Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The adaptation and application of the Escherichia coli T7 RNA polymerase system for regulated and promoter-specific gene expression in Bacillus subtilis is reported. The expression cassette used in Bacillus subtilis was tightly regulated and T7 RnA polymerase (T7 RNAP)appeared 30 minutes after induction. The efficiency of T7 promoter-specific gene expression in B.subtilis was studied using one secretory and two cytosolic proteins of heterologous origin. The accumulation of E. coli beta-galactosidase, as well as a 1,4-beta-glucosidase from Thermoanaerobacter brockii in B. subtilis after T7 RNAP induction was strongly enhanced by rifampicin inhibition of host RNAP activity. The alpha-amylase of Thermactinomyces vulgaris, a secretory protein, was found to accumulate in the culture supernatant up to levels of about 70 mg/l 10-20 h after T7 RNAP induction, but was also deposited in cellular fractions. The addition of rifampicin inhibited chi-amylase secretion, but unexpectedly, after a short period, also prevented its further (intra)cellular accumulation.
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PMID:A T7 promoter-specific, inducible protein expression system for Bacillus subtilis. 862 23

The cytosolic beta-glucosidase (EC 3.2.1.21) present in the livers of mammalian species is distinguished by its broad specificity for sugars and its preference for hydrophobic aglycones. We purified the cytosolic beta-glucosidase from guinea pig liver and sequenced 142 amino acid residues contained within 12 trypsin digest fragments. Using degenerate oligonucleotide primers deduced from the peptide sequences, a 622 bp cytosolic beta-glucosidase cDNA was amplified by reverse-transcriptase PCR, using total guinea pig liver RNA as template. The 'rapid amplification of cDNA ends (RACE)' method [Frohman (1993) Methods Enzymol. 218, 340-356] was used to synthesize the remaining segments of the full-length cDNA. The complete cDNA contained 1671 nucleotides with an open reading frame coding for 469 amino acid residues. The amino acid sequence deduced from the cDNA sequence included the amino acid sequences of all 12 trypsin digest fragments derived from the purified enzyme. Amino acid sequence analysis indicates that the guinea pig liver cytosolic beta-glucosidase is a Family 1 beta-glycosidase and that it is most closely related to mammalian lactase-phlorizin hydrolase. These results suggest that the cytosolic beta-glucosidase and lactase-phlorizin hydrolase diverged from a common evolutionary precursor.
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PMID:Primary structure of the cytosolic beta-glucosidase of guinea pig liver. 892 Sep 87

Expression systems based on high selectivity and activity of T7 RNA polymerase and presence of a strong T7 promoter have been commonly used for cloning and expression of various recombinant proteins in Escherichia coli. When the expression system is designed in such a way that the produced protein is not being transferred into periplasm, bacterial cells must be lysed in order to isolate and purify the protein. The final yield and quality of the synthesized protein then depend on various factors, protein size, amino acid sequence, solubility in cytoplasm, and folding requirements among them. The yield in the T7 RNA polymerase/promoter system can be positively influenced by use of rifampicin. In this report we demonstrate usefulness of the antibiotic in detail. We describe rifampicin-enhanced expression of a plant cytokinin-specific beta-glucosidase. Two bacterial cultures are compared, one expressing the enzyme without and one in the presence of rifampicin. The antibiotic not only increased the yield of the recombinant protein, which seems to be a general phenomenon, but also favored the final assembly of the protein's subunits into a catalytically active dimer form.
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PMID:Use of rifampicin in T7 RNA polymerase-driven expression of a plant enzyme: rifampicin improves yield and assembly. 1042 61

Listeria monocytogenes is an important food-borne pathogen that can tolerate a wide range of stress conditions. However, its stress adaptation processes are still poorly understood. Real-time-based quantitative RT-PCR (qRT-PCR) provides a tool to probe gene expression changes underlying stress adaptation. But, a limitation to study mRNA levels by real-time qRT-PCR is that validated reference genes are required for normalization. Such genes are currently lacking for experimental models that may be applied to evaluate stress-related gene expression changes in L. monocytogenes. Therefore, five housekeeping genes (HKG) were studied as potential reference genes. Their expression stability was evaluated across 16 L. monocytogenes strains. Three experimental models designed to assess gene expression changes induced by cold, acid and high NaCl concentration stress adaptation were applied. The 16S rRNA gene was consistently the most stably expressed HKG across the different L. monocytogenes strains under all the experimental conditions. While the expressions of beta-glucosidase (bglA), Glyceraldehyde-3P-dehydrogenase (gap), RNA polymerase beta subunit (rpoB) and Ribosomal protein L4 (rplD) was stable amongst the different L. monocytogenes strains, they were prone to significant variations under the different stress adaptation models.
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PMID:Evaluation of housekeeping genes in Listeria monocytogenes as potential internal control references for normalizing mRNA expression levels in stress adaptation models using real-time PCR. 1726 45

Polycyclic aromatic hydrocarbons (PAHs) are potentially carcinogenic and toxic to humans through ingestion of contaminated food crops. PAHs can enter crop roots through proton/PAH symporters; however, to date, the symporter remains unclear. Here we reveal, for the first time, the plasma membrane proteome of Triticum aestivum seedling roots in response to phenanthrene (a model PAH) exposure. Two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF/TOF-MS and protein database search engines were employed to analyze and identify phenanthrene-responsive proteins. Over 192 protein spots are reproducibly detected in each gel, while 8 spots are differentially expressed under phenanthrene treatment. Phenanthrene induces five up-regulated proteins distinguished as 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase 2, enolase, heat shock protein 80-2, probable mediator of RNA polymerase II transcription subunit 37e (heat shock 70-kDa protein 1), and lactoylglutathione lyase. Three proteins identified as adenosine kinase 2, 4-hydroxy-7-methoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl glucoside beta-D-glucosidase 1c, and glyceraldehyde-3-phosphate dehydrogenase 3 are down-regulated under exposure to phenanthrene. The up-regulated proteins are related to plant defense response, antioxidant system, and glycolysis. The down-regulated proteins involve the metabolism of high-energy compounds and plant growth. Magnesium, which is able to bind to enolase, can enhance the transport of phenanthrene into wheat roots. Therefore, it is concluded that phenanthrene can induce differential expression of proteins in relation to carbohydrate metabolism, self-defense, and plant growth on wheat root plasma membrane. This study not only provides novel insights into PAH uptake by plant roots and PAH stress responses, but is also a good starting point for further determination and analyses of their functions using genetic and other approaches.
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PMID:Proteomic analysis of plasma membrane proteins in wheat roots exposed to phenanthrene. 2689 80