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)

We developed a reverse transcription-polymerase chain reaction (RT-PCR) method which permits the simultaneous amplification of several mRNAs, even though their relative levels may be very different. First-strand cDNAs were synthesized from total RNA by MMLV reverse transcriptase with oligo(dT)15 priming. Analysis was performed in the linear phase of PCR, allowing the detection of the products by polyacrylamide gel electrophoresis followed by ethidium bromide staining. In order to obtain a similar amplification of multiple targets in the same PCR system, it was necessary: (i) to adjust the relative concentration of each set of primers and (ii) to use high-stringency conditions (annealing temperature and addition of organic solvent). These conditions allowed the rapid quantitation of several mRNAs in multiple samples, minimizing experimental variations. The reliability of the method was established by measuring variations of c-Ki-Ras, ornithine decarboxylase, alpha-amylase, and beta-actin mRNA levels during the growth of pancreatic tumoral AR4-2J cells. Glyceraldehyde-6-phosphate dehydrogenase expression showed very small variations which confirm that it represents a reliable internal standard for studies of gene expression. Results from competitive PCR amplification of target cDNA and internal competitive template were in agreement with those of simultaneous amplification, validating the quantitative aspect of the method.
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PMID:Quantitation of changes in the expression of multiple genes by simultaneous polymerase chain reaction. 750 50

We report the characterization and cDNA cloning of two alpha-amylase isozymes from larvae of the Western corn rootworm (Diabrotica virgifera virgifera LeConte). Larvae raised on artificial media have very low levels of amylase activity, and much higher levels are found in larvae raised on maize seedlings. At pH 5.7, the optimum pH for enzyme activity, the alpha-amylases are substantially but not completely inhibited by amylase inhibitors from the common bean (Phaseolus vulgaris) and from wheat (Triticum aestivum). Using the reverse transcriptase polymerase chain reaction (RT-PCR), we cloned two cDNAs with 83% amino acid identity that encode alpha-amylase-like polypeptides. Expression of one of the two cDNAs in insect cells with a baculovirus vector shows that this cDNA encodes an active amylase with a mobility that corresponds to that of one of the two isozymes present in larval extracts. The expressed enzyme is substantially inhibited by the same two inhibitors. We also show that expression in Arabidopsis of the cDNA that encodes the amylase inhibitor AI-1 of the common bean results in the accumulation of active inhibitor in the roots, and the results are discussed with reference to the possibility of using amylase inhibitors as a strategy to genetically engineer maize plants that are resistant to Western corn rootworm larvae.
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PMID:cDNA cloning, biochemical characterization and inhibition by plant inhibitors of the alpha-amylases of the Western corn rootworm, Diabrotica virgifera virgifera. 1089 64

Functional analyses of a number of hydrolase gene promoters, induced by gibberellin (GA) in aleurone cells following germination, have identified a GA-responsive complex as a tripartite element containing a pyrimidine box motif 5'-CCTTTT-3'. We describe here that BPBF, a barley (Hordeum vulgare) transcription factor of the DOF (DNA-Binding with One Finger) class, previously shown to be an activator of reserve protein encoding genes during development, also has a role in the control of hydrolase genes following seed germination. Northern-blot, reverse transcriptase-polymerase chain reaction, and in situ hybridization analyses evidenced that the transcripts of the BPBF-encoding gene (Pbf), besides being present during endosperm development, are also expressed in aleurone cells of germinated seeds where they are induced by GA, an effect counteracted by abscisic acid. Electrophoretic mobility shift assays have shown that the BPBF protein binds specifically to the pyrimidine box motif in vitro within the different sequence contexts that naturally occur in the promoters of genes encoding a cathepsin B-like protease (Al21) and a low-isoelectric point alpha-amylase (Amy2/32b), both induced in the aleurone layers in response to GA. In transient expression experiments, BPBF repressed transcription of the Al21 promoter in GA-treated barley aleurone layers and reverted the GAMYB-mediated activation of this protease promoter.
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PMID:A role for the DOF transcription factor BPBF in the regulation of gibberellin-responsive genes in barley aleurone. 1222 91

