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)

The early protein P35 from the baculovirus Autographa californica nucleopolyhedrovirus is a direct inhibitor of caspases and can block apoptosis in a wide variety of systems. In addition, it has been linked to the regulation of viral gene expression, shut-down of protein synthesis in infected insect cells and malignant transformation of mouse fibroblasts. By yeast-two-hybrid screening we identified the RPB11a subunit of human RNA polymerase II as an interaction partner of P35. Specificity of the interaction was confirmed by affinity blotting. By immunocytology, P35 was in part found in the nucleus of transfected cells. Homology searches further revealed that P35 has structural similarity with RPB3, the subunit of RNA polymerase II that has been demonstrated to interact directly with RPB11a. When transfected into human colon carcinoma cells, P35 was able to enhance the activity of E-cadherin and beta-actin promoters by about a factor of two as measured by luciferase reporter assay. P35 and hRPB11a together enhanced the E-cadherin activity about three- to fourfold. These data suggest an additional role for P35 in the regulation of cellular transcription.
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PMID:Baculovirus P35 interacts with a subunit of human RNA polymerase II and can enhance promoter activity in human cells. 1457 6

Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and beta-actin have been used for that purpose. However, it has been reported that these genes as well as alternatives, like rRNA genes, are unsuitable references, because their transcription is significantly regulated in various experimental settings and variable in different tissues. Therefore, quantitative real-time PCR was used to determine the mRNA transcription profiles of 13 putative reference genes, comparing their transcription in 16 different tissues and in CCRF-HSB-2 cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and ionomycin. Our results show that "Classical" reference genes are indeed unsuitable, whereas the RNA polymerase II gene was the gene with the most constant expression in different tissues and following stimulation in CCRF-HSB-2 cells.
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PMID:Guideline to reference gene selection for quantitative real-time PCR. 1470 21

Uncoupling proteins (UCPs) present in the inner mitochondrial membrane are involved in uncoupling respiration from ATP synthesis. Five UCP isoforms have been identified but information about their presence and level of expression in the central nervous system remains incomplete. To determine the nature and proportion of UCP isoform mRNAs present in brain cortex, we developed and optimized a specific quantitative reverse-transcription polymerase chain reaction procedure. Optimal range of RNA concentrations to be used in the reverse-transcriptase reaction was determined. Primer design and concentration were optimized for each target gene while polymerase chain reaction efficiency was assessed for a range of reverse-transcriptase dilutions. Genomic contribution to the quantitative signal was evaluated for each isoform and minimized. Three reference genes were tested for normalization, and beta-actin was found to be the most stable among tissues. Results indicate that brain cortex contains significant amounts of all UCP mRNAs, with UCP5 and UCP4 being the most abundant, as opposed to brown adipose tissue and skeletal muscle, which predominantly express UCP1 and UCP3, respectively. These data provide a first quantitative assessment of UCP mRNA expression in mouse brain, showing the presence of all five isoforms with distinct proportions, thus suggesting specific roles in the central nervous system.
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PMID:Quantitative rt-PCR analysis of uncoupling protein isoforms in mouse brain cortex: methodological optimization and comparison of expression with brown adipose tissue and skeletal muscle. 1524 Nov 86

Actin is abundant in the nucleus and has been implicated in transcription; however, the nature of this involvement has not been established. Here we demonstrate that beta-actin is critically involved in transcription because antibodies directed against beta-actin, but not muscle actin, inhibited transcription in vivo and in vitro. Chromatin immunoprecipitation assays demonstrated the recruitment of actin to the promoter region of the interferon-gamma-inducible MHC2TA gene as well as the interferon-alpha-inducible G1P3 gene. Further investigation revealed that actin and RNA polymerase II co-localize in vivo and also co-purify. We employed an in vitro system with purified nuclear components to demonstrate that antibodies to beta-actin block the initiation of transcription. This assay also demonstrates that beta-actin stimulates transcription by RNA polymerase II. Finally, DNA-binding experiments established the presence of beta-actin in pre-initiation complexes and also showed that the depletion of actin prevented the formation of pre-initiation complexes. Together, these data suggest a fundamental role for actin in the initiation of transcription by RNA polymerase II.
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PMID:Actin is part of pre-initiation complexes and is necessary for transcription by RNA polymerase II. 1551 91

