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)

It has previously been demonstrated that the epidermis is a rich source of proinflammatory cytokines and growth factors and that complex interactions between these factors may affect inflammatory responses in skin. To investigate whether IL-10 (cytokine synthesis inhibitory factor) is part of this complex process, RNA was extracted from normal epidermis at various times after application of various chemicals to murine skin and mRNA signals for IL-10 were sought using a quantitative reverse-transcriptase-polymerase chain reaction technique. IL-10 signal strength was normalized to that of beta-actin in each sample. IL-10 mRNA signals were occasionally identified in normal epidermis but were uniformly enhanced 4 h after hapten application, and were maximal after 12 h. Contact allergens induced IL-10 mRNA signals whereas vehicles and irritants did not. Depletion of Langerhans cells, Thy-1+ dendritic epidermal cells, and T lymphocytes demonstrated that keratinocytes were the main source of IL-10 mRNA. IL-10 signals were also detected in mRNA derived from PAM 212 (spontaneously transformed keratinocyte) cells. IL-10 protein could be detected by immunoprecipitation, with an IL-10 mAB, of supernatants obtained 16 h after cultured epidermal cells were coupled with hapten. This study demonstrates that murine keratinocytes are capable of producing IL-10 mRNA and protein, and that signal strength of IL-10 mRNA is enhanced by hapten application.
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PMID:Identification and induction of keratinocyte-derived IL-10. 160 65

For most genetic deficiencies manifested in the liver, maximization of gene expression in hepatocytes will be an important factor in achieving successful gene therapy. A rapid, highly efficient, and nontoxic method for transfecting DNA into hepatocytes was used to compare directly promoter strengths of various cellular and viral promoters. Conditions are described here for transfecting 5-10% of primary hepatocytes using the positively charged liposomes, Lipofectin. Cells are not damaged by this method as they continue to transcribe genes controlled by liver specific promoters and can survive for over 2 weeks in culture. We find that the cytomegalovirus, SR alpha, and beta-actin promoters are more active than the SV40, RSV, RNA polymerase II, albumin, alpha 1-antitrypsin, or phosphoenolpyruvate carboxykinase promoters. A simple TK promoter and a TK promoter with the polyoma enhancer (MCI) were almost completely inactive. This information will be useful in the construction of vectors designed to express genes efficiently in primary hepatocytes for purposes of gene therapy, although the stability of expression from these promoters will need to be demonstrated in hepatocytes in vivo.
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PMID:Evaluation of relative promoter strength in primary hepatocytes using optimized lipofection. 186 38

The anthracycline antibiotic doxorubicin produces a characteristic myopathy in cardiac muscle that limits its use in cancer therapy. We have shown in cultured neonatal rat cardiac muscle cells that doxorubicin treatment resulted in a rapid, selective decrease in the expression of muscle-specific genes, which preceded other changes characteristic of doxorubicin cardiomyopathy. Doxorubicin selectively and dramatically decreased the levels of mRNA for the sarcomeric genes, alpha-actin, troponin I, and myosin light chain 2, as well as the muscle-specific, but nonsarcomeric M isoform of creatine kinase. However, doxorubicin did not affect nonmuscle gene transcripts (pyruvate kinase, ferritin heavy chain, and beta-actin). Actinomycin D, an inhibitor of DNA-dependent RNA polymerase, did not show a similar selective decrease of muscle-specific mRNAs but, rather, produced a nonspecific, dose-dependent decrease of muscle and nonmuscle transcripts. The doxorubicin effect on muscle gene expression was limited to cardiac muscle; cultured skeletal myocytes were resistant to the effects of doxorubicin at 100-fold greater doses than those causing changes in mRNA levels in cardiac muscle cells. These effects of doxorubicin were reproduced in vivo; rats injected with doxorubicin showed a dose-dependent decrease in the levels of mRNAs for alpha-actin, troponin I, myosin light chain 2, and M isoform of creatine kinase in cardiac but not skeletal muscle. These selective changes in gene expression in cardiocyte cultures and cardiac muscle precede classical ultrastructural changes and may explain the myofibrillar loss that characterizes doxorubicin cardiac injury.
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PMID:Doxorubicin selectively inhibits muscle gene expression in cardiac muscle cells in vivo and in vitro. 234 36

Eight eukaryotic promoters have been tested for their activity in vivo in Escherichia coli. The rat beta-actin, rat amylase, rat chymotrypsin B, mouse metallothionein I, rat insulin I, human insulin, Rous sarcoma virus long terminal repeat (RSV LTR) and hepatitis B viral precore promoter activities were measured by using the bacterial chloramphenicol acetyltransferase coding sequences as the reporter function and by primer extension RNA analysis. All eight promoter-chloramphenicol acetyltransferase constructs produce chloramphenicol acetyltransferase activity with the following relative strengths: RSV LTR greater than rat beta-actin greater than rat insulin I greater than rat amylase greater than hepatitis B virus precore greater than human insulin greater than rat chymotrypsin B greater than mouse metallothionein I. A primer extension analysis indicates that transcription from the RSV LTR, rat insulin I, and rat beta-actin promoters initiates at the sites expected for eukaryotic rather than prokaryotic promoters. Thus the site of initiation is determined by the DNA sequence rather than by the RNA polymerase.
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PMID:Eukaryotic promoters drive gene expression in Escherichia coli. 268 Nov 82

