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)

Copper (Cu) is an essential element required in many biological processes including cellular growth and development. The molecular mechanisms involved in copper homeostasis include proteins that play a role in Cu uptake. Genes encoding high affinity copper transporters (Ctr) have been identified in yeast, plant and mammalian cells. Analysis of copper and zinc content in growing ovarian follicles and ovulated eggs of the reptilian Podarcis sicula demonstrated that the levels of both metals rise during oocyte growth, reaching the maximum in ovulated eggs. By exploiting the remarkable evolutionary conservation of the primary structure of Ctr proteins, cDNA encoding a Ctr was isolated from the liver of the lizard P. sicula by reverse transcriptase PCR and RACE strategy by using primers designed based on consensus motifs present in mammalian Ctr. The predicted protein sequence contains three transmembrane domains and a putative hydrophilic extracellular amino-terminal domain. Besides complementing the respiratory deficiency of yeast cells defective in high affinity Cu transport, expression of lizard Ctr1(1) in Hek293 cells stimulates Cu uptake.Gene expression assessed by Northern blot hybridization of RNA from different tissues of P. sicula shows the highest levels of transcript in both intestine and liver. The profile of Ctr1 mRNA in growing ovarian follicles and eggs demonstrates that the transcript accumulates during the oocyte growth and reaches the highest levels in ovulated eggs. These results suggest that lizard Ctr1 protein may function in Cu acquisition in growing oocytes and eggs.
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PMID:High affinity copper transport protein in the lizard Podarcis sicula: molecular cloning, functional characterization and expression in somatic tissues, follicular oocytes and eggs. 1203 92

Heat shock transcription factors (HSFs) regulate expression of heat shock proteins (Hsps). We have previously shown that in zebrafish a unique isoform, zHSF1b, disappears concomitant with heat shock-induced Hsp70 expression. To characterize the role of zHSF1a and zHSF1b isoforms in the regulation of the stress response in vivo, we have carried out cadmium (10-100 microM) and copper (10-30 microM) exposures in order to specify whether the disappearance of HSF1b is specific for heat stress. After 4-h metal exposures we analyzed the expression of hsp70, zHSF1a, zHSF1b and metallothionein (MT) by reverse transcriptase polymerase chain reaction in zebrafish liver, gonads and gills. Although cadmium is a known inducer of Hsps, it did not affect hsp70 expression significantly in the studied tissues. Induction of hsp70 was observed upon copper exposure in liver and gonads, but not in gills. Neither metal affected the zHSF1a/b ratio. Both cadmium and copper exposure caused upregulation of MT, regulator of metal homeostasis and detoxification, confirming that the tissues were subjected to metal loads. Thus, hsp70 appears to be more weakly induced upon metal exposure than in response to heat shock and HSF1 isoforms may participate in stressor-specific regulation of hsp70.
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PMID:Stressor-dependent regulation of the heat shock response in zebrafish, Danio rerio. 1281 92

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

SUMMARY. Discriminating viable from dead cells is of importance in the development of bacterial detection methods. A positive reverse transcriptase-polymerase chain reaction (RT-PCR) amplification signal was tested as a potential predictor of chick colonization. Some researchers have suggested that the presence of messenger RNA (mRNA) may not correlate with cell viability. Chicken colonization by cells that have positive mRNA signal but that are noncultivable would provide a correlation in cell viability and persistence of mRNA. The role of a viable but noncultivable (VBNC) form of Campylobacter spp. for colonization of poultry could be verified by such an mRNA signal. The levels of four strains of Campylobacter spp., previously isolated from poultry feces, declined progressively over time, and loss of cultivability occurred after 6 to 7 wk incubation in phosphate-buffered saline (PBS) at 4 C. Cold-stored, noncultivable and heat-inactivated (60 C for 10 min) Campylobacter spp. produced inconsistent amplified products from RT-PCR assay, depending on the target transcripts and strains used, although all fresh cultures showed mRNA signals. For the most part, signals of mRNA species from VBNC and heat-killed Campylobacter spp. AH-1, AH-2, and CH-3 persisted. RT-PCR amplification of transcripts originating from the tkt and cmp genes and a 256-base pair amplicon (from a previously described putative haem-copper oxidase) provided consistent signals, whereas transcripts from the flaA gene did not. Presumed VBNC and heat-inactivated Campylobacter spp., which produced positive mRNA signal but was not cultivable by conventional culture-based methods, did not establish colonization in the intestine of chicks 7 days after challenge. These results lead us to question the correlation between mRNA durability with cell viability as well as the significance of the VBNC cells in environmental transmission of Campylobacter spp.
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PMID:Relationship of messenger RNA reverse transcriptase-polymerase chain reaction signal to Campylobacter spp. viability. 1528 12

