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)

Transcription and replication of hepatitis delta virus (HDV) RNA is thought to be performed by host RNA polymerase II. The mechanism which enables polymerase II to use RNA as a template is unclear. However, since extensive intramolecular complementarity allows HDV RNA to form a rod-shaped structure, it is possible that the mostly double-stranded HDV RNA may resemble double-stranded DNA in structure, and can thus be used by RNA polymerase II as a template. To investigate this possibility, we examined whether the cDNA counterpart of HDV RNA contains a promoter and thus can drive the transcription and replication of HDV RNA. Circularized monomers of HDV cDNA, when transfected into various cell lines, were found to generate both monomeric and dimeric forms of HDV RNA and hepatitis delta antigen at levels comparable to those generated with HDV cDNA multimers under the control of a SV40 late promoter, suggesting that HDV cDNA contains endogenous promoters. Using chloramphenicol acetyltransferase and human growth hormone as reporter genes, the specific promoter activity for the synthesis of antigenomic HDV RNA was localized to a 29-nucleotide region (nucleotides 1650-1679), although an additional 224-nucleotide upstream region was also necessary for maximum activity. Similarly, promoter activity for the synthesis of genomic RNA was localized to a 160-nucleotide region around position 1679 that overlapped with the antigenomic promoter region. Since these regions are in a highly conserved double-stranded region of HDV RNA, they may represent RNA promoters recognized by RNA polymerase II. This result also suggests a convenient method, using circularized monomer HDV cDNA, to study HDV RNA replication.
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PMID:Endogenous promoters can direct the transcription of hepatitis delta virus RNA from a recircularized cDNA template. 837 36

A phage T7 class-III promoter (pT7), which is highly specific for T7 RNA polymerase in bacteria, was tested in mammalian cells for its specificity. After having shown that T7 RNA polymerase can transcribe from pT7 in the nucleus of stably transformed cells [Lieber et al., Nucleic Acids Res. 17 (1989) 8485-8493], we describe here that pT7 could also direct efficient intracellular gene expression in the absence of T7 RNA polymerase. Using the genomic human growth hormone-encoding gene and the firefly luciferase-encoding gene as reporters, we found expression levels comparable with those obtained with the Rous sarcoma viral promoter. Inhibition of expression with alpha-amanitin suggests that transcription is by RNA polymerase II. Binding studies with HeLa cell extracts clearly show that synthetic pT7 sequences are specifically bound (gel retardation) and that the promoter region is protected from DNase degradation. The experimental data, as well as the nucleotide sequence, suggest that pT7 has properties of an initiator element. Indeed, the activity of pT7 can be stimulated by the presence of an upstream element or an enhancer. These results have practical implications for the use of pT7 in mammalian expression vectors. Commercial pT7 plasmids can be used for both prokaryotic and eukaryotic expression systems.
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PMID:A phage T7 class-III promoter functions as a polymerase II promoter in mammalian cells. 840 19

All the angiotensin peptides originate from angiotensinogen, a glycoprotein synthesized by several tissues, including the brain and the anterior pituitary. In the rat, immunohistochemistry has been used to localize angiotensinogen in gonadotropes and in uncharacterized cells surrounding sinusoids. Both cell types are capable of secreting angiotensinogen in cell culture; only the gonadotropes contain angiotensin II (AngII) and are capable of secreting it in culture. It has been asserted that the perisinusoidal cells are the only source of angiotensinogen for the generation of AngII by gonadotropes. Our current data favor the existence of a complete intracellular renin-angiotensin system (RAS) in gonadotropes and a separate extracellular system which utilizes the high concentration of angiotensinogen from perisinusoidal cells. Furthermore, we postulate that gonadotrope AngII serves mainly reproductive functions, while the proximity of angiotensinogen-secreting cells to folliculostellate cells, and their access to the intercellular sinusoidal and follicular spaces, places the extracellular RAS in a strategic position to affect pituitary growth and the mediation of acute-phase immune responses. In the rat brain, angiotensinogen is expressed by the 16-18th day of fetal life and by areas generally concerned with vasopressor, electrolyte, and fluid homeostasis. Antisense deoxyoligonucleotides to angiotensinogen mRNA lower blood pressure in hypertensive rats and inhibit in vitro growth of neuroblastoma cells, indicating a significant role for angiotensinogen in mitogenic and homeostatic functions. It is commonly agreed that astrocytes express angiotensinogen. Neuronal angiotensinogen has also been demonstrated by immunohistochemistry, as a secretion from neuronal cell cultures, and by reverse-transcriptase polymerase chain reaction. The fate of secreted astrocytic and neuronal angiotensinogen remains obscure. Angiotensinogen is regulated in a tissue-specific manner with smaller or absent responses observed for brain tissue. By using astrocyte and neuronal cultures the actions on angiotensinogen production of growth hormone, IGF-1, inflammatory lipopolysaccharide, and phorbol ester have been examined. Recent observations show that angiotensinogen is regulated positively or negatively by glucocorticoids and that a positive synergism between cAMP and glucocorticoids exists. On the basis of analogous systems for other proteins, a scheme involving glucocorticoid receptors, CREB, and AP-1 transcription factors is formulated to explain glucocorticoid-cAMP interactions. These transcriptional interactions may form a significant functional link between the RAS and adrenergic mechanisms.
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PMID:Novel perspectives on pituitary and brain angiotensinogen. 910 Dec 59

