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)

The complete amino acid sequence of the fungus Syncephalastrum racemosum (Sr-) nuclease has been delineated on the basis of protein sequencing of the intact protein and its protease-digested peptides. The resulting 250-residue sequence shows a carbohydrate side chain attached at Asn134 and two half-cystine residues (Cys242 and Cys247) cross-linked to form a small disulphide loop. On the basis of the sequence of Sr-nuclease, a computer search in the sequence database yielded 60% and 48% positional identities with the sequences of Cunninghamella echinulata nuclease C1 and yeast mitochondria nuclease respectively, and very little similarity to those of several known mammalian DNases I. Sequence alignment of the three similar nucleases reveals that the single small disulphide loop is unchanged but the carbohydrate attachment in Sr-nuclease is absent from the other two nucleases. Alignment also shows a highly conserved region harbouring Sr-nuclease His85, which is assigned as one of the essential residues in the active site. The cDNA encoding Sr-nuclease was amplified by using reverse transcriptase-mediated PCR with degenerate primers based on its amino acid sequence. Subsequently, specific primers were synthesized for use in the 3' and 5' rapid amplification of cDNA ends (RACE). Direct sequencing of the RACE products led to the deduction of a 1.1 kb cDNA sequence for Sr-nuclease. The cDNA contains an open reading frame of 320 amino acid residues including a 70-residue putative signal peptide and the 250-residue mature protein. Finally, the recombinant Sr-nuclease was expressed in Escherichia coli strain BL21(DE3) in which the recombinant protein, after solubilization with detergent and renaturation, showed both DNase and RNase activities. The assignment of His85 to the active site was further supported by evidence that the mutant protein Sr-nuclease (H85A), in which His85 was replaced by Ala, was not able to degrade DNA or RNA.
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PMID:Protein structure and gene cloning of Syncephalastrum racemosum nuclease. 1019 Dec 56

A nested reverse transcriptase (RT) PCR assay detected mRNA of the salmonid pathogen Renibacterium salmoninarum in samples of RNA extracts of between 1 and 10 cells. Total RNA was extracted from cultured bacteria, Atlantic salmon (Salmo salar L.) kidney tissue and ovarian fluid seeded with the pathogen, and kidney tissue from both experimentally challenged and commercially raised fish. Following DNase treatment, extracted RNA was amplified by both RT PCR and PCR by using primers specific for the gene encoding the major protein antigen of R. salmoninarum. A 349-bp amplicon was detected by polyacrylamide gel electrophoresis and silver stain. Inactivation of cultured bacteria by rifampin or erythromycin produced a loss of nested RT PCR mRNA detection corresponding to a loss of bacterial cell viability determined from plate counts but no loss of DNA detection by PCR. In subclinically diseased fish, nested RT PCR identified similar levels of infected fish as determined by viable pathogen culture. Higher percentages of fish testing positive were generated by PCR, particularly in samples from fish previously subjected to antibiotic chemotherapy where 93% were PCR positive, but only 7% were nested RT PCR and culture positive. PCR can generate false-positive data from amplification of target DNA from nonviable pathogen cells. Therefore, nested RT PCR may prove useful for monitoring cultured Atlantic salmon for the presence of viable R. salmoninarum within a useful time frame, particularly samples from broodstock where antibiotic chemotherapy is used prior to spawning to reduce vertical pathogen transmission.
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PMID:A sensitive nested reverse transcriptase PCR assay to detect viable cells of the fish pathogen Renibacterium salmoninarum in Atlantic salmon (Salmo salar L.). 1038 1

