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)

The longitudinal expression of two brush-border enzymes, lactase-phlorizin hydrolase (EC 3.2.1.23/62) and aminopeptidase N (EC 3.4.11.2), was studied in the small intestine of the post-weaned pig. Whereas the level of mRNA, encoding aminopeptidase N (relative to that of beta-actin), only varied moderately from the duodenum to the terminal ileum, the amount of lactase-phlorizin hydrolase mRNA exhibited a sharp maximum in the proximal jejunum. For both enzymes, the level of protein synthesis, studied in cultured mucosal explants, correlated well with the level of mRNA, and no major variation in post-translational processing or intracellular transport was observed along the intestine. The mRNA/specific-activity ratio for both enzymes was markedly (3-5-fold) higher in the duodenum and proximal jejunum, compared with the ileum. This indicates an increased proximal turnover rate, most likely caused by the presence in the gut lumen of pancreatic proteases. In neonatal animals, the level of mRNA for lactase-phlorizin hydrolase in both proximal and distal regions of the intestine was of the same magnitude as in the proximal jejunum of the post-weaned pigs. Our results point to two mechanisms that affect the expression of lactase-phlorizin hydrolase in the pig during development: (1) a primary regulation at the level of mRNA (predominantly in the ileum); (2) an increased rate of turnover of the enzyme, mainly in the duodenum and proximal jejunum, and most likely due to an increased secretion into the gut lumen of pancreatic proteases (a mechanism also affecting aminopeptidase N and probably other brush-border enzymes as well).
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PMID:Lactase-phlorizin hydrolase and aminopeptidase N are differentially regulated in the small intestine of the pig. 810 80

The plasmid DNA, pAcZ, containing Escherichia coli beta-galactosidase (lacZ) gene under the control of chicken beta-actin gene promoter was injected in a linearized form into the germinal disc of fertilized chick ova at the single-cell stage. The manipulated embryos were cultured by the method of Naito et al. (1990) until hatching. The rate of hatching was 11.8% (31/263), and 19 males and 6 females were matured. DNA from blood and semen samples of the 25 matured chickens was analyzed for the presence of the injected DNA by Southern blot hybridization. The injected DNA was detected in the blood DNA of one male and in the sperm DNA of another male up to 13 months after hatching, indicating that the injected DNA was stably maintained in these chickens. Restriction digestion analysis of the injected DNA suggested that it was not rearranged and was organized as head-to-tail multimers. The copy numbers of the DNA were 0.07-0.02 in the blood DNA of one male per diploid genome, and 0.02-0.015 in the sperm DNA of another male, indicating that the exogenous DNA was present in limited populations of blood and sperm cells.
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PMID:Introduction of exogenous DNA into somatic and germ cells of chickens by microinjection into the germinal disc of fertilized ova. 817

The formation of transgenic chimeric chickens for use in developmental studies and as intermediates in the production of transgenic chickens requires the incorporation of stably transfected blastodermal cells into a chimera. To obtain blastodermal cells, area pellucidae of stage X (Eyal-Giladi and Kochav, Dev. Biol. 49:321-337, 1976:E.-G.&K.) embryos were collected from unincubated, freshly oviposited Barred Plymouth Rock eggs and dissociated in 0.25% trypsin/0.04% EDTA (w/v) and 2% (v/v) chicken serum in phosphate-buffered saline (Ca2+ and Mg2+ free) at 4 degrees C for 10 min. The blastodermal cells were suspended in Dulbecco's Modified Eagle's Medium (DMEM) and transfected by lipofection with superhelical pmiwZ, a plasmid containing a hybrid lacZ gene encoding bacterial beta-galactosidase (beta-gal) under the control of a chicken beta-actin/Rous sarcoma virus promoter. A mixture of 2.5 micrograms Lipofectin and 1.56 micrograms pmiwZ in 250 microliters DMEM was incubated for 30 min at 37 degrees C and added to 500 microliters of 20-40,000 cells in suspension. Cells incubated with the transfection reagents in the presence or absence of pmiwZ were either plated and cultured for 48 h at 37 degrees C in 5% CO2/95% air, or injected through a shell window into the subgerminal cavity of White Leghorn stage X (E.-G.&K.) embryos previously exposed to 500-600 rads from a 60Co source, after which the window was sealed and the egg incubated at 38 +/- 1 degrees C for 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Efficient incorporation of transfected blastodermal cells into chimeric chicken embryos. 829 32

