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)

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

An improved baculovirus expression vector was developed to expedite screening and facilitate oligonucleotide-directed mutagenesis. This vector contained twin promoters derived from the P10 and polyhedrin genes of Autographica californica nuclear polyhedrosis virus. The P10 promoter directed the synthesis of beta-galactosidase, whereas the polyhedrin promoter controlled the synthesis of foreign gene products. These two genes recombined with wild-type virus genome to yield recombinants which were polyhedrin negative, produced the foreign gene product, and formed blue plaques when beta-galactosidase indicator was present in the agarose overlay. An origin of replication derived from M13 or f1 bacteriophage was also included in the plasmid to permit the synthesis of single-stranded DNA. This template DNA was used to introduce or delete sequences through the process of site-specific mutagenesis. The measles virus virion possesses a membrane envelope which contains two glycoproteins: the hemagglutinin (H) and membrane fusion (F) proteins. The H polypeptide has receptor-binding and hemagglutinating activity, whereas the F protein mediates virus penetration of the host cell, formation of syncytia, and hemolysis of erythrocytes. Genes for these two glycoproteins were inserted into the NheI cloning site of the modified expression vector described above. The vector and purified wild-type viral DNA were introduced into Sf9 insect cells by calcium phosphate precipitation. A mixture of wild-type and recombinant virus was generated and used to infect Sf9 cells, which were subsequently overlaid with agarose. After 3 days, 0.1 to 1% of the plaques became blue in the presence of beta-galactosidase indicator. At least 70% of these blue viral colonies contained the foreign gene of interest as determined by dot blot analysis. Recombinant virus was separated from contaminating wild-type virus through several rounds of plaque purification. Insect cells were then infected with the purified recombinants, and synthesis of H and F proteins were verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblot detection and Coomassie blue staining. Glycosylation of the proteins appeared to be impaired somewhat, and the precursor to the F protein was not completely cleaved by the proteases present in insect host cells. On the other hand, both proteins appeared to be active in hemagglutination, hemolysis, and cell fusion assays. Levels of synthesis were in the order of 50 to 150 mg of protein per 10(8) cells.
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PMID:Synthesis of the membrane fusion and hemagglutinin proteins of measles virus, using a novel baculovirus vector containing the beta-galactosidase gene. 210 44

Proliferation-competent and differentiation-competent adult rat hepatocytes in primary culture were investigated for their ability to express reporter genes (firefly luciferase, bacterial chloramphenicol acetyltransferase, and bacterial beta-galactosidase) driven by tumor virus or eucaryotic promoters that vary in transcriptional efficiency and tissue specificity. Supercoiled plasmid DNA molecules were introduced into the cells by the calcium phosphate coprecipitation protocol of C. Chen and H. Okayama (Mol. Cell. Biol. 7:2745-2752, 1987). Reporter gene expression was virtually restricted to hepatocytes and was efficient (2 to 20% of the cells). The patterns and absolute levels of reporter gene expression depended on assay conditions employed (plasmid concentration [optimal at 2.4 micrograms of DNA per ml] and duration of exposure [optimal between 5 and 10 h]), culture growth cycle stages (lag, log, or stationary phase), properties and tissue specificity of the promoter(s) tested, and composition (and timing of fluid change) of the culture medium with or without the hepatocyte mitogen human transforming growth factor-alpha. Initial observations suggest that during hepatocellular growth transitions, human transforming growth factor-alpha differentially regulates exogenously introduced promoters associated with hepatocyte-specific function and proliferation. These findings provide a simple, fast, and powerful approach to analyzing the molecular and cellular biology of hepatocyte growth control.
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PMID:DNA-mediated gene transfer into adult rat hepatocytes in primary culture. 210 58

The nutrient lactose acts as a potential dietary fibre. Depending on the composition of diet, lactose can partially escape from digestion and absorption in the small intestine and is utilised by the colonic microflora. The suggested mechanism, i.e. different rates of enzymatic hydrolysis of the lactose anomers and an influence of food ingredients on lactose mutarotation, seems to be supported by experimental results. The Michaelis constant of the mucosal beta-galactosidase of rats is 14 mmol/l with alpha-lactose and 50 mmol/l with beta-lactose as substrates. During intestinal perfusion of human infants, alpha-lactose is digested at a significantly higher rate than beta-lactose. The mutarotation of lactose is influenced by general acid-base catalysis. Milk formulae containing a high concentration of weak-acid anions, e.g. cow milk which is rich in phosphate and citrate, accelerate mutarotation, but milk formulae with a low content of these anions do not. With these experimental results a mathematical model has been set up simulating lactose digestion in the small intestine of human infants and of rats. The data show that certain dietary components may influence the ratio between lactose anomers, but, surprisingly, they do not influence the amount of digested lactose.
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PMID:Does mutarotation influence lactose digestion? Experimental investigations and a mathematical model. 212 25

