of a typical B.t.-toxin gene, cryIA(c), which accumulates very little mRNA in tobacco (Nicotiana tabacum) cells. Most cell lines transformed with the cryIA(c) B.t.-toxin gene accumulate short, polyadenylated transcripts. The abundance of these transcripts can be increased by treating the cells with cycloheximide, a translation inhibitor that can stabilize many unstable transcripts. Using a series of hybridizations, reverse-transcriptase polymerase chain reactions, and RNase-H-digestion experiments, poly(A+) addition sites were identified in the B.t.-toxin-coding region corresponding to the short transcripts. A fourth polyadenylation site was identified using a chimeric gene. These results demonstrate for the first time to our knowledge that premature polyadenylation can limit the expression of a foreign gene in plants. Moreover, this work emphasizes that further study of the fundamental principles governing polyadenylation in plants will have basic as well as applied significance.
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PMID:Premature polyadenylation at multiple sites within a Bacillus thuringiensis toxin gene-coding region. 970 99


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