Influenza virus infection has adverse effects on the metabolism of two representative RNA polymerase II transcripts in chicken embryo fibroblasts, those coding for beta-actin and for avian leukosis virus (ALV) proteins. Proviral ALV DNA was integrated into host cell DNA by prior infection with ALV. Within 1 h after influenza virus infection, the rate of transcription of beta-actin and ALV sequences decreased 40 to 60%, as determined by labeling the cells for 5 min with [3H]uridine and by in vitro, runoff assays with isolated nuclei. The transcripts that continued to be synthesized did not appear in the cytoplasm as mature mRNAs, and the kinetics of labeling of these transcripts strongly suggest that they were degraded in the nucleus. By S1 endonuclease assay, it was confirmed that nuclear ALV transcripts disappeared very early after infection, already decreasing ca. 80% by 1 h postinfection. A plausible explanation for this nuclear degradation is that the viral cap-dependent endonuclease in the nucleus cleaves the 5' ends of new polymerase II transcripts, rendering the resulting decapped RNAs susceptible to hydrolysis by cellular nucleases. In contrast to the nuclear transcripts, cytoplasmic beta-actin and ALV mRNAs, which are synthesized before infection, were more stable and did not decrease in amount until after 3 h postinfection. Similar stability of cytoplasmic host cell mRNAs was observed in infected HeLa cells, in which the levels of actin mRNA and two HeLa cell mRNAs (pHe 7 and pHe 28) remained at undiminished levels for 3 h of infection and decreased only slightly by 4.5 h postinfection. The cytoplasmic actin and pHe 7 mRNAs isolated from infected HeLa cells were shown to be translated in reticulocyte extracts in vitro, indicating that host mRNAs were not inactivated by a virus-induced modification. Despite the continued presence of high levels of functional host cell mRNAs, host cell protein synthesis was effectively shut off by about 3 h postinfection in both chicken embryo fibroblasts and HeLa cells. These results are consistent with the establishment of an influenza virus-specific translational system that selectively translates viral and not host mRNAs.
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PMID:Metabolism and expression of RNA polymerase II transcripts in influenza virus-infected cells. 609 46

Normal C57BL/10SnJ myoblasts were transplanted into the tibialis anterior of C57BL/10SnJ, C57BL/ScSn mdx, or BALB/c mice. These transplantations allowed us to investigate the immune response not only against MHC but also against dystrophin introduced in the dystrophic muscles by such transplantations. Recently, our group reported following myoblast transplantations cellular infiltration of the host muscle by class II MHC cells, macrophages, and lymphocytes expressing CD4 or CD8 and IL-2 receptors. In the present study, activation of these infiltrating lymphocytes was investigated by measuring the expression of granzyme B mRNA. We used reverse-transcriptase polymerase chain reaction to detect granzyme B mRNA at various intervals after myoblast transplantations. To standardize the results, the mRNA were reverse transcribed using an oligo (dt) so that beta-actin mRNA could also be amplified from the same cDNA preparation. Granzyme B mRNA was increased for at least 3 weeks after MHC alloincompatible grafts. The absence of increased granzyme B expression after allocompatible transplantation in mdx mice suggests that dystrophin is not sufficiently immunogenic to induce short term acute rejection. These results indicate that lymphocytes infiltrated in muscles injected with histoincompatible myoblasts are activated and sustain the requirement for an adequate immunosuppression after such transplantations.
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PMID:Increased granzyme B mRNA after alloincompatible myoblast transplantation. 749 74

Thirty-five patients with Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) were classified on the basis of the fusion pattern of bcr-abl mRNA determined by the reverse-transcriptase-polymerase chain reaction (RT-PCR) method. Semiquantitative assay of the bcr exon 2/abl exon 2 fused mRNA (b2-a2) and bcr exon 3/abl exon 2 fused mRNA (b3-a2) resulted in 21 patients showing b3-a2 type mRNA, seven showing b2-a2 type and seven showing coexpression. Quantification of the autoradiographic signals of amplified products was estimated using an MCID image analysis system. The relative intensity was defined as the ratio of bcr-abl signal to that of beta-actin. The relationship between the semiquantified bcr-abl mRNA and the platelet/megakaryocyte counts was analyzed. A possible correlation was found between the semiquantified b3-a2 type mRNA and the platelet (p < .05, N = 28) and megakaryocyte (p < .05, N = 13) counts of these patients. This finding suggests the possibility that b3-a2 mRNA may affect the thrombopoietic activity in Ph1-positive CML in a dose-response manner.
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PMID:Possible correlation of b3-a2-type bcr-abl messenger RNA defined by semiquantitative RT-PCR to platelet and megakaryocyte counts in Philadelphia-positive chronic myelogenous leukemia. 752 Jul 86

