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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In the primitive eukaryotic parasite, Trypanosoma brucei, most of the enzymes of glycolysis are located within microbody organelles called glycosomes. Proteins destined for the glycosome are synthesized on free ribosomes and post-translationally translocated into the organelle. The gene, gPGK, encoding the glycosomal isozyme of phosphoglycerate kinase (gPGK), was cloned adjacent to a T7 promoter and cotransformed with a plasmid encoding T7 RNA polymerase into Escherichia coli Pgk-cells. Functional complementation occurred, but only after the creation of a ribosome-binding site by mutagenesis. This represents the first example of complementation of an E. coli mutant with a gene encoding a microbody protein. Enzymatically active recombinant gPGK was purified to near homogeneity by ion exchange chromatography from highly expressing E. coli. The recombinant protein will aid in studies of glycosomal biogenesis.
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PMID:Microbody phosphoglycerate kinase of Trypanosoma brucei: expression and complementation in Escherichia coli. 220 31

The expression of a number of genes was measured in P1798 cells treated for various periods of time with 0.1 microM dexamethasone. Thymidine kinase (TK) activity decreased under these conditions with 50% inhibition achieved within approximately 8 h. Decreased TK activity was associated with reduced abundance of TK mRNA. Analysis of nuclear transcription indicated that this was attributable to a decrease in the number of RNA polymerase II molecules engaged in transcription of the TK gene. With respect to TK, there was an overall correlation between enzyme activity, mRNA, and nuclear transcription. The data are consistent with the hypothesis that glucocorticoid inhibition of expression of TK is primarily due to inhibition of transcription. Transcription of the TK gene was also reduced by greater than 90% after inhibition of protein synthesis for 6 h. This suggests that transcription of this gene requires a protein of short biological half-life. It is proposed that this hypothetical transcription factor is regulated by glucocorticoids. The amount of thymidylate synthase and dihydrofolate reductase remained constant for at least 24 h in dexamethasone-treated P1798 cells. Dihydrofolate reductase mRNA likewise remained constant. However, the mRNA encoding thymidylate synthase decreased 80-90% within 24 h. The mRNA encoding ornithine decarboxylase also decreased. In neither case did this appear to be primarily due to inhibition of transcription of the respective genes. The abundance of the mRNAs encoding hypozanthine-guanine phosphoribosyl transferase and phosphoglycerate kinase did not decrease in dexamethasone-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glucocorticoid regulation of the genes encoding thymidine kinase, thymidylate synthase, and ornithine decarboxylase in P1798 cells. 339 44

The DNA sequence of the gene for the yeast glycolytic enzyme, 3-phosphoglycerate kinase (PGK), has been obtained by sequencing part of a 3.1 kbp HindIII fragment obtained from the yeast genome. The structural gene sequence corresponds to a reading frame of 1251 bp coding for 416 amino acids with no intervening DNA sequences. The amino acid sequence is approximately 65 percent homologous with human and horse PGK protein sequences and is in general agreement with the published protein sequence for yeast PGK. As for other highly expressed structural genes in yeast, the coding sequence is highly codon biased with 95 percent of the amino acids coded for by a select 25 codons (out of 61 possible). Besides structural DNA sequence, 291 bp of 5'-flanking sequence and 286 bp of 3'-flanking sequence were determined. Transcription starts 36 nucleotides upstream from the translational start and stops 86-93 nucleotides downstream from the translational stop. These results suggest a non-polyadenylated mRNA length of 1373 to 1380 nucleotides, which is consistent with the observed length of 1500 nucleotides for polyadenylated PGK mRNA. A sequence TATATATAAA is found at 145 nucleotides upstream from the translational start. This sequence resembles the TATAAA box that is possibly associated with RNA polymerase II binding.
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PMID:The primary structure of the Saccharomyces cerevisiae gene for 3-phosphoglycerate kinase. 629 91

The synthesis and characterization of guanosine 5'-O-(1-thiotriphosphate) (GTP alpha S) and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) using chemical and enzymatic methods are described. GTP alpha S A (SP diastereomer) can be prepared enzymatically from a chemically synthesized mixture of the diastereomers of guanosine 5'-O-(1-thiodiphosphate) (GDP alpha S) with phosphoglycerate kinase. GTP alpha S B (RP diastereomer) can be similarly synthesized with succinyl-CoA synthetase and by back-digesting the small amounts of GTP alpha S A formed with phosphoglycerate kinase. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) serves as the precursor for both GTP beta S A (SP diastereomer), prepared with pyruvate kinase and by back-digesting with glycerol kinase, and GTP beta S B (RP diastereomer), obtained with acetate kinase and by back-digesting with myosin. These analogues can be gamma-32P labeled by 32Pi exchange with either phosphoglycerate kinase-phosphoglyceraldehyde dehydrogenase or succinyl-CoA synthetase. Finally, the interaction of these four nucleotides with acetate kinase, RNA polymerase, and succinyl-CoA synthetase is described.
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PMID:Synthesis and characterization of diastereomers of guanosine 5'-O-(1-thiotriphosphate) and guanosine 5'-O-(2-thiotriphosphate). 709 23