Improved methods of bone regeneration are needed in the craniofacial rehabilitation of patients with significant bone deficits secondary to tumor resection, congenital deformities, and prior to prosthetic dental reconstruction. In this study, a gene-enhanced tissue-engineering approach was used to assess bone regenerative capacity of Sonic hedgehog (Shh)-transduced gingival fibroblasts, mesenchymal stem cells, and fat-derived cells delivered to rabbit cranial bone defects in an alginate/collagen matrix. Human Shh cDNA isolated from fetal lung tissue was cloned into the replication-incompetent retroviral expression vector LNCX, in which the murine leukemia virus retroviral LTR drives expression of the neomycin-resistance gene. The rat beta-actin enhancer/promoter complex was engineered to drive expression of Shh. Reverse transcriptase-polymerase chain reaction analysis demonstrated that the transduced primary rabbit cell populations expressed Shh RNA. Shh protein secretion was confirmed by enzyme-linked immunosorbent assay (ELISA). Alginate/ type I collagen constructs containing 2 x 10(6) Shh-transduced cells were introduced into male New Zealand White rabbit calvarial defects (8 mm). A total of eight groups (N=6) were examined: unrestored empty defects, matrix alone, matrix plus the three cell populations transduced with both control and Shh expression vectors. The bone regenerative capacity of Shh gene enhanced cells was assessed grossly, radiographically and histologically at 6 and 12 weeks postimplantation. After 6 weeks, new full thickness bone was seen emanating directly from the alginate/collagen matrix in the Shh-transduced groups. Quantitative two-dimensional digital analysis of histological sections confirmed statistically significant (P<0.05) amounts of bone regeneration in all three Shh-enhanced groups compared to controls. Necropsy failed to demonstrate any evidence of treatment-related side effects. This is the first study to demonstrate that Shh delivery to bone defects, in this case through a novel gene-enhanced tissue-engineering approach, results in significant bone regeneration. This encourages further development of the Shh gene-enhanced tissue-engineering approach for bone regeneration.
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PMID:Sonic hedgehog gene-enhanced tissue engineering for bone regeneration. 1551 Jan 77

When transcription from the human U6 snRNA gene is reconstituted with recombinant factors and purified RNA polymerase III (pol III), pol III must be treated with CK2 to be active. We show that highly purified pol III contains associated beta-actin, and beta-actin localizes to an active U6 promoter in vivo. Pol III immunoprecipitated from IMR90 cells treated with a genotoxic agent lacks associated beta-actin and is inactive in the reconstituted assay. Transcription is regained upon treatment of pol III with CK2 and addition of beta-actin. This suggests that beta-actin associated with pol III is essential for basal pol III transcription.
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PMID:A role for beta-actin in RNA polymerase III transcription. 1557 86

The renin-angitensin system (RAS) plays an important role as a growth factor in cardiac development. Angiotensin converting enzyme is involved in converting angiotensin I to angiotensin II (Ag-II). The effects of Ag-II are mediated by two primary receptors, type 1 (AT1) and type 2 (AT2). Ag-II stimulates transforming growth factor-beta1(TGF-beta1) and acts as a potent stimulant of myocyte growth and fetal contractile protein gene transcription. The aim of this study was to determine the expression of Ag-II receptor subtypes and TGF-beta1 in the hypoplastic heart of nitrofen-induced congenital diaphragmatic hernia (CDH). CDH was induced in pregnant rats following administration of 100 mg nitrofen on day 9.5. The fetuses were divided into three groups: normal controls (n=16), nitrofen-treated without CDH (n=16), and nitrofen-induced CDH (n=16). Reverse transcriptase-polymerase chain reaction was performed to evaluate mRNA expression of AT1, AT2, and TGF-beta1. Levels of mRNA were expressed as a ratio of the band density divided by that of beta-actin. AT1 and AT2 mRNA expressions were significantly decreased in CDH heart compared with controls (0.43+/-0.33 vs. 1.0+/-0.48 and 0.62+/-0.23 vs. 1.4+/-0.43, respectively). TGF-beta1 mRNA expressions were also significantly decreased in CDH heart compared with controls (0.38+/-0.17 vs. 0.72+/-0.26). No significant difference was found between the hearts of controls and nitrofen-treated rats without CDH. The decreased expression of AT1, AT2, and TGF-beta1 mRNA in the hypoplastic heart suggests that the downregulation of RAS may be involved in the pathogenesis of cardiac hypoplasia in nitrofen-induced CDH.
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PMID:Altered expression of angiotensin II receptor subtypes and transforming growth factor-beta in the heart of nitrofen-induced diaphragmatic hernia in rats. 1557 92

