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)

Using liposomes as the mediator of DNA transfer, we were successful in the transfection of human hepatocytes isolated from surgical samples with an E. coli beta-galactosidase gene (beta-gal). A comparison of transfection efficiency showed that of the four promoters used, cytomegalovirus (CMV) promoter yielded higher transfection efficiencies than Rous sarcoma virus (RSV), Simian virus-40 (SV-40) and human alpha-1 antitrypsin (AAT) promoters. These studies represent the first report on the successful transfection of primary cultures of human hepatocytes.
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PMID:Gene transfer in primary cultures of human hepatocytes. 131 38

Northern-blot analysis was used to demonstrate that an increase in extracellular glucose concentration increased the content of preproinsulin mRNA 2.3-fold in the beta-cell line HIT T15. A probe for the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase was used as a control. Mannoheptulose blocked this effect of glucose. A stimulatory effect on preproinsulin mRNA levels was also observed in response to mannose and to 4-methyl-2-oxopentanoate. However, galactose and arginine were ineffective. Glucagon, forskolin and dibutyryl cyclic AMP also elicited an increase in HIT-cell preproinsulin mRNA. The ability of the 5' upstream region of the preproinsulin gene to mediate the effect of glucose and other metabolites on transcription was studied by using a bacterial reporter gene technique. HIT cells were transfected with a plasmid, pOK1, containing the upstream region of the rat insulin-1 gene (-345 to +1) linked to chloramphenicol acetyltransferase (CAT). Co-transfection with a plasmid pRSV beta-gal containing beta-galactosidase driven by the Rous sarcoma virus promoter was used as a control for the efficiency of transfection; expression of CAT activity in transfected HIT cells was normalized by reference to expression of beta-galactosidase. Glucose caused a dose-dependent increase in expression of CAT activity, with a half-maximal effect at 5.5 mM and a maximum response of 4-fold. Mannoheptulose blocked this effect of glucose. Other metabolites (mannose, 4-methyl-2-oxopentanoate and leucine plus glutamine) were also able to increase insulin promoter-driven CAT expression, but galactose and arginine were ineffective. The stimulatory effect of glucose on CAT expression was not blocked by verapamil and was inhibited by increasing extracellular Ca2+ from 0.4 to 5 mM. Both dibutyryl cyclic AMP and forskolin caused an increase in insulin promoter-driven gene expression in the presence of 1 mM-glucose, but neither agent further increased the level of expression occurring in the presence of a maximally stimulating glucose concentration. The phorbol ester phorbol 12-myristate 13-acetate (PMA) also increased insulin promoter-driven CAT expression in the presence of 1 mM-, but not 11 mM-glucose. Staurosporine blocked the stimulatory effect not only of PMA but also of glucose and of dibutyryl cyclic AMP. We conclude that the 5' upstream region of the insulin gene contains sequences responsible for mediating the stimulatory effect of glucose on insulin-gene transcription.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Control of insulin gene expression by glucose. 132 37

As an approach to gene therapy for the respiratory manifestations of cystic fibrosis (CF), in vivo plasmid-mediated direct transfer of the normal CF transmembrane conductance regulator (CFTR) gene to the airway epithelium was investigated in mice. To evaluate the feasibility of this strategy, pRSVL, a plasmid composed of a firefly luciferase gene driven by the Rous sarcoma virus long terminal repeat (RSV-LTR), along with cationic liposomes was instilled into the trachea of C57BI/6NCR mice. With administration of 200-400 micrograms plasmid DNA, luciferase expression could be detected in the mouse lung homogenates for at least 4 wk. With this background, a CFTR expression plasmid vector (pRSVCFTR) constructed by replacing the luciferase cDNA from pRSVL with the normal human CFTR cDNA was evaluated in vivo in mice. Intratracheal instillation of pRSVCFTR with cationic liposomes followed by analysis of mouse lung RNA by polymerase chain reaction amplification (after conversion of mRNA to cDNA) using a RSV-LTR specific sense primer and a human CFTR-specific antisense primer demonstrated human CFTR mRNA transcripts from one day to 4 wk after instillation. Further, in vivo evaluation of beta-galactosidase activity after intratracheal administration of an E. coli lacZ gene expression plasmid vector directed by the cytomegalovirus promoter (pCMV beta) demonstrated that the airway epithelium was the major target of transfer and expression of the exogenous gene. These observations demonstrate successful plasmid-mediated gene transfer to the airway epithelium in vivo. This strategy may be feasible as a form of gene therapy to prevent the pulmonary manifestations of CF.
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PMID:Expression of the human cystic fibrosis transmembrane conductance regulator gene in the mouse lung after in vivo intratracheal plasmid-mediated gene transfer. 137 20

