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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report here for the first time direct injection of genes into fish muscle in vivo. Plasmids used contain either SV40 early promoter, rabbit beta-cardiac myosin heavy chain promoter, human MxA promoter or an artificial promoter, fused to a chloramphenicol acetyltransferase (CAT) or beta-galactosidase reporter gene. CAT assays revealed that most gene constructs were highly expressed. Histochemical analysis showed that beta-galactosidase was strongly expressed at the site of injection within muscle fibres. This method provides an excellent system for testing expression of gene constructs, including those of mammalian origin, in fish muscle in vivo and has the potential for fish vaccination.
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PMID:Strong expression of foreign genes following direct injection into fish muscle. 191 96

A cDNA clone coding for an internal fragment of slow-cardiac beta-myosin heavy chain was isolated from a lambda gt10 human skeletal muscle library. Six overlapping cDNA subclones, which span myosin heavy chain subregions and presumably interact with actin, were derived from this clone, fused to a beta-galactosidase vector and expressed in Escherichia coli. Three of the subclones were obtained by PCR (polymerase chain reaction) which enables gene or cDNA fragments to be amplified independently of preexisting restriction sites. Initially, various experiments were carried out using a long MHC (myosin heavy chain) fusion protein containing the 50 kDa-20 kDa connecting region, the whole 20 kDa region and the short subfragment 2 region. This MHC fusion protein was chemically or proteolytically cleaved in the same conditions as the native myosin molecule. Whole and truncated forms of the MHC fusion protein were separated on polyacrylamide gels, electroblotted on nitrocellulose sheets and renatured. They were then assayed in overlay experiments with F-actin and/or myosin light chains in solution. Specific antibodies were used to detect interactions between heavy chain fragments and F-actin or light chains. We thus observed that one long heavy chain fragment synthesized by E. coli behaved like proteolytic or chemical MHC preparations made from native myosin molecules. Two chymotryptic fragments of the MHC fusion protein, which are soluble at low ionic strength, cosedimented with F-actin in solution. Our results demonstrate that, in actin overlay experiments with whole fusion proteins, interactions seem to be due to the heavy chain fragment, not to the bacterial component. All interactions were non ATP-sensitive. We further investigated the possible participation of the six recombinant MHC fragments in contributing to the actomyosin interfaces on the 50 kDa-20 kDa regions of the human cardiac beta-MHC. The present procedure, which enables the synthesis of any MHC fragment independent of any protease site, is a powerful new tool for studying structure-function relationships within the myosin molecule family.
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PMID:Expression of human beta-myosin heavy chain fragments in Escherichia coli; localization of actin interfaces on cardiac myosin. 226 65

The nematode Caenorhabditis elegans produces four distinct myosin heavy chain (MHC) isoforms, A, B, C, and D. The MHC A and MHC B proteins are coordinately expressed in the body wall muscle and are incorporated into different regions of a single kind of thick filament. MHC C and MHC D are exclusively produced in the pharyngeal muscle. Previous studies of mutations that affect MHC B have shown that this isoform is encoded by the unc-54 gene. Three other MHC genes, myo-1, myo-2, and myo-3, were isolated from a C. elegans genomic library by hybridization with fragments of the unc-54 gene. We have now established the MHC isoform encoded by each gene. Restriction fragments from each of these genes were cloned in the plasmid expression vector pUR288, producing fusion proteins between Escherichia coli beta-galactosidase and portions of the MHC rod domains of each gene. The hybrid proteins were screened with a panel of 18 isoform-specific monoclonal antibodies. The results demonstrate that myo-1 encodes MHC D, myo-2 encodes MHC C, and myo-3 encodes MHC A.
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PMID:Immunological identification of the genes encoding the four myosin heavy chain isoforms of Caenorhabditis elegans. 242 55