Tissue kallikrein (TK) and kinin receptors have been immuno-localized in various areas of the human nervous system, suggesting that the kallikrein-kinin system (KKS) may be functionally active in the brain. The aim of this study was to determine the cellular expression of TK and kinin B1 and B2 receptor mRNAs in specific regions of the human brain by in situ reverse transcriptase polymerase chain reaction. Autopsy samples of the brain, spinal cord, kidney and salivary gland were embedded in paraffin. Sections (5 microm), adhered onto silane coated glass slides, were treated with Proteinase K and DNase, followed by reverse transcription polymerase chain reaction with specific KKS primers and digoxigenin-dUTP. Detection of the digoxigenin-label demonstrated localization of TK, B1 and B2 mRNAs in the cytoplasm of some neuronal cell bodies in the hypothalamus, thalamus, frontal cortex and spinal cord. TK mRNA was also observed in the ependymal cells lining the cerebral ventricles and epithelial cells of the choroid plexus. In the choroid plexus, only B1 gene expression was observed in some choroidal epithelial cells while no B2 labeling was detected. The identification of mRNAs to TK, B1 and B2 kinin receptors in human nervous tissue supports previous evidence for the presence of the KKS in the brain and confirms localized protein synthesis.
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PMID:Detection of tissue kallikrein and kinin B1 and B2 receptor mRNAs in human brain by in situ RT-PCR. 1138 57

In this work, we characterize genes in Mycobacterium tuberculosis that are regulated by IdeR (iron-dependent regulator), an iron-responsive DNA-binding protein of the DtxR family that has been shown to regulate iron acquisition in Mycobacterium smegmatis. To identify some of the genes that constitute the IdeR regulon, we searched the M. tuberculosis genome for promoter regions containing the consensus IdeR/DxR binding sequence. Genes preceded by IdeR boxes included a set encoding proteins necessary for iron acquisition, such as the biosynthesis of siderophores (mbtA, mbtB, mbtI), aromatic amino acids (pheA, hisE, hisB-like) and others annotated to be involved in the synthesis of iron-storage proteins (bfrA, bfrB). Some putative IdeR-regulated genes identified in this search encoded proteins predicted to be engaged in the biosynthesis of lipopolysaccharide (LPS)-like molecules (rv3402c), lipids (acpP) and peptidoglycan (murB). We analysed four promoter regions containing putative IdeR boxes, mbtA-mbtB, mbI, rv3402c and bfrA-bfd, for interaction with IdeR and for iron-dependent expression. Gel retardation experiments and DNase footprinting analyses with purified IdeR showed that IdeR binds to these IdeR boxes in vitro. Analysis of the promoters by primer extension indicated that the IdeR boxes are located near the -10 position of each promoter, suggesting that IdeR acts as a transcriptional repressor by blocking RNA polymerase binding. Using quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) coupled to molecular beacons, we showed that mRNA levels of mbtA, mbtB, mbtI, rv3402c and bfd are induced 14- to 49-fold in cultures of M. tuberculosis starved for iron, whereas mRNA levels of bfrA decreased about threefold. We present evidence that IdeR not only acts as a transcriptional repressor but also functions as an activator of bfrA. Three of the IdeR- and iron-repressed genes, mbtB, mbtI and rv3402c, were induced during M. tuberculosis infection of human THP-1 macrophages.
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PMID:The Mycobacterium tuberculosis IdeR is a dual functional regulator that controls transcription of genes involved in iron acquisition, iron storage and survival in macrophages. 1172 47

Endothelins (ETs) are a family of potent vasoconstrictor and comitogenic polypeptides consisting of 21-amino acids. Using in situ hybridization, ET-1 mRNA has previously been localized to neuronal cell bodies in fourteen human brain regions. However, because in situ hybridization has a limited detection sensitivity of 20 mRNA copies per cell, ET-1 mRNA may be present in previously undetected areas. Hence, our objective was to localize ET-1 mRNA in specific human brain regions and astrocytic tumours using the more sensitive in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR). Human brain autopsy tissue and surgical cerebral tumour tissue were treated with proteinase K and DNase, followed by RT-PCR using primers specific for ET-1 mRNA and digoxygenin-labelled dUTP in the PCR mixture. The DIG-dUTP was localized with an immunodetection system. We demonstrate ET-1 mRNA labelling in twenty two of the twenty four brain regions studied including those regions in which ET-1 mRNA has been observed by in situ hybridization. In addition, the localization of ET-1 mRNA observed in astrocytomas suggests a role for ET-1 in tumour pathogenesis. In situ RT-PCR has proven to be highly sensitive in its ability to detect low mRNA expression at the cellular level. Our results confirm a role for ET-1 in the human nervous system.
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PMID:Cellular distribution of endothelin-1 mRNA in human brain by in situ RT-PCR. 1176 33