The conformational space of the most biologically significant backbone folds of a suitable methionine peptide model was explored by density functional computational method. Using a medium [6-31G(d)] and a larger basis set [6-311++G(2d,2p)], the systematic exploration of low-energy backbone structures restricted for the "L-region" in the Ramachandran map of N-formyl-L-methioninamide results in conformers corresponding to the building units of an extended backbone structure (betaL), an inverse gamma-turn (gammaL), or a right-handed helical structure (alphaL). However, no poly-proline II type (epsilonL) fold was found, indicating that this conformer has no intrinsic stability, and highlighting the effect of molecular environment in stabilizing this backbone structure. This is in agreement with the abundance of the epsilonL-type backbone conformation of methionine found in proteins. Stability properties (DeltaE) and distinct backbone-side-chain interactions support the idea that specific intramolecular contacts are operative in the selection of the lowest energy conformers. Apart from the number of different folds, all stable conformers are within a 10 kcal x mol(-1) energy range, indicating the highly flexible behavior of methionine. This conformational feature can be important in supporting catalytic processes, facilitating protein folding and dimerization via metal ion binding. In both of the biological examples discussed (HIV-1 reverse transcriptase and PcoC copper-resistant protein), the conformational properties of Met residues were found to be of key importance. Spatial proximity to other types of residues or the same type of residue seems to be crucial for the structural integrity of a protein, whether Met is buried or exposed.
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PMID:Peptide models XLV: conformational properties of N-formyl-L-methioninamide and its relevance to methionine in proteins. 1561 85

Pentobarbitone sodium (Sodium 5-ethyl-5[1-methylbutyl]-pentobarbitone) is a short-acting barbiturate that is commonly used to euthanase animals. As part of our studies into the molecular genetics of copper toxicosis in Bedlington terrier dogs, reverse-transcription (RT)-PCR was noted to always fail on RNA samples collected from livers of dogs sacrificed by pentobarbitone injection. When samples were collected without pentobarbitone, however, RT-PCR was always successful. We suspected the possible inhibition by pentobarbitone sodium of either reverse transcriptase or Taq polymerase. In vitro studies showed that pentobarbitone interference of PCR occurred at >4 microg/microl. To identify if pentobarbitone produced competitive inhibition, each components (Taq polymerase, MgCl(2), dNTP, etc.) of the PCR was individually altered. However, inhibition still persisted, suggesting that multiple PCR components may be affected. Also it was shown that pentobarbitone interference was not dependent on the PCR product size. Simple dilution of pentobarbitone contaminated DNA solutions, and the addition of bovine serum albumin (BSA) to the PCR mix overcame pentobarbitone interference. In vivo, PCR by pentobarbitone was found to be compounded by high DNA concentration and pentobarbitone contamination. In addition, both high DNA concentration and pentobarbitone contamination could be overcome through dilution and the addition of BSA. Further work is required to quantify pentobarbitone concentration in the liver-extracted DNA and RNA samples before this inhibition effect on PCR can be fully elucidated.
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PMID:The inhibitory effect of pentobarbitone on reverse transcription-PCR. 1565 44

A ribonuclease, with a molecular mass of 9 kDa and an N-terminal sequence resembling the sequence of a fragment of tRNA/rRNA cytosine-C5-methylase and a fragment of a alanyl-tRNA synthetase, was isolated from fresh fruiting bodies of the brown oyster mushroom Pleurotus ostreatus. The ribonuclease was purified using a very simple protocol that comprised ion-exchange chromatography on carboxymethyl (CM)-cellulose and affinity chromatography on Affi-gel blue gel. Subsequent gel filtration by fast protein liquid chromatography on Superdex 75 and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis revealed that the ribonuclease was purified after the first two chromatographic steps. The ribonuclease was adsorbed on CM-cellulose and Affi-gel blue gel. The ribonuclease exhibited the highest activity toward poly A, lower activity toward poly C, slight activity toward poly G, and indiscernible activity toward poly U. The enzyme was stimulated upon exposure to 1 microm Mg2+ and 10 microm Zn2+, but was inhibited by the following ions at 10 mm: Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Mn2+, and Fe3+. The ribonuclease required a pH of 8.0 and a temperature of 50-70 degrees C to express maximal activity. It had a Km of 60 microm toward yeast tRNA. It lacked mitogenic and HIV-1 reverse transcriptase inhibiting activities, but exerted antiproliferative activity toward leukemia L1210 cells.
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PMID:A low-molecular mass ribonuclease from the brown oyster mushroom. 1594 90