We have established a ricin-resistant glycosylation-defective PC12 pheochromocytoma cell line to study biochemically glycoprotein traffic from the cell surface to the Golgi apparatus in regulated secretory cells. The strategy employed in this study is a modification of that used previously (Duncan, J. R., and Kornfeld, S. (1988) J. Cell Biol. 106, 617-628) to demonstrate transport of the cation-independent and -dependent mannose 6-phosphate receptors from the cell surface to the trans-Golgi network in nonsecretory cell types. In ricin-resistant PC12 cells, radiolabeled galactose was incorporated enzymatically into surface glycoconjugates, primarily glycoproteins. Resistance to beta-galactosidase was acquired upon reculture at 37 degrees C due to further terminal glycosylation of the galactose residues. Treatment of N-linked oligosaccharides isolated from recultured cells with a variety of glycosidases in conjunction with beta-galactosidase demonstrated the addition of sialic acid N-acetylglucosamine and fucose residues to the galactose residues in recultured cells. Resistance to beta-galactosidase was not acquired in cells recultured at 19 degrees C, indicating that subsequent glycosylation of galactose residues did not occur at the cell surface or in endosomes. While glycosylation of galactose incorporated into asparagine oligosaccharides in Chinese hamster ovary clone 13 cells was not significant (less than 1%) after 6 h of reculture, approximately 10% of the galactose incorporated into surface oligosaccharides was further glycosylated in PC12 cells in this time. Analysis of total labeled versus beta-galactosidase-resistant proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that endocytic traffic to the site of glycosylation activity in mutant PC12 cells was highly selective, but included a much greater number of proteins than were detected in Chinese hamster ovary clone 13 fibroblasts.
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PMID:Endocytic membrane traffic to the Golgi apparatus in a regulated secretory cell line. 212 89

To ascertain whether mannose 6-phosphate-containing peptides that bind to the insulin-like growth factor II (IGF II)/mannose 6-phosphate receptor activate phospholipase C, we determined the effect of proliferin, transforming growth factor-beta 1 (TGF-beta 1) precursor, and beta-galactosidase on production of inositol trisphosphate (Ins-P3) in basolateral membranes isolated from the renal proximal tubule of dogs. Both proliferin and TGF-beta 1 precursor stimulated Ins-P3 production in a concentration-dependent manner. Maximal production was stimulated by approximately 10(-13) M of each peptide. beta-Galactosidase had no effect on Ins-P3 generation. Neither proliferin nor TGF-beta 1 precursor potentiated IGF II-stimulated Ins-P3 production. Mannose 6-phosphate itself had no effect on Ins-P3 generation. However, mannose 6-phosphate potentiated production stimulated by 10(-11) M proliferin or 10(-11) M TGF-beta 1 precursor while inhibiting production stimulated by 10(-14) M of either peptide. Addition of anti-mannose 6-phosphate receptor antibodies to basolateral membranes abolished proliferin and TGF-beta 1 precursor-stimulated Ins-P3 generation. We conclude that, in addition to IGF II, mannose 6-phosphate-containing ligands for the IGF II/mannose 6-phosphate receptor activate basolateral membrane phospholipase C. Such activation could reflect a common mechanism for signal transduction by these peptides mediated via the IGF II/mannose 6-phosphate receptor.
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PMID:Mannose 6-phosphate-containing peptides activate phospholipase C in proximal tubular basolateral membranes from canine kidney. 216 41

We are attempting to develop methods for the sequencing of glycosaminoglycans from their reducing end. Here we describe a procedure for the analysis of dermatan sulphate from pig skin. The glycosaminoglycan is released from its parent proteoglycan by exhaustive proteolysis by using both endo- and exo-peptidases. The amino group of the residual serine residue is conjugated with a p-hydroxyphenyl group, which in turn is iodinated with 125I (the Bolton-Hunter reagent, BHR). The ion-exchange-purified end-labelled dermatan sulphate is then degraded partially or completely by various enzymic or chemical means to yield fragments extending from the labelled serine residue to the point of cleavage. The various products are separated by gradient PAGE, detected by autoradiography and quantified by videodensitometry. Complete digestion with chondroitin ABC lyase affords the labelled fragment delta HexA-GalNAc(-SO4)-GlcA-Gal-Gal-Xyl-Ser(-BHR). The structure was confirmed by sequential degradation from the non-reducing end by chondroitin AC lyase, HgCl2, and beta-galactosidase. Periodate oxidation cleaves most of the Xyl even without treatment with alkaline phosphatase, showing that Xyl is not substituted with phosphate. Results from partial and selective periodate oxidation indicate that most of the non-sulphated IdoA residues are located towards the non-reducing end. Partial or complete digestions with testicular hyaluronidase (in the presence of an excess of beta-glucuronidase) or chondroitin AC lyase identify the positions of GlcA residues. The results confirm that HexA next to Gal is always GlcA. Moreover, GlcA is common in the first three disaccharide repeats. Results with testicular hyaluronidase indicate that the distribution of clustered GlcA-GalNAc repeats is periodic and peaks at positions 1-3, 8-9 and around 25. Although there must be chains that contain IdoA in nearly all of the available positions, regions that have not been fully processed during biosynthesis are markedly non-random.
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PMID:A method for the sequence analysis of dermatan sulphate. 216 67

