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)

There are various techniques to detect mRNA in tissue specimens. Among these in situ hybridization is widely applied, and for the detection of small quantities of RNA in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR) has been applied. Furthermore in situ transcription, where signal is produced by direct incorporation of labeled nucleotides during production of a cDNA by reverse transcription, has been shown by a few investigators. We present a non-radioactive in situ reverse transcriptase (in situ RT) protocol which is at least as sensitive as in situ hybridization but avoids probe production and long procedures of preincubation, incubation, and washing. Digoxigenin-labeled UTP is incorporated into a cDNA produced by in situ reverse transcription of mRNA. This method is combined with the fast and sensitive immunogold-silver detection system allowing demonstration of the mRNA within 7 h compared to days in the case of in situ hybridization. Contrary to in situ RT-PCR this new method of in situ RT has no background problems due to non-specific amplification or diffusion of the reaction product.
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PMID:Detection of mRNA by non-radioactive direct primed in situ reverse transcription. 1168 47

Silver compounds are used as antimicrobial agents in medicine and bacteria that develop resistance to silver cations (Ag(+)) pose problems similar to those of antibiotic-resistant bacteria. The first set of Ag(+) resistance genes (sil) was from plasmid pMG101, now assigned to the IncHI incompatibility group. Questions of whether sil genes are unique to pMG101 or are more widely found, and whether they are associated with a specific incompatibility group or occur in many plasmid groups and on bacterial chromosomes were addressed. sil genes were identified in five IncH plasmids, but not in plasmids of the IncP incompatibility group. Three sil genes (silP, silR and silE) from these plasmids were PCR-amplified, cloned, sequenced and compared to those of pMG101. Differences of 0-50 nt per kb of sequence were found. Predicted gene products were 0-6% different in amino acid sequence, but the differences did not alter residues thought to be involved in protein function (see supplementary data at http://mic.sgmjournals.org or http://www.uic.edu/depts/mcmi/individual/gupta/index.htm). For representative IncH plasmid R476b and pMG101 the effects of Ag(+) exposure on resistance levels were measured by growth. The inducibility of silC, silR and silE gene expression after Ag(+) exposure was studied by reverse transcriptase (RT)-PCR. Silver resistance increased after Ag(+) exposure for strains carrying plasmid R476b. silC and silE expression from R476b was inducible after Ag(+) exposure and was constitutive and high from pMG101. The mRNA levels for the regulatory gene silR was constitutive for both pMG101 and R476b. Close homologues for silABC(ORF96)RS from pMG101 are clustered on the chromosomes of Escherichia coli strains K-12 and O157:H7, without contiguous silP and silE homologues. Insertion deletions of the E. coli K-12 chromosomal homologues for silA and silP gave Ag(+) hypersensitivity for growth. The silA homologue knockout was complemented back to wild-type resistance by the same gene cloned on a plasmid. Homologues of sil genes have also been identified on other enterobacterial genomes.
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PMID:Diversity of silver resistance genes in IncH incompatibility group plasmids. 1173 72

The purpose of this investigation was to determine the role played by pretranslational events in the decreased rate of myosin heavy-chain (MyHC) protein synthesis in old age. It was hypothesized that the decreased rate of MyHC protein synthesis reported in the elderly population is, at least in part, related to lower MyHC messenger RNA (mRNA) in old age. MyHC protein expression and mRNA levels for the three MyHC isoforms expressed in human muscle, that is, types I, IIa, and IIx/d, were measured in percutaneous vastus lateralis muscle biopsies from 16 young and 16 old healthy men. The MyHC isoform mRNA content was determined by quantitative, real-time reverse transcriptase polymerase chain reaction, and it was normalized to 18S ribosomal RNA; the relative MyHC protein isoform content was measured on silver-stained 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. The old men demonstrated signs of sarcopenia, such as loss of muscle force, a trend toward a loss in lean body mass, and an increased percentage of body fat. Statistically significant correlations were observed between the isoform expression of different MyHCs at the protein and mRNA levels. However, the expression of the different MyHC isoforms at the mRNA and protein levels did not differ between the young and old men. Thus, the present results do not support the hypothesis that pretranslational events in MyHC protein synthesis are playing a significant role in the development of sarcopenia.
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PMID:Effects of aging on human skeletal muscle myosin heavy-chain mRNA content and protein isoform expression. 1202 59