A cDNA expression strategy was used to localize amino acid sequences which were specific for fast, as opposed to slow, isoforms of the chicken skeletal muscle myosin heavy chain (MHC) and which were conserved in vertebrate evolution. Five monoclonal antibodies (mAbs), termed F18, F27, F30, F47, and F59, were prepared that reacted with all of the known chicken fast MHC isoforms but did not react with any of the known chicken slow nor with smooth muscle MHC isoforms. The epitopes recognized by mAbs F18, F30, F47, and F59 were on the globular head fragment of the MHC, whereas the epitope recognized by mAb F27 was on the helical tail or rod fragment. Reactivity of all five mAbs also was confined to fast MHCs in the rat, with the exception of mAb F59, which also reacted with the beta-cardiac MHC, the single slow MHC isoform common to both the rat heart and skeletal muscle. None of the five epitopes was expressed on amphioxus, nematode, or Dictyostelium MHC. The F27 and F59 epitopes were found on shark, electric ray, goldfish, newt, frog, turtle, chicken, quail, rabbit, and rat MHCs. The epitopes recognized by these mAbs were conserved, therefore, to varying degrees through vertebrate evolution and differed in sequence from homologous regions of a number of invertebrate MHCs and myosin-like proteins. The sequence of those epitopes on the head were mapped using a two-part cDNA expression strategy. First, Bal31 exonuclease digestion was used to rapidly generate fragments of a chicken embryonic fast MHC cDNA that were progressively deleted from the 3' end. These cDNA fragments were expressed as beta-galactosidase/MHC fusion proteins using the pUR290 vector; the fusion proteins were tested by immunoblotting for reactivity with the mAbs; and the approximate locations of the epitopes were determined from the sizes of the cDNA fragments that encoded a particular epitope. The epitopes were then precisely mapped by expression of overlapping cDNA fragments of known sequence that covered the approximate location of the epitopes. With this method, the epitope recognized by mAb F59 was mapped to amino acids 211-231 of the chicken embryonic fast MHC and the three distinct epitopes recognized by mAbs F18, F30, and F47 were mapped to amino acids approximately 65-92. Each of these epitope sequences is at or near the ATPase active site.
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PMID:Evolutionarily conserved sequences of striated muscle myosin heavy chain isoforms. Epitope mapping by cDNA expression. 247 86

The Saccharomyces yeast plasmid, 2-micron circle, encodes a partitioning system that ensures equidistribution of plasmid molecules to both progeny following cell division. This system consists of two proteins encoded in plasmid genes REP1 and REP2 and a cis-active noncoding locus, designated REP3. We have raised antibodies against a REP1 beta-galactosidase fusion protein and used them to identify the authentic REP1 protein in plasmid-bearing yeast cells. We find that REP1 protein is located exclusively in the nucleus and co-purifies with a karyoskeletal protein subfraction operationally and morphologically equivalent to the nuclear matrix-pore complex-lamina fraction of higher cells. The carboxyl half of the REP1 protein exhibits strong sequence homology to myosin heavy chain, vimentin, and nuclear lamins A and C, indicating a fibrous structure for the protein. From these observations, we suggest that REP1 protein may promote plasmid partitioning by intercalating into the nuclear lamina of the host cell to provide dispersed anchorage sites for attachment of plasmid molecules.
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PMID:A yeast plasmid partitioning protein is a karyoskeletal component. 354 94

Cardiomyocytes differentiated in vitro from pluripotent embryonic stem (ES) cells of line D3 via embryo-like aggregates (embryoid bodies) were characterized by the whole-cell patch-clamp technique during the entire differentiation period. Spontaneously contracting cardiomyocytes were enzymatically isolated by collagenase from embryoid body outgrowths of early, intermediate, and terminal differentiation stages. The early differentiated cardiomyocytes exhibited an outwardly rectifying, transient K+ current sensitive to 4-aminopyridine and an inward Ca2+ current but no Na+ current. The Ca2+ current showed all features of L-type Ca2+ current, being highly sensitive to 1,4-dihydropyridines but not to omega-conotoxin. Cardiomyocytes of intermediate stage were characterized by the additional expression of cardiac-specific Na+ current, the delayed K+ current, and If current. Terminally differentiated cardiomyocytes expressed a Ca2+ channel density about three times higher than that of early stage. In addition, two types of inwardly rectifying K+ currents (IK1 and IK,Ach) and the ATP-modulated K+ current were found. During cardiomyocyte differentiation, several distinct cell populations could be distinguished by their sets of ionic channels and typical action potentials presumably representing cardiac tissues with properties of sinus node, atrium, and ventricle. Reverse transcription polymerase chain reaction revealed the transcription of alpha- and beta-cardiac myosin heavy chain (MHC) genes synchronously with the first spontaneous contractions. Transcription of embryonic skeletal MHC gene at intermediate and terminal differentiation stages correlated with the expression of Na+ channels. The selective expression of alpha-cardiac MHC gene in ES cell-derived cardiomyocytes was demonstrated after ES cell transfection of the LacZ construct driven by the alpha-cardiac MHC promoter region followed by ES cell differentiation and beta-galactosidase staining. In conclusion, our data demonstrate that ES cell-derived cardiomyocytes represent a unique model to investigate the early cardiac development and permit pharmacological/toxicological studies in vitro.
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PMID:Cardiomyocytes differentiated in vitro from embryonic stem cells developmentally express cardiac-specific genes and ionic currents. 803 37