Novel metallothionein (MT) complementary DNA and genomic sequences were isolated from a cartilaginous shark species, Scyliorhinus torazame. The full-length open reading frame (ORF) of shark MT cDNA encoded 68 amino acids with a high cysteine content (29%). The genomic ORF sequence (932 bp) of shark MT isolated by polymerase chain reaction (PCR) comprised 3 exons with 2 interventing introns. Shark MT sequence shared many conserved features with other vertebrate MTs: overall amino acid identities of shark MT ranged from 47% to 57% with fish MTs, and 41% to 62% with mammalian MTs. However, in addition to these conserved characteristics, shark MT sequence exhibited some unique characteristics. It contained 4 extra amino acids (Lys-Ala-Gly-Arg) at the end of the beta-domain, which have not been reported in any other vertebrate MTs. The last amino acid residue at the C-terminus was Ser, which also has not been reported in fish and mammalian MTs. The MT messenger RNA levels in shark liver and kidney, assessed by semiquantitative reverse transcriptase PCR and RNA blot hybridization, were significantly affected by experimental exposures to heavy metals (cadmium, copper, and zinc). Generally, the transcriptional activation of shark MT gene was dependent on the dose (0-10 mg/kg body weight for injection and 0-20 microM for immersion) and duration (1-10 days); zinc was a more potent inducer than copper and cadmium.
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PMID:Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and expression analysis. 1597 34

The detection method of reverse transcriptase-polymerase chain reaction (RT-PCR), which specifically targets mRNA, was developed and tested for detection of cultivable Campylobacter spp. The expression of four DNA targets- flaA, tkt, porA, and a putative haem-copper oxidase domain-were assayed in heat-inactivated Campylobacter spp. to determine an optimum target for RT-PCR amplification. A diversity of Campylobacter spp. was tested; however, the presented RT-PCR technique was specific for C. jejuni, C. coli, and C. lari. The durability of mRNA species detected by our RT-PCR technique was dependent upon the individual Campylobacter spp. examined, the condition of heat treatment and post-treatment holding time, as well as the transcript targeted. The putative oxidase was determined to be the most stable mRNA species for this assay. The mRNA of the putative oxidase gene was detectable even after Campylobacter spp. had been treated at temperatures of 95-99 degrees C. Using DNA-based PCR, the four DNA targets could be amplified after heat inactivation followed by a 48 h holding time, indicating that the chromosomal DNA was not substantially degraded by the heat treatment. PCR products from the putative oxidase gene were detected at 10(2) to 10(3) C. jejuni CFU per mL, exhibiting the highest level of sensitivity among the genes tested. The results in the present study indicate that mRNA from Campylobacter spp. may persist in a form that is detectable by RT-PCR amplification for an extended period after heat treatment, demonstrating a poor correlation between mRNA detection and cell cultivability. With these results in mind, further investigations are necessary to determine the correlation between RT-PCR amplification and viability.
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PMID:Heat-treated Campylobacter spp. and mRNA stability as determined by reverse transcriptase-polymerase chain reaction. 1599 7

Three novel laccase isozyme genes, lacA, lacB, and lacC, have been identified from basidiomycete Trametes sp. AH28-2. These genes display a high similarity with other basidiomycete laccases at the amino acid level. An inferred TATA box and several putative CAAT, MRE, XRE, and CreA consensus sequences were identified in the lacA, lacB, and lacC promoter regions. Different from the TATA boxes of lacA and lacB at about -100, the TATA box of lacC is located at -172. For all the isozymes, copper ion is essential for laccase synthesis in Trametes sp. AH28-2. More interestingly, different aromatic compounds can selectively induce the production of distinct laccase isozymes, with o-toluidine inducing the expression of laccase A (LacA) while 3,5-dihydroxytoluene mainly stimulating the production of laccase B (LacB). Quantitative reverse transcriptase-polymerase chain reaction showed that the accumulation of laccase messenger RNA transcripts is accompanied by the increase of corresponding enzyme activity in cultures. The glucose-repression effect on laccase expression in Trametes sp. AH28-2 was also observed. Furthermore, lower Cu2+ concentration (lower than 0.5 mM) can induce LacA and a novel laccase (LacC), and the latter will disappear when Cu2+ concentration is increased up to 1-2 mM. Upon induction by 3,5-dihydroxytoluene, the ratio of LacA to LacB decreased in the later phase of induction.
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PMID:Cloning of novel laccase isozyme genes from Trametes sp. AH28-2 and analyses of their differential expression. 1628 98


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