We have constructed two sets of Escherichia coli lacZ-based vectors for use in studies of general mitotic recombination, both in somatic mammalian cells grown in culture and in transgenic animals. The vectors use two mutant copies of the E. coli lacZ gene as their recombination substrates and contain a neo gene for selection of stable transformants. In one vector, pLrec, an SV40 promoter drives lacZ, while the other vector, pArec, utilizes a human non-muscle beta-actin promoter for lacZ expression. Gene conversions, unequal sister chromatid exchanges and reciprocal exchanges between the two lacZ genes result in expression of beta-galactosidase, which can be detected in situ by histochemical staining. These vectors yield rates and frequencies of mitotic intrachromosomal recombination in human and rodent cell lines which are similar to rates reported using conventional recombination vectors. Molecular analysis of recombinational events involving the lacZ-based vectors can be carried out on genomic DNA isolated from clonally expanded populations and individual LacZ+ cells and cell clusters can be analyzed using PCR amplification. These reporter gene-based vectors may facilitate the study of recombination in cells with limited proliferative capacities, allow for analysis of both products of an unequal sister chromatid exchange, and permit in situ detection of recombination in the tissues of transgenic animals.
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PMID:Novel lacZ-based recombination vectors for mammalian cells. 829 44

We have investigated whether three-dimensional cultivation of cells to multicell spheroids influences the expression of a transfected gene. Ltk- cells (mouse fibroblasts, thymidine kinase negative) have been transfected with a bacterial lacZ gene which was coupled to a beta-actin promoter. The transfected cells synthesize beta-galactosidase, a cytoplasmic enzyme which can easily be stained for histology with 5-bromo-4-chloro-3-indoxyl beta-D-galactoside and for cytometry with fluorescein di-(beta-D-galactopyranoside). As we have shown with monolayer cells, beta-galactosidase is produced independently of cell density, medium condition, and cell cycle. In multicell spheroids, however, the portion of producing cells was reduced from approximately 98% to approximately 2% within a week. This reduction is also independent of cell density, medium condition, and cell cycle. Nonproducing multicell spheroid cells, however, regained their ability to synthesize beta-galactosidase within a few days when the cells were recultivated as monolayers. Since the lacZ gene was not lost, its expression might have been regulated by its beta-actin promoter. We, therefore, investigated whether the endogenous synthesis of beta-actin was similarly regulated. A correlation between the distinct reduction in beta-galactosidase-producing cells and filamentous or total actin concentration was not unequivocally observed.
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PMID:Beta-galactosidase activity in transfected Ltk- cells is differentially regulated in monolayer and in spheroid cultures. 831 68

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat beta-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli beta-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli beta-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli beta-galactosidase gene under a beta-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes.
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PMID:Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with Gibbon ape leukemia virus envelopes. 839 Dec 77

We demonstrate that in differentiating myoblasts, the mRNAs encoding two actin isoforms, beta-cytoplasmic, and alpha-cardiac, can occupy different cytoplasmic compartments within the same cytoplasm. beta-actin mRNA is localized to the leading lamellae and alpha-actin mRNA is associated with a perinuclear compartment. This was revealed by co-hybridizing, in situ, fluorochrome-conjugated oligonucleotide probes specific for each isoform. To address the mechanism of isoform-specific mRNA localization, molecular chimeras were constructed by insertion of actin sequences between the Lac Z coding region and SV-40 3'UTR in a reporter plasmid. These constructs were transiently expressed in a mixed culture of embryonic fibroblasts, myoblasts and myotubes, beta-galactosidase activity within transfectants was revealed by a brief incubation with its substrate (X-gal). Since the blue-insoluble reaction product co-localized with the specific mRNAs expressed from each construct, it was used as a bioassay for mRNA localization. Transfectants were scored as either perinuclear, peripheral or nonlocalized with respect to the distribution of the blue product. The percentage of transfectants within those categories was quantitated as a function of the various constructs. This analysis revealed that for each actin mRNA its 3'UTR is necessary and sufficient to direct reporter transcripts to its appropriate compartment; beta-actin peripheral and alpha-actin perinuclear. In contrast, sequences from the 5'UTR through the coding region of either actin gene did not localize the blue product. Therefore, 3'UTR sequences play a key role in modulating the distribution of actin mRNAs in muscle cells. We propose that the mechanism of mRNA localization facilitates actin isoform sorting in the cytoplasm.
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PMID:Isoform-specific 3'-untranslated sequences sort alpha-cardiac and beta-cytoplasmic actin messenger RNAs to different cytoplasmic compartments. 840 95