Hodgkin's disease (HD) and Ki-1 positive anaplastic large cell lymphoma (Ki-1 ALCL) appear pathologically and immunohistochemically related, and a common histogenesis has been postulated in at least some cases. The breakpoints of the t(2;5) (p23;q35) [corrected] translocation, which is reported in about 40% of Ki-1 ALCL, have recently been cloned. They involve a novel tyrosine kinase gene, ALK, at 2p23 and the nucleophosmin gene, NPM, at 5q35. Reverse transcriptase polymerase chain reaction (RT-PCR) using NPM and ALK primers consistently detects a fusion product in Ki-1 ALCL cases with the translocation. To determine if this tumor-specific genetic alteration also occurs in HD, we performed NPM-ALK RT-PCR on RNA samples extracted from 40 lymph node biopsies of HD (25 nodular sclerosis, 11 mixed cellularity, 2 lymphocyte depleted, 2 lymphocyte predominant). Using control samples, the sensitivity of the NPM-ALK RT-PCR assay was shown to be at least 1:10(4). Amplifiable template was confirmed in all samples by RT-PCR using beta-actin primers. None of the 40 cases showed the expected 177-bp RT-PCR product indicative of the translocation. We conclude that the most common primary genetic alteration in Ki-1 ALCL, the t(2;5), is absent or very infrequent in typical cases of HD. These results further support the concept that HD and Ki-1 ALCL are pathogenetically distinct entities.
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PMID:Reverse transcriptase polymerase chain reaction for the Ki-1 anaplastic large cell lymphoma-associated t(2;5) translocation in Hodgkin's disease. 752 17

We developed a non-radioisotopic quantitative RT-PCR method with high sensitivity and reproducibility. The results of this RT-PCR were in agreement with those of the Northern blot analysis. We measured the mRNA levels of beta-actin, transferrin receptor, and two cell cycle-related genes, cyclin B and cdc25, in early mouse embryos by the RT-PCR. In late two-cell stage embryos, beta-actin, transferrin receptor and cyclin B mRNA levels were 10-20% of those in MII stage oocytes. In contrast, the cdc25 mRNA levels were not different between these stages. When we cultured mouse embryos, the presence of an RNA polymerase inhibitor, alpha-amanitin, in the medium did not affect the mRNA levels at the two-cell stage, indicating that most of the detected mRNAs in two-cell embryos were maternally derived. These results suggest that the rate of mRNA degradation is different between cyclin B and cdc25 during early embryogenesis.
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PMID:Non-radioisotopic quantitative RT-PCR to detect changes in mRNA levels during early mouse embryo development. 769 May 61

Spontaneous production of insulin-like growth factor-I (IGF-I) by inflammatory macrophages contributes to aberrant wound healing, but little is known about regulation of IGF-I synthesis in myeloid cells. The T cell-derived cytokine interferon-gamma (IFN gamma) inhibits several fibrogenic and angiogenic components of the wound-healing response. We have used metabolic labeling of primary colony stimulating factor-1 (CSF-1)-derived macrophages and a transformed macrophage cell line (PU5-1R) followed by immunoprecipitation to demonstrate that synthesis of the 17 kilodalton (kDa) prepro-IGF-I protein by these cells is substantially inhibited by IFN gamma. An exon 4 IGF-I/beta-actin riboprobe expression cassette was used in RNase protection assays to show that IFN gamma also reduces steady state levels of IGF-I mRNA in three different populations of macrophages in a time- and dose-dependent manner. This effect is specific for IFN gamma because neither the IFNs-alpha/beta nor lipopolysaccharide (LPS) affects expression of steady state IGF-I transcripts. Down-regulation of IGF-I mRNA by IFN gamma is dependent on de novo protein synthesis and is abrogated by coculture with cycloheximide. Nuclear run-on assays revealed that elongation of IGF-I transcripts is absent in fresh bone marrow cells but is induced several-fold after cells are cultured for 6 days with CSF-1. Treatment of these CSF-1-derived macrophages with IFN gamma for 6 h substantially inhibits synthesis of IGF-I mRNA. Studies on the decay of IGF-I mRNA in PU5-1R macrophages treated with an RNA polymerase inhibitor confirmed that the decline in IGF-I steady state mRNA in IFN gamma-treated cultures arises from an inhibition of transcription rather than from a reduction in mRNA stability. Since a variety of inflammatory mediators can induce expression of IGF-I in macrophages, inhibition of macrophage IGF-I synthesis by IFN gamma provides a mechanism by which leukocytes regulate levels of this growth factor in their microenvironment.
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PMID:Interferon-gamma inhibits macrophage insulin-like growth factor-I synthesis at the transcriptional level. 777 81


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