In order to screen for developmentally active chromosomal domains during zebrafish embryogenesis, we generated transgenic fish by microinjecting two different lacZ reporter constructs into fertilized eggs. Transgenic fish were screened among the progeny of injected fish (F0) crossed to non-injected fish. Groups of 15 to 20 progeny of each cross were tested for lacZ expression and/or transmission of injected sequences using PCR and Southern hybridizations. Progeny from 2 of 102 fish injected with supercoiled constructs containing Rous sarcoma virus promoter sequences showed apparently spatially regulated beta-galactosidase (beta-Gal) activity. However, we were not able to detect this reporter construct in DNA from fins of F1 fish. Injections of a linear reporter construct containing mouse heat-shock promoter sequences revealed transmission of injected sequences to F1 progeny in about 6% of cases (8 of 129 fish, tested with PCR). We found one lacZ-expressing line that showed a spatially and temporally restricted expression of lacZ and, therefore, features typical characteristics of "enhancer trap" lines. In this line, lacZ expression starts at 16 hours post-fertilization in trigeminal ganglion cells. At about 24 hours lacZ expression can be detected in trigeminal ganglion neurons and Rohon-Beard neurons, indicating that the development of these two cell types shows common features. The reporter gene has integrated as a single copy. The founder fish was mosaic: 19% of its offspring (3 of 16 tested animals) carried the reporter construct in their fins; about 51% (13 of 27 tested animals) of the progeny of F1 fish were beta-Gal positive indicating full hemizygosity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A transgene containing lacZ is expressed in primary sensory neurons in zebrafish. 142 33

Chicken blastodermal cells (CBCs) and primary chicken fibroblasts (PCFs) have been lipofected with a variety of lacZ constructs encoding Escherichia coli beta-galactosidase (beta-gal). A reporter construct (phspPTlacZpA) containing a mouse heat-shock protein 68 gene (hsp 68) promoter was used to establish conditions for efficient lipofection. The construct, in circular or linear plasmid form or as reporter sequences alone, was transferred efficiently by incubating the cells for 3.5 h in a mixture of 6.2 micrograms Lipofectin (a cationic liposome preparation from Bethesda Research Laboratories) and 1.55-3.1 micrograms DNA per mL DMEM. These lipofection conditions were used to transfer a reporter construct (pCBcMtlacZ) containing a Zn(2+)-inducible chicken metallothionein (cMt) promoter, and constructs showing constitutive expression due to Rous sarcoma virus plus chicken beta-actin (pmiwZ) or cytomegalovirus (pMaori3) promoters. Endogenous chicken beta-gal and transferred bacterial beta-gal activity could be distinguished clearly by incubating the cells with the substrate, Xgal, at pH 4.3 or 7.4, respectively. Expression of phspPTlacZpA in chicken cells did not appear to require specific induction of the mouse hsp68 promoter, whereas expression of pCBcMtlacZ required treatment of the cells for 6-12 h with 150 microM ZnCl2. Bacterial beta-gal activity was observed following lipofection of CBCs that were cultured in suspension or plated. The efficiency of lipofection was at least 1 in 25 for CBCs, judging by the proportion of cells shown to have beta-gal activity 16-24 h after lipofection treatment began; these events could represent transient or stable incorporation of the construct.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Efficient transfection of chicken cells by lipofection, and introduction of transfected blastodermal cells into the embryo. 175 Oct 34

A DNA fragment encoding the polyhedrin promoter of Autographa californica multiple nuclear polyhedrosis virus (AcMNPV strain) was constructed using overlapping oligodeoxyribonucleotides (oligos), which included the 5'-untranslated leader sequence of the polyhedrin-encoding gene. This DNA fragment was cloned into an intermediate transfer vector (pKX105) providing a unique BamHI site for the insertion of foreign genes. The Escherichia coli lacZ gene was first cloned at the BamHI site of pKX105 and the XhoI-KpnI fragment containing the lacZ gene was transferred to another plasmid vector (pEI) consisting of flanking AcMNPV sequences (pEI-lacZ). The E. coli beta-galactosidase that was produced in the infected insect cells using the recombinant virus constituted about 10% of the total cytoplasmic proteins. The pKX105 plasmid was also modified to give rise to pTT-lacZ which consisted of the lacZ gene under the control of the Rous sarcoma virus long terminal repeat promoter to facilitate rapid screening of the baculoviral recombinants in which the gene of interest was cloned under the control of the polyhedrin promoter. The efficiency of these transfer vectors was verified by obtaining high levels of expression of the adenovirus(Ad)-encoded preterminal protein (pTP) which is involved as a protein primer in the initiation of Ad DNA replication. The baculovirus-produced pTP was immunoprecipitable using rabbit polyclonal antibodies raised against a hydrophilic domain of pTP. The pTP protein was localized in the nucleus of the infected insect cells, and was biologically active in the in vitro Ad type 2 (Ad2) replication initiation assay.
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PMID:Synthesis of biologically active adenovirus preterminal protein in insect cells using a baculovirus vector. 190 55