We showed that a U5-U3 junction was reproducibly detected by a PCR assay as early as 1 to 2 h postinfection with a DNase-treated murine leukemia virus (MLV)-containing supernatant in aphidicolin-arrested NIH 3T3 cells, as well as in nonarrested cells. Such detection is azidothymidine sensitive and corresponded to neosynthesized products of the reverse transcriptase. This observation was confirmed in two additional human cell lines, TE671 and ARPE-19. Using cell fractionation combined with careful controls, we found that a two-long-terminal-repeat (two-LTR) junction molecule was detectable in the cytoplasm as early as 2 h post virus entry. Altogether, our data indicated that the neosynthesized retroviral DNA led to the early formation of structures including true two-LTR junctions in the cytoplasm of MLV-infected cells. Thus, the classical assumption that two-LTR circles are a mitosis-dependent dead-end product accumulating in the nucleus must be reconsidered. MLV-derived products containing a two-LTR junction can no longer be used as an exclusive surrogate for the preintegration complex nuclear translocation event.
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PMID:Early detection of a two-long-terminal-repeat junction molecule in the cytoplasm of recombinant murine leukemia virus-infected cells. 1516 12

Two antifungal peptides (designated alpha- and beta-basrubrins) with molecular masses of 4-5 kDa and distinct N-terminal sequences, and a peptide and a protein with N-terminal sequences resembling heat shock protein (hsp) and serine-threonine kinase, respectively, were isolated from seeds of the Ceylon spinach Basella rubra. The purification procedure entailed saline extraction, (NH4)2SO4 precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and FPLC-gel filtration on a Superdex peptide column. alpha- and beta-basrubrins inhibited mycelial growth in Botrytis cirerea with an IC50 value of 7.5 and 14.7 microM, respectively, Mycosphaerella arachidicola with an IC50 of 12.4 and 6.9 microM, and Fusarium oxysporum with an IC50 of 5.8 and 6.2 microM. Neither alpha-basrubrin nor beta-basrubin exhibited DNase, RNase, lectin or protease activity, indicating that their antifungal action is not due to these activities. HIV-1 reverse transcriptase was inhibited by alpha- and beta-basrubrins with an IC50 of 246 and 370 microM, respectively. Translation in rabbit reticulocyte lysate was inhibited by alpha- and beta-basrubrins with an IC50 of 400 and 100 nM. The heat shock protein-like peptide and serine-threonine kinase-like protein exhibited a molecular mass of 3 and 30 kDa, respectively. They inhibited neither translation in a rabbit reticulocyte system at concentrations up to 50 microM nor HIV-1 reverse transcriptase activity at concentrations up to 400 microM. They did not exert antifungal activity toward B. cinerea, M. arachidicola, and F. oxysporum when tested up to 16 microg. None of the aforementioned proteins demonstrated DNase, RNase, protease or lectin activity.
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PMID:Antifungal peptides, a heat shock protein-like peptide, and a serine-threonine kinase-like protein from Ceylon spinach seeds. 1524 82

Oligonucleotides can mediate sequence-specific gene modification that results in the correction and/or alteration of genomic DNA. There is evidence to suggest that the polymerase chain reaction (PCR)-based analytical methods usually used to analyze oligonucleotide-mediated modification can generate artifacts. To investigate the conditions under which a PCR artifact can be generated and eliminated when analyzing small fragment homologous replacement (SHFR)-mediated modification, cells homozygous for the DeltaF508 mutation (CFBE41o-) were mixed with small DNA fragments (SDFs) containing the wild-type CFTR (wt-CFTR) sequence. An artifact could be generated after wild-type allele-specific PCR (wtAS-PCR) if the genomic DNA was not gel purified. Without gel purification, the amount of SDF/cell required to generate the artifact was dependent to the AS primer pairs used. When the genomic DNA was gel purified, no artifact could be detected with any of the wtAS-PCR primers whether the SDF was mixed with the cells or transfected into the cells. Furthermore, treatment of cellular mRNA with DNase was sufficient to eliminate potential artifacts in the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Thus, it is critical to gel purify genomic DNA and DNase treat mRNA when analyzing SFHR-mediated modification by PCR.
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PMID:Gel purification of genomic DNA removes contaminating small DNA fragments interfering with polymerase chain reaction analysis of small fragment homologous replacement. 1715 12