This study was designed to quantitate cardiac mRNA levels encoding components of the local renin-angiotensin system during the development of volume overload-induced cardiac hypertrophy. Changes in cardiac renin mRNA levels were measured in relation to renin activity in the left ventricle (LV) and in plasma after acute passive stretch of the heart caused by an aortovenocaval shunt in the rat. A quantitative reverse-transcriptase polymerase chain reaction method with competitive internal standards was used to measure mRNA levels in total RNA derived from cardiac tissues after shunt. Seven days after shunt surgery, LV weight was increased by 23%. Renin activities were elevated four- and twofold in plasma and LV, respectively. LV angiotensinogen mRNAs were not significantly increased by shunt surgery; they were twofold higher than phosphoglycerate kinase mRNA from the housekeeping gene PGK-1. By day 7, LV levels for renin mRNA were significantly increased from well below 0.25% to approximately 1% of PGK-1 mRNA. Identity between renin polymerase chain reaction products from kidney and heart cDNAs and absence of "reninlike" amplification products were supported by Southern blotting. Volume overload caused increased expression of the renin gene in the stretched myocardium. This finding is consistent with the concept of a myocardial renin-angiotensin system that can be activated by locally produced renin and contributes to the hypertrophy of cardiac muscle.
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PMID:Stretch-mediated activation of cardiac renin gene. 794 10

Trypanosoma brucei is a unicellular parasite that is transmitted from one mammalian host to the next by tsetse flies. The expression of many trypanosome genes is regulated during the life cycle but there is no evidence for developmental control of transcription by RNA polymerase II. T. brucei expresses at least two hexose transporter mRNAs that are developmentally regulated; we show here that specific portions of the 3'-untranslated regions are responsible for the differential expression. Different trypanosome 3'-untranslated regions, from surface protein, phosphoglycerate kinase and aldolase genes as well as the hexose transporter genes, conferred a spectrum of levels of reporter gene expression, and these activities differed between bloodstream forms and the procyclic forms that replicate in the tsetse vector. Experiments with permanently transformed cell lines showed that regulation occurs at the mRNA level. The results suggest that post-transcriptional control of mRNAs in trypanosomatids operates at several levels, and that it will not always be possible to attribute all the regulation to short RNA motifs.
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PMID:Role of 3'-untranslated regions in the regulation of hexose transporter mRNAs in Trypanosoma brucei. 872 Jan 70

Reovirus mu 2 protein can be expressed via the mouse phosphoglycerate kinase promoter to low levels in stably transfected L cells. To increase mu 2 expression, the terminal regions of the M1 gene cDNA constructs were modified and the effect on mu 2 expression was analyzed. The M1 gene has a single large open reading frame beginning at nucleotide 14 with another, in frame, AUG codon at nucleotide 161 reported to be used for translation initiation. Unexpectedly, deletions of the M1 5' terminal sequence upstream of the reported translation initiation codon, AUG161, resulted in loss of detection of mu 2 expression. When expression was driven by the stronger T7 promoter in the presence of recombinant vaccinia virus expressing the T7 RNA polymerase, constructs with the M1 5'-terminal deletion produced a smaller protein product of approximately 68 kDa, compared to approximately 73 kDa for the protein produced from the full-length M1-containing constructs consistent with the loss of 49 amino acids. The amount of shorter mu 2 product was increased by producing an improved 'Kozak' consensus sequence around the AUG codon at nucleotide 161 or by introducing an internal ribosome entry site at this location. Full-length M1 gene constructs produced a protein of the same size as the authentic mu 2 protein from virus-infected cells. It was further shown that the approximately 73 kDa product was expressed when the M1 gene was in different plasmid backgrounds and even when the M1 gene transcript was preceded by a 1 kb gene. This study demonstrated that translation of the reovirus M1 gene initiates from the first AUG codon in both infected and transfected cells.
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PMID:Translation of the reovirus M1 gene initiates from the first AUG codon in both infected and transfected cells. 872 23