The enzymatic activities of alpha-amylase and its corresponding messenger RNA levels in developing sea bass (Lates calcarifer) larvae were studied from hatching until 27 days post hatching (dph). An increasing activity of amylase enzyme was measured until 5 dph, and the activity gradually decreased thereafter and reached a constant level by 12 dph. To achieve a better understanding of the molecular mechanisms underlying amylase expression, we have cloned and sequenced a 318-bp fragment of alpha-amylase complementary DNA. Based on this sequence, a real-time reverse transcriptase polymerase chain reaction technique to monitor the changes in the mRNA levels in the larvae was developed. A correlation between enzymatic activity and mRNA level of alpha-amylase could be demonstrated during the early development of sea bass larvae. This suggests that the changes in alpha-amylase are controlled at least at the transcriptional level during early larval development of sea bass.
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PMID:Ontogeny of alpha-amylase gene expression in sea bass larvae (Lates calcarifer). 1496 39

A metabolic engineering approach was exploited to improve growth and protein secretion in the non-conventional yeast, Schwanniomyces occidentalis. Vitreoscilla hemoglobin (VHb) gene was expressed in S. occidentalis under the control of the native alpha-amylase (AMY1) promoter. Expression of VHb was confirmed by reverse transcriptase polymerase chain reaction and Western blot hybridization analysis. Effect of VHb on growth and protein secretion was studied in synthetic medium under both limiting and non-limiting dissolved oxygen conditions. Under both conditions, VHb-expressing cells exhibited higher oxygen uptake and higher specific growth rates. Levels of extracellular alpha-amylase were also elevated in the VHb-transformed strain relative to the control strain. In amylase production medium, VHb-expressing cells showed 3-fold elevated levels of alpha-amylase and a 31% increase in the total secreted protein under oxygen-limiting environment. VHb was found to localize in the mitochondria in addition to its cytoplasmic location. Inhibition of respiration by antimycin A resulted in the loss of the growth-enhancing effects of VHb. A 2.5-fold increase in the cytochrome c oxidase (COX) activity was observed in VHb-expressing cells relative to the control. In addition to this, exogenously added VHb in the assay mixture augmented COX activity.
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PMID:Expression of Vitreoscilla hemoglobin enhances growth and levels of alpha-amylase in Schwanniomyces occidentalis. 1664 33

Full-length starch branching enzyme II (SBE, EC 2.4.1.18) cDNA from mungbean ( Vigna radiata L. cv. Tainan no. 5), VrsbeII, was cloned, characterized, and expressed as an active enzyme in Escherichia coli . Gene-specific primers first amplified an internal cDNA by reverse transcriptase Polymerase Chain Reaction (RT-PCR), followed by obtaining 5' and 3' fragments by RT-PCR and rapid amplification of cDNA ends (RACE). VrsbeII possesses a complete open reading frame (ORF) of 2571 bp, and the deduced polypeptide includes the common catalytic (beta/alpha)(8)-barrel domain and conserved regions of the alpha-amylase family. Phylogenetic analysis classified VrsbeII into SBE family A. Its partial 3D structure and functional features were predicted. VrsbeII has a shorter N-terminal among SBEs; however, two 6 bp (CCAGTT) direct repeat sequences (DRS) were found. A 24 bp shortened VrsbeII at the 3' end, skipping one DRS, was ligated into pET21b vector and expressed as His(6)-rVrSBEII in E. coli BL21 (DE3) cells. The optimal expression condition for rVrSBEII was evaluated and detected by Western blot with a molecular size of 108 kDa and activity of 6.4 U/mg.
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PMID:Cloning, characterization, and expression of mungbean ( Vigna radiata L.) starch branching enzyme II cDNA in Escherichia coli. 1914 23