The freshwater prawn Macrobrachium rosenbergii was experimentally infected with white spot syndrome virus (WSSV) by intramuscular injection. Infection was confirmed by positive, single-step, WSSV polymerase chain reaction (PCR) assays targeting the VP28 gene from Day 2 up to Day 90 post injection (p.i.). Although no mortality of WSSV-infected prawns was observed, bioassays with the black tiger shrimp Penaeus monodon using hemolymph from Day 90 PCR-positive prawns resulted in white spot disease (WSD). Transcriptional analysis of the VP28 gene of WSSV by reverse transcriptase (RT)-PCR assays and Western blot assays revealed transient expression of the VP28-specific transcript in DNase-treated total RNA from hemolymph, gills, head soft tissue and eyestalks at 2 d p.i. By 3 d p.i., the VP28 transcript could no longer be detected in eyestalks and hemolymph but was still lightly detectable in head soft tissue and gills. It became undetectable there from 5 d p.i. onwards, despite the undiminished presence of the virus shown by single-step PCR targeting of the VP28 gene. VP28 was not detected by the less sensitive Western blot in hemolymph at any time during the study period, but it was detectable in all other tested tissues from Days 2 to 4 p.i. Our results demonstrated that M. rosenbergii is tolerant to a relatively constant level of persistent WSSV infection characterized by a low expression of VP28 and, possibly, other virion proteins. Despite this, M. rosenbergii can carry a level of infectious WSSV sufficient to represent a feasible threat to cultivated penaeid shrimp such as P. monodon. It remains to be seen whether a very low virion protein expression relative to the viral copy number may constitute a general decapod characteristic for persistent viral infections that produce no signs of disease.
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PMID:Tolerance to white spot syndrome virus (WSSV) in the freshwater prawn Macrobrachium rosenbergii is associated with low VP28 envelope protein expression. 1733 Jul 38

Feline allergic skin disease is thought to be associated with dermal infiltration of Th(2) lymphocytes and synthesis of associated cytokines. In this study, real-time RT-PCR assays were developed to measure feline interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10, IL12 (p35 and p40), IL-18, tumour necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in the skin of healthy control cats, and in the lesional and non-lesional skin of cats with allergic skin disease. Total RNA was extracted from skin biopsies using the RNeasy Mini Kit with on-column and in-solution DNase digestion steps. cDNA was synthesised using Improm-II reverse transcriptase and random hexamers. Real-time PCR was carried out using an iCycler IQ system (Bio-Rad), and gene-specific primers were designed to span an exon/exon junction of each cytokine gene. Taq-man probes were used to add specificity to the system. Messenger RNA from the housekeeping gene GAPDH was used for normalisation of the cytokine threshold cycle. The eleven cytokine mRNA transcripts quantified were present at varying levels, but there was no apparent difference in expression between normal, non-lesional and lesional skin. TGF-beta represented the most abundant transcript while IL-4, IL-5, IL-6, IL-10, IL-12, IL-18 and TNF-alpha were present at levels approximately 1000-fold less. IL-2 and INF-gamma represented the least abundant templates with no detectable copies in most RNA samples. This quantitative analysis of cytokine mRNA expression in feline skin biopsies has suggested that there is not a simple Th(2) bias in lesional skin of cats with allergic dermatopathies.
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PMID:Quantitative real-time RT-PCR measurement of cytokine mRNA expression in the skin of normal cats and cats with allergic skin disease. 1819 Dec 30


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