The post-translational processing of beta-glucuronidase in BW5147 mouse lymphoma cells is slow relative to other newly synthesized lysosomal enzymes. To characterize this slow maturation the acid hydrolase was immunoprecipitated from cells pulse-labeled with [2-3H]mannose. Radiolabeled beta-glucuronidase migrated as the precursor form of the enzyme for up to 4 h of chase, whereas another acid hydrolase, beta-galactosidase, was processed completely to its mature form within this same time period. Both beta-glucuronidase and beta-galactosidase obtained high levels of mannose 6-phosphate (Man 6-P) within 60 min of their biosynthesis. The Man 6-P content of beta-galactosidase declined rapidly during a subsequent chase while that of beta-glucuronidase remained high during the first 4 h of chase and then slowly declined. 3H-Labeled phosphorylated high mannose-type oligosaccharides isolated from beta-glucuronidase after 1 h of chase were composed primarily of species with one or two phosphodiester groups, but oligosaccharides with one and two phosphomonoesters became the predominant phosphorylated species with longer chase times. The phosphorylated oligosaccharides attached to other newly synthesized acid hydrolases, on the other hand, contained primarily phosphodiester species at all chase times. When BW5147 cells were pulsed with [3H]mannose and chased in the presence of monensin to disrupt transport, the number of phosphorylated oligosaccharides recovered from beta-glucuronidase was comparable to the quantity recovered from the enzyme produced by non-drug-treated cells. The number of phosphorylated units recovered from all other newly synthesized acid hydrolases, however, was greater in the presence of the ionophore than in its absence. Nondenaturing gel electrophoresis studies indicated that beta-glucuronidase existed in two forms at steady state within BW5147 cells and, as such, was similar to liver beta-glucuronidase in which a large percentage of the enzyme was present as a complex bound to egasyn. These data suggest that newly synthesized beta-glucuronidase produced by BW5147 cells complexes with an egasyn-like protein within the endoplasmic reticulum. This interaction retards the enzyme's migration through the secretory apparatus but does not prevent its access to Golgi-associated processing enzymes.
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PMID:beta-Glucuronidase is transported slowly to lysosomes in BW5147 mouse lymphoma cells: evidence that the prelysosomal enzyme is not restricted to the endoplasmic reticulum. 222 12

Transcription of the genes in the phosphate regulon in Escherichia coli is activated by PhoB protein, which is phosphorylated by PhoR protein under phosphate-limiting conditions. In the absence of the phoR function, the genes in the phosphate regulon are expressed constitutively and the expression is dependent on the function of phoM and phoB. We constructed a plasmid with a lacZ'-'phoM fusion gene, which encoded a hybrid protein (PhoM1206) in which the hydrophobic amino-terminal half of the native PhoM was replaced by beta-galactosidase. The phoM1206 gene could complement the phoM mutation in vivo. We purified PhoM1206 from the overproducing strain carrying the plasmid; it was autophosphorylated at a histidine residue in the presence of ATP, and the phospho-PhoM1206 phosphorylated PhoB. PhoM1206 could also transphosphorylate the product of phoM-orf2, which is structurally homologous to phoB and located immediately upstream of phoM. Although PhoR1084 that lacked the hydrophobic amino-terminal region of the native PhoR protein transphosphorylated PhoB, it could not phosphorylate PhoM-open reading frame 2. Therefore, cross talk by protein phosphorylation appears to occur from PhoM to PhoB but not from PhoR to PhoM-open reading frame 2.
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PMID:Cross talk to the phosphate regulon of Escherichia coli by PhoM protein: PhoM is a histidine protein kinase and catalyzes phosphorylation of PhoB and PhoM-open reading frame 2. 222 61

The Saccharomyces cerevisiae SNF5 gene affects expression of both glucose- and phosphate-regulated genes and appears to function in transcription. We report the nucleotide sequence, which predicts that SNF5 encodes a 102,536-dalton protein. The N-terminal third of the protein is extremely rich in glutamine and proline. Mutants carrying a deletion of the coding sequence were viable but grew slowly, indicating that the SNF5 gene is important but not essential. Evidence that SNF5 affects expression of the cell type-specific genes MF alpha 1 and BAR1 at the RNA level extends the known range of SNF5 function. SNF5 is apparently required for expression of a wide variety of differently regulated genes. A bifunctional SNF5-beta-galactosidase fusion protein was localized in the nucleus by immunofluorescence. No DNA-binding activity was detected for SNF5. A LexA-SNF5 fusion protein, when bound to a lexA operator, functioned as a transcriptional activator.
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PMID:The SNF5 protein of Saccharomyces cerevisiae is a glutamine- and proline-rich transcriptional activator that affects expression of a broad spectrum of genes. 223 8


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