Metallocarboxypeptidase D (CPD), is a 180-kDa protein that contains three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail and which functions in the processing of proteins that transit the secretory pathway. An initial report on the Drosophila melanogaster silver gene indicated a CPD-like protein with only two and a half carboxypeptidase-like domains with no transmembrane region (Settle, S. H., Jr., Green, M. M., and Burtis, K. C. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9470-9474). A variety of bioinformatics and experimental approaches were used to determine that the Drosophila silver gene corresponds to a CPD-like protein with three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail. In addition, two alternative exons were found, which result in proteins with different carboxypeptidase-like domains, termed domains 1A and 1B. Northern blot, reverse transcriptase PCR, and sequence analysis were used to confirm the presence of the various mRNA forms. Individual domains of Drosophila CPD were expressed in insect Sf9 cells using the baculovirus expression system. Media from domain 1B- and domain 2-expressing cells showed substantial enzymatic activity, whereas medium from domain 1A-expressing cells was no different from cells infected with wild-type virus. Domains 1B and 2 were purified, and the enzymatic properties were examined. Both enzymes cleaved substrates with C-terminal Arg or Lys, but not Leu, and were inhibited by conventional metallopeptidase inhibitors and some divalent cations. Drosophila domain 1B is more active at neutral pH and greatly prefers C-terminal Arg over Lys, whereas domain 2 is more active at pH 5-6 and slightly prefers C-terminal Lys over Arg. The differences in pH optima and substrate specificity between Drosophila domains 1B and 2 are similar to the differences between duck CPD domains 1 and 2, suggesting that these properties are essential to CPD function.
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PMID:Characterization of Drosophila carboxypeptidase D. 1239 82

To ascertain some of the important biochemical and molecular events that take place during early larval development of silver sea bream (Sparus sarba), we undertook a study of changes in the morphology as well as the ontogeny of the RNA-DNA ratio, growth hormone (GH), insulin-like growth factor I (IGF-I) messenger RNA abundance, Na(+)-K(+)-ATPase subunit mRNA abundance, and Na(+)-K(+)-ATPase enzyme activity. Larvae samples were collected at 1 to 46 days posthatch (dph). At 7 dph the yolk sac was fully absorbed, and from 28 dph onward larvae underwent rapid developmental changes to the juvenile stage. The RNA-DNA ratio was highest at 1 dph, decreased to low levels between 7 and 21 dph, then increased by 28 dph, and then again by 46 dph. The ontogenetic profiles of GH, IGF-I, and Na(+)-K(+)-ATPase alpha1 and beta1 subunits were studied using reverse transcriptase polymerase chain reaction, coupled with radioisotope hybridization of immobilized DNA. Growth hormone abundance reached a constant and high level from 35 dph onward, whereas the IGF-I level reached a peak at 35 dph and then significantly decreased. Both Na(+)-K(+)-ATPase alpha1 and beta1 subunit mRNAs increased up to 35 dph, however, at 46 dph the alpha1 subunit remained high whereas the beta1 subunit decreased. Na(+)-K(+)-ATPase activity was low in 1-dph larvae but increased rapidly as development progressed. The importance of these findings is discussed within the context of larval development.
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PMID:Larval development of silver sea bream (Sparus sarba): ontogeny of RNA-DNA ratio, GH, IGF-I, and Na(+)-K(+)-ATPase. 1292 22

Trophoblast cell migration is unusual in epitheliochorial placentae but occurs in placentomes of cows as "restricted" trophoblast invasion of binucleated trophoblast giant cells (TGC). Migration may be induced by integrin binding to the extracellular matrix initiating two pathways: (1) conformational changes of the actin cytoskeleton induced by an accumulation of its associated proteins and (2) integrin-dependent phosphorylation of various protein kinases. In cow placentomes, actin, its associated proteins (alpha-actinin, vinculin) and a key protein kinase of the signal transduction cascade (phosphorylated mitogen-activated protein kinase, pMAPK) were localized by immunogold-silver enhancement and immunoperoxidase staining at the light- and transmission electron-microscopical levels. Findings were confirmed by amplification of specific mRNA transcripts by reverse transcriptase/polymerase chain reaction. Actin and alpha-actinin were co-localized apically in mononuclear trophoblast cells, along the cytoplasmic membrane of TGC and apically in maternal crypt cells. The actin and alpha-actinin immunoreaction occurred as a band of electron-dense particles beneath the cytoplasmic membrane. Vinculin labelling was membrane-associated in TGC and in fetal and maternal endothelial cells. MAPK was observed as nuclear clusters in both kinds of trophoblast cells and was less dense in single uterine epithelial cells. Most MAPK immunoreactivity was detected in the nuclei of the trophoblast epithelium but was also sometimes membrane-associated in the cytoplasm. Thus, actin, alpha-actinin, MAPK and vinculin may be involved in the regulation of TGC migration. "Restricted" trophoblast invasion could serve as a model for invasive processes.
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PMID:Cytoskeletal filaments and associated proteins during restricted trophoblast invasion in bovine placentomes: light and transmission electron microscopy and RT-PCR. 1472 75

RNA was extracted from spleens of diarrhea and non-diarrhea piglets of Jiuyang Pig, Jianbai Pig and Landrace. RNA pools were established and DDRT-PCR using three anchored and seven arbitrary primers combined with silver staining was conducted to identify ESTs differentially expressed in diarrhea resistance to E. coli K88. Five cDNA were identified in the non-diarrhea RNA pools. Among them two are new sequences and three are highly identical to the ESTs in the GeneBank, of which two have known functions. One is the mammalian Nck adaptor protein 1 and the other is the human LINE-1 reverse transcriptase.
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PMID:Screening specifically expressed ESTs related to piglet diarrhea resistance. 1519 63

