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)

A specific method for in situ-hybridization of slow myosin heavy chain MHCI (beta-cardiac MHC) mRNA was established with the use of a nonradioactively labeled cRNA probe. The digoxigenin-labeled probe was the T7-RNA polymerase transcript from a 350 bp SacI fragment of a rabbit beta-cardiac MHC cDNA. Northern blot analyses of RNA preparations from skeletal and cardiac muscles with homologous and complementary RNA proved the specificity of the hybridization. The in situ-hybridization was applied for studying the distribution of MHCI mRNA in normal fast- and slow-twitch muscles, as well as in muscles undergoing fast-to-slow transformation by chronic low-frequency stimulation. The majority of soleus muscle fibers was intensely stained, whereas fast-twitch muscles contained only a few positive fibers. The intracellular distribution of the hybridization product showed a clear relationship to the nuclei with intense staining of the perinuclear regions within the subsarcolemmal space. The more intensely stained fibers of transforming muscle displayed hybridization product also within the nuclei. As revealed by inspection of longitudinal sections at high magnification and polarized light, MHCI mRNA was also detectable in the myofibrils in a cross-striational pattern resulting from staining of the I-bands.
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PMID:In situ hybridization of slow myosin heavy chain mRNA in normal and transforming rabbit muscles with the use of a nonradioactively labeled cRNA. 170 75

Two genomic fragments were isolated from a normal and a dystrophic library containing the 3'OH terminus of the fast isoform of myosin heavy chain gene. Restriction map analysis confirmed that the genes were similar. The sequences coding for myosin were defined and shown to be the same in each genomic fragment. However, using a cDNA clone for tcRNA102 and two specific oligomers for tcRNA102 sequences, we determined that only the genomic fragment from normal chick contained homologous sequences to tcRNA102. Dystrophic chick DNA did not contain these regions of homology. In addition, the normal genomic fragment transcribes tcRNA102 in vitro via RNA polymerase III while the corresponding fragment of DNA from dystrophic chick was inactive. These results suggest that there are detectable differences between the normal and dystrophic genomes in this regard.
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PMID:The localization of a tcRNA102 gene near the 3' OH terminus of a fast myosin heavy chain gene. A comparison between normal and dystrophic chickens. 242 21

Miller-Dieker syndrome (MDS), a disorder manifesting the severe brain malformation lissencephaly ("smooth brain"), is caused, in the majority of cases, by a chromosomal microdeletion of the distal short arm of chromosome 17. Using human chromosome 17-specific DNA probes, we have begun a molecular dissection of the critical region for MDS. To localize cloned DNA sequences to the MDS critical region, a human-rodent somatic cell hybrid panel was constructed which includes hybrids containing the abnormal chromosome 17 from three MDS patients with deletions of various sizes. Three genes (myosin heavy chain 2, tumor antigen p53, and RNA polymerase II) previously mapped to 17p were excluded from the MDS deletion region and therefore are unlikely to play a role in its pathogenesis. In contrast, three highly polymorphic anonymous probes, YNZ22.1 (D17S5), YNH37.3 (D17S28), and 144-D6 (D17S34), were deleted in each of four patients with visible deletions, including one with a ring chromosome 17 that is deleted for a portion of the single telomeric prometaphase subband p13.3. In two MDS patients with normal chromosomes, a combination of somatic cell hybrid, RFLP, and densitometric studies demonstrated deletion for YNZ22.1 and YNH37.3 in the paternally derived 17's of both patients, one of whom is also deleted for 144-D6. The results indicate that MDS can be caused by submicroscopic deletion and raises the possibility that all MDS patients will prove to have deletions at a molecular level. The two probes lie within a critical region of less than 3,000 kb and constitute potential starting points in the isolation of genes implicated in the severe brain maldevelopment in MDS.
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PMID:Molecular detection of microscopic and submicroscopic deletions associated with Miller-Dieker syndrome. 318 30

The complete amino acid sequence (1961 amino acids) of a vertebrate cellular myosin heavy chain-A was deduced from cDNA clones of a secretory rat mast cell line, the RBL-2H3 cell. The rat, human and chicken cellular myosin heavy chain-A exhibited high similarity in domains that allow binding of ATP and actin. The amino acid sequence of non-muscle myosin heavy chain-A from rat was 96% identical to that in human and 92% identical to that in chicken. Northern blot analysis of mRNA indicated the presence of single message of 7.4 kilobases. Northern blot, reverse-transcriptase polymerase chain reaction, and Western blot with isoform-specific antibodies indicated that RBL-2H3 cells expressed exclusively myosin heavy chain-A. Unlike rat PC12 cells, as well as a wide variety of other cultured cells and tissues, myosin heavy chain-B mRNA and protein were not detectable in RBL-2H3 cells. Because RBL-2H3 cells can be stimulated to release secretory granules as well as newly generated arachidonic acid and cytokines but lack myosin heavy chain-B, this cell line may provide a unique model to study the role of myosin heavy chain-A in cellular responses to antigen and other stimulants.
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PMID:Cloning of the cDNA encoding rat myosin heavy chain-A and evidence for the absence of myosin heavy chain-B in cultured rat mast (RBL-2H3) cells. 874 Apr 33

