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)

We have cloned and determined the nucleotide (nt) sequence of a 6.5-kb genomic DNA fragment containing the rat MyoD gene (encoding a muscle regulatory factor, MyoD). Mouse fibroblasts transfected with this DNA display a high degree of conversion to a muscle phenotype, suggesting that this genomic clone contains sufficient sequence information to allow the production of the rat MyoD protein in these cells. The 6.5-kb genomic fragment contains the complete coding region of MyoD, distributed over three exons, plus 2.3 kb of 5'-noncoding sequence and 1.4 kb of 3'-noncoding sequence. Based on RNase protection assays, the major transcription start point of MyoD is located 210 nt 5' to a methionine start codon and 26 nt 3' to a TAAATA motif which bears similarity to a consensus recognition sequence (TATA) utilized by eukaryotic RNA polymerase II transcription complexes. The high degree of identity between the amino acid sequence of rat MyoD and the MyoD proteins isolated from other vertebrates indicates that this muscle regulatory protein has been evolutionarily conserved.
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PMID:Isolation and structural analysis of the rat MyoD gene. 132 78

Expression of the p53 gene plays an important role in the regulation of cellular proliferation and malignant transformation. Overexpression of mutant forms of p53 is in fact a common feature of many transformed cells. Studies dealing with the transcriptional regulatory regions of the p53 gene indicate that, unlike most promoters transcribed by RNA polymerase II, the p53 promoter contains no TATA-like sequence upstream of the transcription start site. Here we demonstrate that the murine p53 promoter contains a cis-acting element that maps downstream to the transcription initiation site. The integrity of this element is required for high-level expression from the promoter in transformed cells. By DNase I protection and mobility-shift analysis, we show that a nuclear factor binds to this downstream element through the consensus recognition sequence for the helix-loop-helix (HLH)-containing proteins of the myc/MyoD family of transcriptional regulators. We propose that the activity of one or more members of this family of transcription factors is an important determinant in the expression of p53 and that at least one level of p53 overexpression in transformed cells may thus be due to aberrant expression of the relevant factor(s). Furthermore, the possibility that the regulation of expression of p53 occurs, in part, by means of a potential HLH-containing factor provides a possible mechanism for the suppression of proliferation by the MyoD family of transcriptional regulators.
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PMID:Expression from the murine p53 promoter is mediated by factor binding to a downstream helix-loop-helix recognition motif. 185 94

A non-differentiating myoblastic cell line, INC2, and a differentiating cell line, COM3, were established from the mouse myoblastic cell line C2C12. Under differentiation conditions, both COM3 and INC2 cells stopped proliferation in a similar manner. The COM3 cells then differentiated into myotubes during the 4-day differentiation culture. In contrast, almost none of the INC2 cells differentiated into myotubes even in differentiation medium. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunoblot analyses showed that the levels of myogenin and MyoD proteins were significantly decreased in INC2 cells. The differentiation marker sarcomeric myosin heavy chain (MHC) was expressed in COM3 but not in INC2 cells. In contrast, both INC2 and COM3 cells expressed another myogenic regulatory factor, muscle LIM protein (MLP), in a differentiation condition-dependent manner. These results suggest that MLP gene expression is regulated in a myogenin/MyoD-independent manner. Enforced expression of the myogenin gene induced MHC expression in INC2 cells. Thus, the signaling pathway situated downstream is assumed to be intact in INC2 cells and suppression of myogenin, gene expression may be a primary defect in INC2 cells.
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PMID:Isolation of a differentiation-defective myoblastic cell line, INC-2, expressing muscle LIM protein under differentiation-inducing conditions. 922 3

A highly sensitive method of reverse-transcriptase polymerase chain reaction (RT-PCR) was established to quantify transcript levels of the myogenic regulatory factors MyoD, myogenin and MRF4 (muscle regulatory factor 4) and for Id-1 (inhibitor of differentiation), a putative negative regulator of myogenesis. The method was sensitive enough to detect mRNA amounts as low as 20 molecules. Measurements in 10 different skeletal muscles of the rat revealed that the amounts of the four factors differ by almost three orders of magnitude. Id-1 is expressed at lowest levels (approximately 4x10(5) molecules/microg RNA) and MRF4 at highest levels (approximately 9x10(7) molecules/microg RNA). In general, myogenin and MyoD mRNAs were inversely distributed in slow and fast muscles. A correlation seemed to exist between the levels of MyoD and myosin heavy chain (MHC) IIb, the fastest MHC isoform. However, as revealed by changes in the expression levels of these two regulatory factors under conditions of hypothyroidism and chronic low-frequency stimulation (CLFS), MyoD and myogenin did not seem to be strictly correlated with fast and slow myosins, respectively. Hypothyroidism led to pronounced depressions of MyoD, but only to small increases in myogenin mRNA in fast muscles. These changes were only slightly increased by CLFS. However, as previously shown, CLFS in combination with hypothyroidism induces in rat muscle pronounced fast to slow transitions in myosin expression [Kirschbaum, B. J., Kucher. H.-B., Termin, A., Kelly, A. M. & Pette, D. (1990) J. Biol. Chem. 265, 13974-13980]. These findings suggest that MyoD and myogenin may not be causally related to the development and maintenance of fiber-type diversities.
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PMID:Quantification of MyoD, myogenin, MRF4 and Id-1 by reverse-transcriptase polymerase chain reaction in rat muscles--effects of hypothyroidism and chronic low-frequency stimulation. 924 14