Real-time PCR is frequently used for gene expression quantification due to its methodological sensitivity and reproducibility. The gene expression is quantified by normalization to one or more reference genes, usually beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPD) or to ribosomal RNA (18S). However, different environmental or pathological conditions might also influence the expression of normalizing genes, which could severely skew the interpretation of quantitative results. This study evaluates whether 16 genes frequently used as endogenous controls in expression studies, can serve as such for comparison of human brain tissues of chronic alcoholics and control subjects. The prefrontal and motor cortices that are affected differently by chronic alcohol consumption were analyzed. The reference genes that have no or small differences in expression in alcoholics and control subjects, were found to be specific for each region: beta-actin (ACTB) and ribosomal large P0 (RPLP0) for the prefrontal cortex while importin 8 (IPO8) and RNA polymerase II (POLR2A) for the motor cortex. Four out of sixteen analyzed genes demonstrated significant differences in expression between alcoholics and controls: phosphoglycerate kinase (PGK1), hypoxanthine phosphoribosyl transferase (HPRT1) and peptidylprolyl isomerase A (PPIA) in the motor cortex and beta-2-microglobulin (B2M) in the prefrontal cortex. Our study demonstrates the importance of validation of endogenous control genes prior to real-time PCR analysis of human brain tissues. Prescribed and non-prescribed drugs, pathological or environmental conditions along with alcohol abuse may differentially influence expression of reference genes.
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PMID:Validation of endogenous controls for quantitative gene expression analysis: application on brain cortices of human chronic alcoholics. 1718 56

Gene expression changes are used with increasing frequency to assess the effects of exposure to environmental agents. Housekeeping (Hk) genes are essential in these analyses as internal controls for normalizing expression levels evaluated with Real-Time PCR (RT-PCR). Ideal Hk genes are constitutively expressed, do not respond to external stimuli and exhibit little or no sample-to-sample or run-to-run variation. Previous studies indicate that some commonly used Hk genes including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin have differential expression in various cell lines. Here we examine the expression of 11 Hk genes in four normal human lymphoblastoid cell lines and one T-cell leukemia (Jurkat) cell line following exposure to graded doses of ionizing radiation or to varying ratio concentrations of phytohemagglutinin (PHA) and phorbol myristate acetate (PMA). PHA and PMA are known to have synergistic effects on the expression of some genes and have very different effects from those of radiation. There has been no systematic study performed to ascertain the best control genes for radiation and/or PHA/PMA exposures in lymphoblastoid cells. Using a two-step reverse-transcriptase RT-PCR protocol we show that following radiation doses ranging from 0 to 400 cGy, 18S rRNA, acidic ribosomal protein, beta-actin, cyclophilin, GAPDH, phosphoglycerokinase, beta-2 microglobulin (B2M), beta-glucuronidase, hypoxanthine phosphoribosyltransferase and transferrin receptor showed no significant variation in expression in normal lymphoblastoid cells. In contrast, only 18S rRNA levels were unchanged in Jurkat cells. After PHA/PMA treatment of the same normal cell lines, B2M showed no significant variation and 18S rRNA, GAPDH and transcription binding protein (TBP) were minimally responsive, whereas in Jurkat cells all these genes were unresponsive. While our results suggest that the utility of a particular Hk gene should be determined for each experimental condition, 18S rRNA and B2M appear to be excellent candidates for use as internal controls in RT-PCR in human lymphoblastoid cells because they have the most constant levels of expression across cell lines following exposure to ionizing radiation as well as to PHA/PMA.
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PMID:Evaluation and validation of housekeeping genes in response to ionizing radiation and chemical exposure for normalizing RNA expression in real-time PCR. 1790 13

Increasing evidence has shown the complex and dynamic nature of polyploids. Two single copy nuclear genes were used to explore genome evolutionary dynamics and the origin of tetraploid E. ciliaris: the phosphoglycerate kinase (PGK1) and the second largest subunit of RNA polymerase II (RPB2) together with a chloroplast gene encoding ribosomal protein S16 (RPS16). RPS16 data confirmed that the maternal origin of E. ciliaris is the St genome species. Both RPB2 and PGK1 data supported that E. ciliairs has multiple origins, and originated from the Pseudorogneria (St) and unknown donor (Y) diploids. The St genome in E. ciliaris species has a complex evolutionary history. Both RPB2 and PGK1 data suggested the absence of St genome in accession PI 377532 of E. ciliaris. However, cpDNA RPS16 clearly indicated that its maternal origin is the same as other E. ciliaris accessions, and is St genomic diploid species. Results suggest that there are two lineages of St genome present in E. ciliaris species; one is grouped with Pseudoroegneria diploid species, the other is grouped with Hordeum (H) species (named St?). The Japanese accession PI 377532 might have introgression either from HordeumH genome species or from ElymusStH genome species with replacement of at least some nuclear St-loci by H-loci. The correlation between genome differentiation and geographical distribution is also discussed.
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PMID:Phylogenetic analysis revealed reticulate evolution of allotetraploid Elymus ciliaris. 2383 60


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