In this communication we demonstrate that gene transfer methodology can be applied to study gene expression in intact retinal explant cultures. The appropriate enzyme activity is observed in extracts obtained after electroporation of embryonic day-10 chicken retina with plasmids containing the chloramphenicol acetyltransferase-encoding or beta-galactosidase-encoding reporter genes under transcriptional control by the Rous sarcoma virus long terminal repeat. Similar results are obtained using Ca.phosphate-mediated gene transfer. Moreover, it has been previously established that glucocorticoid hormones stimulate transcription of glutamine synthetase (Glns) mRNA in embryonic retina. We report here that, based on the results of gene transfer experiments with chimeric plasmids containing 5'-flanking DNA derived from the cloned chicken Glns-encoding gene (Glns), essential glucocorticoid response elements reside between approx. 1.3 kb and 2.5 kb upstream from the Glns transcription start point. These data show that transfection of explant cultures can provide a useful approach to the study of gene expression in complex systems.
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PMID:Glucocorticoid-inducible expression of a glutamine synthetase-CAT-encoding fusion plasmid after transfection of intact chicken retinal explant cultures. 197 78

The ability to program recombinant gene expression in cardiac myocytes in vivo holds promise for the treatment of many inherited and acquired cardiovascular diseases. In this report, we demonstrate that a recombinant beta-galactosidase gene under the control of the Rous sarcoma virus promoter can be introduced into and expressed in adult rat cardiac myocytes in vivo by the injection of purified plasmid DNA directly into the left ventricular wall. Cardiac myocytes expressing recombinant beta-galactosidase were detected histochemically in rat hearts for at least 4 weeks after injection of the beta-galactosidase gene. These results demonstrate the potential of this method of somatic gene therapy for the treatment of cardiovascular disease.
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PMID:Expression of recombinant genes in myocardium in vivo after direct injection of DNA. 217 47

An evaluation has been made of the E. coli beta-galactosidase (beta-gal) gene for use as a reporter gene in mammalian cells in culture. We have adopted a histochemical procedure which enables identification of those cells within a population that express the introduced bacterial gene. Data is presented concerning the sensitivity of the histochemical method relative to an immunological method of detection. It has been found that several clonal cell lines generated after transfection of human 293 cells with a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter-beta-gal construction are mosaic for expression of the introduced mini-gene. Furthermore, after treatment of these clonal cell lines with the nucleoside analog 5-aza-cytidine (5-aza-C), an increase in production of beta-gal under control of this promoter element was observed.
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PMID:Histochemical staining of clonal mammalian cell lines expressing E. coli beta galactosidase indicates heterogeneous expression of the bacterial gene. 244 Jan 17

We examined the promoter activity of the 1.3-kb chicken beta-actin gene sequence located between the 5' flanking region and the proximal region of the second exon. This promoter region showed higher promoter activity than the simian virus 40 (SV40) early promoter or the Rous sarcoma virus (RSV) long terminal repeat (LTR) as assayed by transient lacZ gene expression in mouse L cells. Furthermore, replacement of the 3' splice sequence in this promoter by that derived from the rabbit beta-globin gene resulted in a approximately 2.5-fold enhancement in the synthesis of beta-galactosidase (beta Gal). Introduction of the SV40 origin of DNA replication (ori) into the vector carrying this hybrid promoter, which we designate the AG promoter, markedly enhanced the production of beta Gal in an SV40 T antigen-producing cell, BMT10. We have constructed a useful vector containing the strong AG promoter, several unique restriction sites, a SV40 polyadenylation signal and the SV40 ori for transient expression of cDNA in BMT10 or COS cells. We demonstrate the use of this vector for efficient production of interleukin-5 in BMT10 cells.
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PMID:Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5. 255 78


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