The aim of this work was to identify a new protein that discriminated CD8 from CD4 and CD19 lymphocytes. Proteins were separated by high-resolution two-dimensional electrophoresis. After silver staining, the gel images were captured with a laser densitometer, and studied with a dedicated software. This study confirmed the presence of two spots that appeared to be preferentially associated with CD8 lymphocytes, and mass spectrometry analyzes (liquid chromatography-tandem mass spectrometry, LC-MS/MS) identified six peptides for one spot and four for the other. The peptide sequences corresponded to an unknown protein that we named swiprosin 1 (Swiss-Prot Q96C19). Molecular analysis (reverse transcriptase-polymerase chain reaction, RT-PCR) and Northern blots confirmed that the gene expression was increased in purified populations of CD8 lymphocytes, when compared to CD19 and CD4 lymphocytes. Database mining revealed that swiprosin 1 contains two potential EF-hand domains, and therefore may have a role in calcium signaling. Its predominant presence in CD8 lymphocytes suggests that it may be involved in functions that are important for cytotoxic lymphocytes.
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PMID:Identification of swiprosin 1 in human lymphocytes. 1527 14

In order to elucidate the mechanism of fungal heterokayosis, a wild type strain of Fusarium oxysporum f. sp. vasinfectum was isolated from the cotton field in Anyang, Henan Province. Through single hyphal-tip isolation, a heterokaryon, Ag149, was obtained, and its two different phenotypic segregants, Ag149-I and Ag149-III, were separated from the mutated sectors on colony of the heterokaryon. They have remarkable differences on color of colony, morphology of hypha and pathogenicity. After analyzing by RAPD on their nuclear DNA with 100 random primers, no polymorphic difference was found among them. On going to find the different expressed gene, the mRNA differential display method was performed. Two kinds of reverse transcriptase AMV and MMLV, and a kit which consists of three kinds of 3' terminal anchor primers and eight kinds of 5' terminal arbitrary primers were used in differential display PCR (DD-PCR). Total RNA as template was reverse transcribed into corresponding cDNA by 3' terminal anchor primers, and the cDNA were amplified by polymerase chain reaction with a set of one same 3' anchor primer and one 5' arbitrary primer. The PCR products were then resolved on denaturing polyacylamimide gel, and the cDNA bands were visualized by silver staining. Among the 144 PCR products, 19 differentially expressed cDNA fragments ranged from 300 bp to 700 bp were purified. All of them were ligated to pGEM-T vector respectively for sequencing and Rev-Northern blotting. Two cDNA fragments (G5 and C6) were observed to be positive after Rev-Northern blotting. The C6 was highly expressed in the heterokaryon Ag149 and its segregant Ag149-I. It is 564 bp in length and can be predicted 77 amino acids from the beginning of the 3rd to 233th base, and then searched from GenBank. The amino acid sequence of C6 shared homologies with the 6th subunit of NADH dehydrogenase found in some bacteria, plants and animals at 30% - 70% level. While the G5 was highly expressed in Ag149 and its segregant Ag149-III. It is 432 bp in length and can be predicted 101 amino acids from the beginning of the 2nd to 304th base, and the amino acid sequence is 35% homologies comparing with the tetracycline efflux protein (OtrB) of Streptomyces rimosus. We also checked these two kinds of the pGEM-T vector harboring cDNA fragments (C6 and G5) by Southern blotting with their nuclear DNA and mitochondrial DNA (mtDNA) separately. The positive identification signals only appeared from nuclear DNA,and it addressed that C6 and G5 locate on their nuclear genome. The result indicated that the difference between the heterokaryon and its segregants is distinct on gene transcriptional level. Thus a molecular evidence for the formation of heterokaryon in filamentous fungi was provided.
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PMID:[Transcriptional differences between a heterokaryon and its segregants of Fusarium oxysporum f. sp. vasinfectum]. 1547 7

In the present study, the gene encoding hepatic glucose-6-phosphate dehydrogenase (G6PDH) was cloned and characterized from silver sea bream (Sparus sarba). The deduced amino acid sequence from sea bream g6pdh shared 78-84% homology with deduced amino acid sequences from previously cloned teleost g6pdh genes. A reverse transcriptase polymerase chain reaction (RT-PCR) coupled with radioisotope hybridization method was used for assessment of g6pdh expression and it was found that administration of growth hormone to sea bream increased g6pdh transcript and G6PDH activity whereas injections of somatostatin decreased both of these parameters. It was also found that sea bream maintained at an isoosmotic salinity (12 ppt) and cold temperature (12 degrees C) displayed upregulated g6pdh expression and enhanced G6PDH activtity. The results from this study demonstrate that g6pdh expression can be mediated by both hormonal and environmental factors in teleosts.
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PMID:Expression studies on glucose-6-phosphate dehydrogenase in sea bream: effects of growth hormone, somatostatin, salinity and temperature. 1601 52


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