Corticotrophin-releasing factor (CRF) and urocortin possess a high-affinity binding protein. Although the CRF binding protein (BP) can sequester these ligands and inhibit their activity, the endogenous activity of this protein is not understood. Therefore, transgenic mouse lines that over-express the CRF-BP were created. The transgene was constructed by ligating rat CRF-BP cDNA (1.1 kb) between a mouse metallothionein-I promoter (1.8 kb) and a nonfunctional human growth hormone gene sequence (2.1 kb) in a modified pBR322 plasmid and microinjecting the transgene into C57BL/6 x SJL hybrid ova. The transgene was expressed in 50% in both male and female progeny. All transgenic lines were maintained by crossing transgenic animals with wild-type C57BL/6 mates. Reverse-transcriptase (RT) PCR of the CRF-BP transgene showed that it is widely expressed not only in the brain and pituitary, but also peripheral tissues including the liver, kidney and spleen. Transgenic animals of both sexes showed significant increases in weight gain as established by analysis of variance; however, the weight gain profiles for each sex were distinct. High levels of circulating CRF-BP were detected in the transgenic animals, but the basal ACTH and corticosterone levels were not significantly decreased compared to wild-type littermates. The hypothalamopituitary-adrenal (HPA) axis was stimulated by systemic inflammation induced with lipopolysaccharide (LPS). An expected increase in transgene expression was observed and was accompanied by a significant attenuation of ACTH secretion at 3 h after LPS injection in the transgenic males but not the females. These data suggest that HPA axis regulation is significantly affected only with very high circulating levels of CRF-BP. Moreover, this work supports previous studies that implicate CRF and urocortin in the regulation of appetite and the binding protein expression may play a sexually dimorphic role in regulating this and other responses.
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PMID:Ectopic expression of the CRF-binding protein: minor impact on HPA axis regulation but induction of sexually dimorphic weight gain. 970 Jun 75

The gene for a 45 kDa merozoite surface protein (MSA-2) of the human malaria parasite Plasmodium falciparum was PCR amplified and cloned into eukaryotic expression vectors VR1012 and pcDNA3 to yield plasmids P1 and P2, respectively. The coding sequences for two N-terminal fragments of the 185 kDa merozoite surface protein (MSA-1) gene were similarly PCR amplified and cloned into vectors VR1020 and VR1012 to yield plasmids P3 and P4, respectively. The MSA-1 signal peptide sequence, present in P4, was replaced with the human tissue plasminogen activator signal sequence in P3. The four plasmids expressed the cloned genes under the control of the cytomegalovirus promoter and carried 3' bovine growth hormone termination/poly A signals. P1, P3 and P4 also contained the cytomegalovirus intron A enhancer sequence. MSA-1 expression was more readily detected than MSA-2 in Cos cells transfected with P3/P4 and P1/P2 respectively. The MSA-2 gene was also cloned into the phagemid pBluescript IISK+ with and without a 3' poly A tail composed of 35 A residues. MSA-2 was synthesised in HeLa cells infected with a recombinant vaccinia virus carrying T7 RNA polymerase when MSA-2 recombinant pBluescript was transfected into the cells. Inoculation with P1 intramuscularly or intradermally and with P2 intradermally into rabbits led to the production of antibodies to MSA-2 detectable by immunofluorescence and Western blotting. Antibodies were also produced against MSA-1 after intramuscular/intradermal inoculation with P3 and P4. Inoculation of rabbits with MSA-2 mRNA yielded better antibody titres when a poly A tail was present. Antibody levels were maintained for > 9 weeks after the final immunisation. However the immune sera failed to inhibit in vitro parasite growth.
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PMID:Mammalian cell expression of malaria merozoite surface proteins and experimental DNA and RNA immunisation. 998 40