In the present study, we investigated the involvement of protein degradation via the 26S proteasome during progesterone receptor (PR)-mediated transcription in T-47D cells containing a stably integrated MMTV-CAT reporter construct (CAT0 cells). Progesterone induced CAT and HSD11beta2 transcription while co-treatment with the proteasome inhibitor, MG132, blocked PR-induced transcription in a time-dependent fashion. MG132 treatment also inhibited transcription of beta-actin and cyclophilin, but not two proteasome subunit genes, PSMA1 and PSMC1, indicating that proteasome inhibition affects a subset of RNA polymerase II (RNAP(II))-regulated genes. Progesterone-mediated recruitment of RNAP(II) was blocked by MG132 treatment at time points later than 1 h that was not dependent on the continued presence of PR, associated cofactors, and components of the general transcription machinery, supporting the concept that proteasome-mediated degradation is needed for continued transcription. Surprisingly, progesterone-mediated acetylation of histone H4 was inhibited by MG132 with the concomitant recruitment of HDAC3, NCoR, and SMRT. We demonstrate that the steady-state protein levels of SMRT and NCoR are higher in the presence of MG132 in CAT0 cells, consistent with other reports that SMRT and NCoR are targets of the 26S proteasome. However, inhibition of histone deacetylation by trichostatin A (TSA) treatment or SMRT/NCoR knockdown by siRNA did not restore MG132-inhibited progesterone-dependent transcription. Therefore, events other than histone deacetylation and stability of SMRT and NCoR must also play a role in inhibition of PR-mediated transcription.
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PMID:Inhibition of the 26S proteasome blocks progesterone receptor-dependent transcription through failed recruitment of RNA polymerase II. 1585 53

The aim of the present study was to estimate the expression of mRNA, specific for thymidine kinase 1 (TK1), deoxycytidine kinase (dCK), and thymidine phosphorylase (dThdPase), i.e. enzymes involved in pyrimidine and purine metabolism in human papillary thyroid carcinoma (PTC) tissue. Additionally, the expression of dCK was estimated, in medullary thyroid carcinoma (MTC). For control, the RNA expression levels for all the enzymes were measured in macroscopically unchanged thyroid tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) and densitometry were employed for mRNA expression measurements, with the beta-actin gene as a control housekeeping gene. The levels of mRNA expression for TK1, dCK and dThdPase in human PTC, as well as mRNA expression for dCK in MTC, were significantly higher than mRNA expressions for those enzymes found in macroscopically unchanged thyroid tissue. It is concluded that an increased expression of mRNA, specific for TK1, dCK and dThdPase, may be involved in carcinogenic processes in the human thyroid.
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PMID:Increased expression of mRNA specific for thymidine kinase, deoxycytidine kinase or thymidine phosphorylase in human papillary thyroid carcinoma. 1597 30

In this study, we show that exposure of human hepatocellular HepG2 cells to SP600125 rapidly and dramatically reduced global histone H3-Ser10 phosphorylation, without significantly affecting the global acetylation of neighboring lysines. The loss of phosphorylation is not due to changes in cell cycle distribution and/or apoptosis and is mediated independent of either p46/54(JNK) or MSK-1/2 inhibition. Moreover, SP600125 repressed the basal expression of the endogenous LDL receptor in a gene-specific manner, whereas the expression of squalene synthase, sterol response element-binding protein-1, and beta-actin was not altered by SP600125. Finally, chromatin immunoprecipitation and in vivo footprinting assays provided direct evidence that localized histone H3-Ser10 dephosphorylation at the low-density lipoprotein receptor promoter was associated with a significant decrease in the occupancy of the Sp1 binding site, with a slight reduction in the occupancy of RNA polymerase II. Together, our findings show that SP600125 is an efficient inhibitor of histone H3-Ser10 phosphorylation in vivo, and our results led us to hypothesize that this modification plays a novel role in regulating transcriptional control by modulating promoter accessibility to maintain basal expression in a gene-specific manner.
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PMID:Selective repression of low-density lipoprotein receptor expression by SP600125: coupling of histone H3-Ser10 phosphorylation and Sp1 occupancy. 1644 44


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