Ankyrin 1, an erythrocyte membrane protein that links the underlying cytoskeleton to the plasma membrane, is also expressed in brain and muscle. We cloned a truncated, muscle-specific ankyrin 1 cDNA composed of novel 5' sequences and 3' sequences previously identified in the last 3 exons of the human ankyrin 1 erythroid gene. Northern blot analysis revealed expression restricted to cardiac and skeletal muscle tissues. Deduced amino acid sequence of this muscle cDNA predicted a peptide of 155 amino acids in length with a hydrophobic NH2 terminus. Cloning of the corresponding chromosomal gene revealed that the ankyrin 1 muscle transcript is composed of four exons spread over approximately 10 kilobase pairs of DNA. Reverse transcriptase-polymerase chain reaction of skeletal muscle cDNA identified multiple cDNA isoforms created by alternative splicing. The ankyrin 1 muscle promoter was identified as a (G + C)-rich promoter located > 200 kilobase pairs from the ankyrin 1 erythroid promoter. An ankyrin 1 muscle promoter fragment directed high level expression of a reporter gene in cultured C2C12 muscle cells, but not in HeLa or K562 (erythroid) cells. DNA-protein interactions were identified in vitro at a single Sp1 and two E box consensus binding sites contained within the promoter. A MyoD cDNA expression plasmid transactivated an ankyrin 1 muscle promoter fragment/reporter gene plasmid in a dose-dependent fashion in both HeLa and K562 cells. A polyclonal antibody raised to human ankyrin 1 muscle-specific sequences reacted with peptides of 28 and 30 kDa on immunoblots of human skeletal muscle.
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PMID:An alternate promoter directs expression of a truncated, muscle-specific isoform of the human ankyrin 1 gene. 943 Jun 67

I-mfa (inhibitor of the MyoD family) is a transcription modulator that binds to and suppresses the transcriptional activity of MyoD family members. I-mfa transcripts are expressed in sclerotome, suggesting a role of I-mfa in skeletogenesis. The aim of this study was to examine the expression and regulation of I-mfa in osteoblasts. We found that I-mfa is expressed at a low level in an osteoblast-like cell line, MC3T3E1, and a pluripotent differentiation modulator, 1,25-dihydroxyvitamin D(3), specifically enhanced I-mfa mRNA expression. This effect was completely blocked by the presence of an RNA polymerase inhibitor, but not by a protein synthesis inhibitor, suggesting that 1,25-dihydroxyvitamin D(3) upregulates transcription of the I-mfa gene without requirement for new protein synthesis. Western blot analysis indicated that 1,25-dihydroxyvitamin D(3) increased the I-mfa protein levels severalfold in MC3T3E1 cells. I-mfa expression was also observed in primary mouse calvaria cells and ROS17/2.8 cells and 1,25-dihydroxyvitamin D(3) enhanced I-mfa expression in these cells. These data indicate that I-mfa is a novel transcriptional regulator gene expressed in osteoblasts and that its level is under the control of 1,25-dihydroxyvitamin D(3).
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PMID:Vitamin D(3) enhances the expression of I-mfa, an inhibitor of the MyoD family, in osteoblasts. 1138 74