Two histochemical marker genes, Drosophila alcohol dehydrogenase (ADH) and human placental alkaline phosphatase (ALP), were cloned into the recombinant retroviral vectors pLJ and pgag beta-actin. The resulting vectors were transfected into retroviral producer cell lines, psi CRE and psi CRIP, and stable recombinant retrovirus producers were isolated. Recombinant virus was harvested and used to transduce genes into several cell lines, singly or in conjunction with lacZ (Escherichia coli beta-galactosidase)-containing retrovirus. Cell lines were then stained using standard histochemical methods for recombinant gene expression. We found that multiple gene products could be identified in the same cell populations and in the case of ALP and beta-galactosidase, in the same cells. The resulting reagents should be useful for a variety of cell-marking studies including those involving multiple clonal analysis and developmental studies for gene therapy.
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PMID:Recombinant retroviruses containing novel reporter genes. 851 8

Recombinant plasmid containing the Drosophila beta-actin promoter coupled to a beta-galactosidase cassette was linearized and introduced in fertilized eggs of the red abalone (Haliotis rufescens) by electroporation. Fertilized abalone eggs tolerated electroporation well with larval survival rates between 70% and 84% of that for non-electroporated siblings. Dot blot and Southern blot analysis were used to detect if abalone retained the foreign gene at various developmental stages. The inserted construct was retained in 70% to 100% of all abalone sampled with an average of 72% retention in the three- to seven-month-old juveniles. Maximal DNA uptake and retention was observed in abalone electroporated at 30-40 min after fertilization. Southern hybridization analysis suggested that the inserted vector was in head-to-tail concantermers integrated in the abalone genome. This preliminary study demonstrates that electroporation is an efficient means of transferring foreign DNA into abalone embryos.
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PMID:Electroporation as an effective means of introducing DNA into abalone (Haliotis rufescens) embryos. 854 86

Transforming growth factor-beta 1 (TGF-beta 1) is known to regulate cardiac cell function and its overexpression in the heart is thought to contribute to the development of cardiac hypertrophy and fibrosis. We wished to develop a high efficiency gene transfer method that could be used both in vitro and in vivo and result in the overexpression of TGF-beta 1. For this purpose, we constructed a replication-deficient human adenovirus 5 vector encoding for human TGF-beta 1 and used for control purposes an adenovirus lacZ vector. The adenovirus 5 construct was capable of infecting neonatal rat cardiac myocytes, fibroblasts and VSMCs. Of the three cell types, cardiac myocytes appear more susceptible to infection by the adenovirus 5 construct as assessed through beta-galactosidase staining. Infection of cardiac fibroblasts, myocytes and VSMCs with the hTGF-beta 1 adenovirus leads to the expression of hTGF-beta 1 mRNA and enhanced levels of bioactive and total TGF-beta 1 protein. Infection with hTGF-beta 1 adenovirus also results in enhanced levels of collagen type III gene expression in VSMCs and fibroblasts whereas in cardiac myocytes it leads to increased levels for sarcomeric and beta-actin. Thus, this adenoviral vector might be used for the exploration of in vivo effects of altered levels of cardiac TGF-beta 1.
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PMID:Adenovirus-mediated overexpression of human transforming growth factor-beta 1 in rat cardiac fibroblasts, myocytes and smooth muscle cells. 873 1


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