In the last decade, as a result of molecular cloning and the reverse-transcriptase polymerase chain reaction, numerous isoforms of the contractile protein myosin have been discovered. What lags behind their discovery is knowledge of their functions. This review focuses on some of my recent work on the structure, function and regulation of isoforms of the heavy chain of vertebrate smooth muscle and nonmuscle myosin II. Reference to related work in the field is included where appropriate. The particular isoforms discussed are those that are generated by alternative splicing near the 5' end of the pre-mRNA, resulting in either an insertion or a deletion of a cassette of amino acids near the amino-terminus of the myosin heavy chain (MHC) protein. In both the smooth muscle and nonmuscle MHCs, this splicing occurs in the exact same region, which begins at amino acid 212 in the primary sequence. In the three-dimensional structure of the molecule, these inserts are located near the ATP-binding pocket in a region of the MHC that was not resolved in the crystal structure and therefore is believed to represent a flexible loop. In the smooth muscle MHC, the insertion of seven amino acids in this loop confers a higher enzymatic activity on the myosin. The potential mechanism by which this occurs and the significance to smooth muscle contractile diversity is discussed. In the nonmuscle MHC, the insert in this region is a different size and sequence of amino acids than that in the smooth muscle MHC. A serine residue (Ser-214) in the nonmuscle loop is phosphorylated by p34cdc2 kinase in Xenopus during meiotic maturation of oocytes to eggs and is dephosphorylated in interphase egg extracts that are equivalent to the interphase after fertilization of the egg. Thus, MHC-B phosphorylation by cdc2 kinase correlates with the cortical reorganization that occurs during meiosis, and dephosphorylation correlates with the cortical contraction that occurs at fertilization, which aids in pronuclear fusion. In summary, these inserts in the MHC molecule, in a flexible loop near the ATP-binding pocket, appear to be important in determining differences in function or regulation among myosin II isoforms.
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PMID:Characterization of isoform diversity among smooth muscle and nonmuscle myosin heavy chains. 918 13

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

We have derived a cardiac muscle cell line, designated HL-1, from the AT-1 mouse atrial cardiomyocyte tumor lineage. HL-1 cells can be serially passaged, yet they maintain the ability to contract and retain differentiated cardiac morphological, biochemical, and electrophysiological properties. Ultrastructural characteristics typical of embryonic atrial cardiac muscle cells were found consistently in the cultured HL-1 cells. Reverse transcriptase-PCR-based analyses confirmed a pattern of gene expression similar to that of adult atrial myocytes, including expression of alpha-cardiac myosin heavy chain, alpha-cardiac actin, and connexin43. They also express the gene for atrial natriuretic factor. Immunohistochemical staining of the HL-1 cells indicated that the distribution of the cardiac-specific markers desmin, sarcomeric myosin, and atrial natriuretic factor was similar to that of cultured atrial cardiomyocytes. A delayed rectifier potassium current (IKr) was the most prominent outward current in HL-1 cells. The activating currents displayed inward rectification and deactivating current tails were voltage-dependent, saturated at >>+20 mV, and were highly sensitive to dofetilide (IC50 of 46.9 nM). Specific binding of [3H]dofetilide was saturable and fit a one-site binding isotherm with a Kd of 140 +/- 60 nM and a Bmax of 118 fmol per 10(5) cells. HL-1 cells represent a cardiac myocyte cell line that can be repeatedly passaged and yet maintain a cardiac-specific phenotype.
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PMID:HL-1 cells: a cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte. 950 Dec 1

We report the identification and cloning of a unique chick myosin heavy chain (CMHC1) that is expressed exclusively in the heart during embryogenesis. Using primers specific to myosin heavy chains, we used reverse transcriptase-polymerase chain reaction to clone and isolate CMHC1 from embryonic day 10 chicken heart RNA. Sequence analysis indicated that CMHC1 was a novel member of the myosin heavy chain family. Expression of the CMHC1 transcripts was detected in Hamburger Hamilton stage 10 chick embryos in the fusing myocardium. Expression of CMHC1 was maintained at high levels throughout the tubular heart of later stage embryos. Reverse transcriptase-polymerase chain reaction and in situ hybridizations failed to detect CMHC1 transcripts in the developing somites, limb buds, or skeletal musculature at any stage of chick development. Genomic CMHC1 clones have been isolated that contain sequences approximately 5.2 kilobase upstream of the presumptive CMHC1 transcription start site. Portions of the upstream regulatory region induced a 21-fold increase in reporter gene expression in primary cardiomyocytes. Because of its unique cardiac-restricted expression, CMHC1 will provide an excellent model system to study the molecular mechanisms required for the early developmental regulation of heart-specific genes.
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PMID:Identification and genomic cloning of CMHC1. A unique myosin heavy chain expressed exclusively in the developing chicken heart. 1063 96

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


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