Temperature influences many aspects of muscle development in herring (Clupea harengus). In Clyde herring, myofibril synthesis occurred later with respect to somite stage in embryos reared at 5 degrees C compared with 12 degrees C. The aim of the present study was to test the hypothesis that the relative timing of expression of myogenic regulatory factors (MRFs) and myosin heavy chain (MyHC) transcripts changes with developmental temperature. Reverse transcriptase/polymerase chain reaction (RT-PCR) was used to clone partial coding regions of MyoD, myogenin and MyHC from juvenile Clyde herring. Embryos were reared at 5, 8 and 12 degrees C, and the spatial and temporal expression patterns of transcripts were investigated using cRNA probes and in situ hybridisation. Antisense probes revealed a rostral-caudal progression of all three transcripts. MyoD transcription initially took place in the adaxial cells of the unsegmented, presomitic mesoderm, whereas myogenin transcription first occurred in newly formed somites. The MyHC gene transcript was not detected until approximately nine somites had formed. Since the somite stage at which the MRFs and MyHC were first expressed was independent of temperature, the hypothesis was rejected. We suggest that the effects of temperature on myofibril synthesis must occur downstream from MyHC transcription either at the level of translation or at the assembly stage.
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PMID:Embryonic temperature and the relative timing of muscle-specific genes during development in herring (Clupea harengus L.). 1171 29

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-specific gene expression at least in part by down-regulating the activity of these cdks. Unlike the other cdks, cdk9 is not serum- or cell cycle-regulated and is instead involved in the regulation of transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. While ectopic expression of cdk2 together with its regulatory subunits (cyclins E and A) inhibits myogenic transcription, overproduction of cdk9 and its associated cyclin (cyclin T2a) strengthens MyoD-dependent transcription and stimulates myogenic differentiation in both MyoD-converted fibroblasts and C2C12 muscle cells. Conversely, inhibition of cdk9 activity by a dominant negative form (cdk9-dn) represses the myogenic program. Cdk9, cyclinT2 and MyoD can be detected in a multimeric complex in C2C12 cells, with the minimal cdk9-binding region of MyoD mapping within 101-161 aa of the bHLH region. Finally, cdk9 can phosphorylate MyoD in vitro, suggesting the possibility that cdk9/cycT2a regulation of muscle differentiation includes the direct enzymatic activity of the kinase on MyoD.
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PMID:Activation of MyoD-dependent transcription by cdk9/cyclin T2. 1203 70

RNA polymerase II core subunit 3 (RPB3) is an a-like core subunit of RNA polymerase II (pol II). It is selectively down-regulated upon treatment with doxorubicin (dox). Due to the failure of skeletal muscle cells to differentiate when exposed to dox, we hypothesized that RPB3 is involved in muscle differentiation. To this end, we have isolated human muscle RPB3-interacting proteins by using yeast two-hybrid screening. It is of interest that an interaction between RPB3 and the myogenic transcription factor myogenin was identified. This interaction involves a specific region of RPB3 protein that is not homologous to the prokaryotic a subunit. Although RPB3 contacts the basic helix-loop-helix (HLH) region of myogenin, it does not bind other HLH myogenic factors such as MyoD, Myf5, and MRF4. Coimmunoprecipitation experiments indicate that myogenin contacts the pol II complex and that the RPB3 subunit is responsible for this interaction. We show that RPB3 expression is regulated during muscle differentiation. Exogenous expression of RPB3 slightly promotes myogenin transactivation activity and muscle differentiation, whereas the region of RPB3 that contacts myogenin, when used as a dominant negative molecule (Sud), counteracts these effects. These results indicate for the first time that the RPB3 pol II subunit is involved in the regulation of tissue-specific transcription.
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PMID:The alpha-like RNA polymerase II core subunit 3 (RPB3) is involved in tissue-specific transcription and muscle differentiation via interaction with the myogenic factor myogenin. 1220 9

To investigate the roles played by MyoD in the terminal differentiation of satellite cell-derived myoblasts, the effect of antisense inhibition of MyoD expression was examined in bovine adult myoblast culture, in which inhibition treatment was limited to the terminal differentiation phase. MyoD antisense oligonucleotide DNA (AS-mD) suppressed the formation of multinucleated myotubes in the cell culture. Myotube formation was suppressed even when AS-mD treatment was limited to the period preceding the onset of myotube formation. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that treatment with AS-mD suppressed the expression of myosin heavy chain embryonic isoform and troponin T isoforms at 4 days after the induction of differentiation. AS-mD also suppressed the expression of MRF4, but did not alter the expression of either Myf5 or myogenin, in contrast to previous results using mouse cells possessing MyoD(-/-) genetic background. These findings suggest that MyoD controls myogenesis but not Myf5 or myogenin mRNA expression during the terminal differentiation phase. Furthermore, among the alpha4, alpha5, alpha6, and alpha7 integrins, alpha4, alpha5, and alpha7 integrin expression was suppressed by AS-mD treatment, in parallel with the suppression of myotube formation, which suggests that MyoD controls myotube formation by regulating the expression of alpha4, alpha5, and alpha7 integrins.
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PMID:Effect of phase limited inhibition of MyoD expression on the terminal differentiation of bovine myoblasts: no alteration of Myf5 or myogenin expression. 1617 75


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