In excitable cells, oscillations in intracellular free calcium concentrations ([Ca(2+)](i)) can arise from action-potential-driven Ca(2+) influx, and such signals can have either a localized or global form, depending on the coupling of voltage-gated Ca(2+) influx to intracellular Ca(2+) release pathway. Here we show that rat pituitary somatotrophs generate spontaneous [Ca(2+)](i) oscillations, which rise from fluctuations in the influx of external Ca(2+) and propagate within the cytoplasm and nucleus. The addition of caffeine and ryanodine, modulators of ryanodine-receptor channels, and the depletion of intracellular Ca(2+) stores by thapsigargin and ionomycin did not affect the global nature of spontaneous [Ca(2+)](i) signals. Bay K 8644, an L-type Ca(2+) channel agonist, initiated [Ca(2+)](i) signaling in quiescent cells, increased the amplitude of [Ca(2+)](i) spikes in spontaneously active cells, and stimulated growth hormone secretion in perifused pituitary cells. Nifedipine, a blocker of L-type Ca(2+) channels, decreased the amplitude of spikes and basal growth hormone secretion, whereas Ni(2+), a blocker of T-type Ca(2+) channels, abolished spontaneous [Ca(2+)](i) oscillations. Spiking was also abolished by the removal of extracellular Na(+) and by the addition of 10 mM Ca(2+), Mg(2+), or Sr(2+), the blockers of cyclic nucleotide-gated channels. Reverse transcriptase-polymerase chain reaction and Southern blot analyses indicated the expression of mRNAs for these channels in mixed pituitary cells and purified somatotrophs. Growth hormone-releasing hormone, an agonist that stimulated cAMP and cGMP productions in a dose-dependent manner, initiated spiking in quiescent cells and increased the frequency of spiking in spontaneously active cells. These results indicate that in somatotrophs a cyclic nucleotide-controlled plasma membrane Ca(2+) oscillator is capable of generating global Ca(2+) signals spontaneously and in response to agonist stimulation. The Ca(2+)-signaling activity of this oscillator is dependent on voltage-gated Ca(2+) influx but not on Ca(2+) release from intracellular stores.
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PMID:Characterization of a plasma membrane calcium oscillator in rat pituitary somatotrophs. 1058 49

Numerous renal abnormalities accompany thyroid disease, most of which have been ascribed to the effects of thyroid hormone on renal metabolism. In the present report, we investigate the renal expression of the nominally thyroid-specific proteins, thyroid-stimulating hormone (TSH) receptor (TSHR) and thyroglobulin (Tg), as potential links between renal and thyroid function. The expression of TSHR has been identified in several extrathyroidal tissues, but its presence in the kidney remains controversial. We have used reverse-transcriptase polymerase chain reaction and DNA sequencing to demonstrate the presence of TSHR transcript in human and mouse kidney, in a primary culture of human kidney, and in a green monkey kidney epithelioid cell line. Furthermore, human kidney cells responded to TSH with a 2.5- fold increase in intracellular cyclic adenosine monophosphate, suggesting the presence of functional TSHR protein. Comparison of renal expression of TSHR in a bovine growth hormone transgenic mouse model of progressive glomerulosclerosis with control mice suggested increased TSHR transcript in the renal cortex of transgenic animals. TSHR transcript was also detected in mouse mesangial cells in vitro which responded to TSH with significant increases in the formation of three-dimensional hillhocks. Polymerase chain reaction also confirmed the presence of Tg transcript in human and mouse kidneys and in mouse mesangial cells, but no effect of either TSH or cyclic adenosine monophosphate on Tg transcript levels could be discerned. Immunofluorescent staining with a monoclonal anti-Tg antibody identified positive staining in the cytoplasm of mesangial cells. These data suggest that the kidney is capable of expressing the thyroid-specific genes, TSHR and Tg, which could conceivably mediate effects of thyroid disease in the kidney.
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PMID:Renal expression of two 'thyroid-specific' genes: thyrotropin receptor and thyroglobulin. 1094 Jul 22