Fetal cardiomyocytes isolated from transgenic mice carrying a fusion gene of the alpha-cardiac myosin heavy chain promoter with a beta-galactosidase reporter were examined for their ability to form stable intracardiac grafts. Embryonic day 15 transgenic cardiomyocytes delivered directly into the myocardium of syngeneic hosts formed stable grafts, as identified by nuclear beta-galactosidase activity. Grafted cardiomyocytes were observed as long as 2 months after implantation, the latest date assayed. Intracardiac graft formation did not induce overtly negative effects on the host myocardium and was not associated with chronic immune rejection. Electron microscopy revealed the presence of nascent intercalated disks connecting the engrafted fetal cardiomyocytes and the host myocardium. These results suggest that intracardiac grafting might provide a useful approach for myocardial repair, provided that the grafted cells can contribute to myocardial function.
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PMID:Formation of nascent intercalated disks between grafted fetal cardiomyocytes and host myocardium. 814 Apr 16

We analyzed, in transgenic mice, the cellular expression pattern of the quail fast skeletal muscle troponin I (TnIfast) gene and of a chimeric reporter construct in which quail TnIfast DNA sequences drive expression of E. coli beta-galactosidase (beta-gal). Both constructs were actively expressed in skeletal muscle and specifically in fast, as opposed to slow, muscle fibers. Unexpectedly, both constructs showed a marked differential expression among the adult fast fiber subtypes according to the pattern IIB > IIX > IIA. This expression pattern was consistent in multiple lines and differed from the endogenous mouse TnIfast pattern, which shows approximately equal expression in all fast fibers. These observations indicate that distinct regulatory mechanisms contribute to high-level expression of TnIfast in the various fast fiber subtypes and suggest that the outwardly simple pattern of equal expression in all fast fiber types shown by the endogenous mouse TnIfast gene is based on an intricate system of counterbalancing mechanisms. The adult expression pattern of the TnIfast/beta-gal construct emerged in a two-stage developmental process. Differential expression in fast versus slow fibers was evident in neonatal animals, although expression in fast fibers was relatively weak and homogeneous. During the first two weeks of postnatal life, expression in maturing IIB fibers was greatly increased whereas that in IIA/IIX fibers remained weak, giving rise to marked differential expression among fast fiber types. Thus at least two serially acting (pre- and post-natal) fiber-type-specific regulatory mechanisms contribute to high-level gene expression in adult fast muscle fibers. Unexpected similarities between TnIfast transgene expression and that of the myosin heavy chain gene family (which includes differentially expressed IIB-, IIX- and IIA-specific members) suggest that similar mechanisms may regulate adult fast muscle gene expression in a variety of unrelated muscle gene families.
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PMID:Complex fiber-type-specific expression of fast skeletal muscle troponin I gene constructs in transgenic mice. 818 38

Myocardial infarcts heal by scar formation because there are no stem cells in myocardium, and because adult myocytes cannot divide and repopulate the wound. We sought to redirect the heart to form skeletal muscle instead of scar by transferring the myogenic determination gene, MyoD, into cardiac granulation (wound repair) tissue. A replication-defective adenovirus was constructed containing MyoD under transcriptional control of the Rous sarcoma virus long terminal repeat. The virus converted cultured cardiac fibroblasts to skeletal muscle, indicated by expression of myogenin and skeletal myosin heavy chains (MHCs). To determine if MyoD could induce muscle differentiation in vivo, we injected 2 x 10(9) or 10(10) pfu of either the MyoD or a control beta-galactosidase adenovirus into healing rat hearts, injured 1 wk previously by freeze-thaw. After receiving the lower viral dose, cardiac granulation tissue expressed MyoD mRNA and protein, but did not express myogenin or skeletal MHC. When the higher dose of virus was administered, double immunostaining showed that cells in reparative tissue expressed both myogenin and embryonic skeletal MHC. No muscle differentiation occurred after beta-galactosidase transfection. Thus, MyoD gene transfer can induce skeletal muscle differentiation in healing heart lesions. Modifications of this strategy might eventually provide new contractile tissue to repair myocardial infarcts.
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PMID:Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD. 894 36

The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp beta-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expressing cells on an expression map. beta-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the beta-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.
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PMID:Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos. 921 24


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