The GAGA box of the rat serine protease inhibitor 2 (spi 2) genes not only acts as a basal promoter element, but also mediates transcriptional activation by growth hormone and interleukin-6. The GAGA box is separated from the TATA box by only 12 bp, and this close association is required for efficient transcription. Hence, the GAGA box may influence transcription efficiency through interactions between GAGA box binding proteins and some components of the RNA polymerase II complex. Here we report the cloning of two GAGA box-binding proteins termed p38 and p40, that belong to the type A/B heterogeneous nuclear ribonucleoprotein subgroup. GAGA box mutations that diminish the affinity for p38 and p40 decrease basal and GH-induced reporter gene expression. Furthermore, nuclear extracts depleted of p38 and p40 can no longer support GAGA box-dependent in vitro transcription. Therefore, two polypeptides previously assigned to a family of RNA processing proteins also act as DNA-binding, promoter-specific transcription factors.
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PMID:Purification and cloning of type A/B hnRNP proteins involved in transcriptional activation from the Rat spi 2 gene GAGA box. 1115 60

Chinese Meishan pigs develop rapidly with onset of puberty at less than 100 days of age, and have a smaller placental size and larger litter size as compared with British/Continental breeds. POU1F1 is a member of the POU-domain family gene and is a positive regulator for growth hormone (GH), prolactin (PRL), and thyroid-stimulating hormone beta (TSHbeta) in several mammalian species. To investigate the role of POU1F1 in controlling pig growth and reproduction traits, Meishan (MS) pigs segregating a MspI POU1F1 polymorphism were used to determine differences of GH and PRL at both mRNA and circulating hormone concentrations. Animals from nine litters were used to collect pituitary (n=60) and/or blood samples (n=80) at day 0, 15, and 30 after birth, and all animals were genotyped (CC, CD, DD) for the MspI POU1F1 polymorphism. Reverse transcriptase-polymerase chain reaction (RT-PCR) with standard curve quantification was used to quantify mRNA levels for GH, PRL, and two alternative POU1F1 transcripts, POU1F1-alpha, and POU1F1-beta. Radioimmunoassays were done to determine the circulating concentration of GH and PRL in blood plasma. Our results indicated a significant effect of POU1F1 genotype on circulating levels of both GH and PRL at birth, but not thereafter. The DD neonates had lower levels of GH, but higher levels of PRL, than other genotypes. POU1F1-alpha mRNA decreased (P<0.05) from days 0 to 30, which paralleled decreases (P<0.05) in GH mRNA as well as PRL and GH plasma levels over the same period. POU1F1-beta mRNA levels did not significantly change over this period. Correlations were significant between POU1F1-alpha mRNA and both GH mRNA and GH plasma concentration levels, as well as between the two POU1F1 mRNA isoforms. Results from this study add to our understanding of the role of POU1F1 in controlling pig development and reproduction.
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PMID:Neonatal Meishan pigs show POU1F1 genotype effects on plasma GH and PRL concentration. 1181 32

The expression of the insulin-like growth factor II (IGF-II) gene (Igf2) in rodents is completely abrogated in almost all adult tissues. A prominent exception are neoplasms in which IGF-II frequently serves as an autocrine growth factor. We have investigated the potential role of Igf2 expression during liver carcinogenesis. After application of diethylnitrosamine (DEN) preneoplastic foci and adenomas emerged in liver tissue of wild-type and phosphoenolpyruvate carboxykinase (PEPCK)-IGF-II transgenic mice. Surprisingly, number and size of preneoplastic foci were not significantly increased in PEPCK-IGF-II mice as compared with wild-type animals. In situ preparation showed that early adenomas expressed Igf2 transcripts. Reverse transcriptase polymerase chain reaction (RT-PCR) and restriction enzyme analysis confirmed that DEN treatment had indeed reactivated the hepatic expression of murine Igf2 in control mice in a dose-dependent manner. This re-expression of Igf2 persisted for at least 18 months. Species-specific RT-PCR analyses also revealed the presence of murine Igf2 mRNAs in some PEPCK-IGF-II mice. A similar reactivation of Igf2 was detected in bovine growth hormone transgenic mice which develop hepatocellular neoplasms with high frequency. Our results suggest that reactivation of Igf2 is an early event during hepatocarcinogenesis in mice. Its appearance in two independent animal models suggests that Igf2 may be important at pivotal checkpoints of hepatocarcinogenesis.
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PMID:Diethylnitrosamine induces long-lasting re-expression of insulin-like growth factor II during early stages of liver carcinogenesis in mice. 1212 4


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