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 rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116 amino acid nuclear regulatory protein (Rev) that increases the cytoplasmic expression of viral mRNAs containing the Rev response element (RRE) and coding for the structural proteins, Gag and Env. To identify the functional domains of Rev, amino acid deletion and chain termination mutations were introduced in the Rev coding region. The ability of these mutants to increase the cytoplasmic expression of a Rev-test plasmid (pSV-AR), containing the RRE cloned into the 3' noncoding region of the CAT gene in plasmid pSV2CAT, was examined in transient expression assays in HeLa cells. Our results indicate that three distinct regions mapping within the N-terminal 98 amino acids of Rev are essential for its activity. The subcellular localization of the various Rev proteins was examined in COS cells by indirect immunofluorescence. Rev was found to localize predominantly in the nucleolus of transfected cells. All mutant Rev proteins, with the exception of a deletion mutant (rev delta 41-44) lacking four Arg residues of a highly basic domain, were found to localize in the nucleolus. Mutant rev delta 41-44 exhibited weak diffuse fluorescence in the nucleus with a tendency to accumulate in the cytoplasm. A 15 amino acid region encompassing this basic domain (38-52) when fused to the Escherichia coli beta-galactosidase gene efficiently directed the fusion gene product to the nucleus and nucleolus, suggesting a role for this domain in the nucleolar localization of Rev.
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PMID:Functional domains of the HIV-1 rev gene required for trans-regulation and subcellular localization. 210 12

COS-1 cells were transfected by electroporation with a cDNA for human acid beta-galactosidase cloned in our laboratory and stable transformants expressing the enzyme activity were selected. The precursor form of the enzyme was secreted in large quantities into the culture medium. The fibroblasts from patients with GM1-gangliosidosis or Morquio B disease showed a remarkable increase of enzyme activity, up to the normal level, after culture in this medium for 2 days; the amount of uptake was essentially the same as that for the precursor form in human fibroblasts. After endocytosis, the precursor molecules were processed normally to the mature form and remained as stable as those produced by human fibroblasts. On the other hand, cells from galactosialidosis patients did not show any increase of enzyme activity in a similar experiment. It was concluded that the transformants are useful as the source of precursor proteins for the study of intracellular turnover of enzyme molecules in mutant cells.
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PMID:Beta-galactosidase-deficient human fibroblasts: uptake and processing of the exogenous precursor enzyme expressed by stable transformant COS cells. 212 39

A full-length cDNA coding for mouse lysosomal acid beta-galactosidase has been isolated on the basis of homology with the human gene. Catalytic activity toward 4-methylumbelliferyl beta-D-galactoside in the COS-1 cell expression system provided positive proof for its authenticity. The sequence analysis showed that the degree of similarity between the human and mouse enzymes was approximately 70% in the nucleotide sequence and nearly 80% in the amino acid sequence. The deduced primary amino acid sequences of the enzymes from the two species indicated that, of the seven possible N-glycosylation sites in the human enzyme, five are conserved in the mouse enzyme. Three additional possible N-glycosylation sites, not present in the human enzyme, are found in the primary amino acid sequence of the mouse enzyme. All seven cysteine residues in the mouse enzyme are conserved in the human enzyme. Although the nucleotide sequence could be aligned to 60% identity with the E. coli beta-galactosidase, similarity in the amino acid sequence was minimal.
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PMID:Molecular cloning of mouse acid beta-galactosidase cDNA: sequence, expression of catalytic activity and comparison with the human enzyme. 212 9

Many stresses, including elevated temperature and exposure to toxins or heavy metals, activate a stereotyped response of cultured cells known as the heat-shock response. The products of several highly conserved heat-shock genes (heat-shock proteins) protect the cells against subsequent stresses. The intracellular signal for the response is unknown, but may include the presence of damaged and abnormal proteins in the cell. Ethanol at high concentration (1.3 mol/L) has been shown to activate the heat-shock response in hamster ovary fibroblasts, suggesting that this response might be an important consequence of exposure of cells to ethanol and might mediate some of its cellular toxicity. To determine if lower concentrations of ethanol or its metabolites could activate a heat-shock response, we transfected COS-1 cells with a reporter gene (the Drosophila 70 kd heat-shock protein promoter fused to the beta-galactosidase gene), then exposed them to various compounds. Exposure to heat induced at least a threefold to fourfold increase in beta-galactosidase activity, whereas 1.3 mol/L ethanol induced a sixfold increase. Lower concentrations of ethanol (100 to 500 mmol/L) or acetaldehyde (100 to 500 mumol/L) did not induce a measurable heat-shock response. Similarly, high concentrations of metabolites generated during ethanol oxidation (10 mmol/L lactate or acetate) did not induce the response. We conclude that the heat-shock response cannot be detected with this assay system in COS-1 cells after short exposure to physiologically achievable concentrations of ethanol or its metabolites. However, it is possible that it is induced at a low level or in tissues directly exposed to alcoholic beverages (e.g., oropharynx, esophagus, and stomach).
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PMID:The heat-shock response in cultured cells exposed to ethanol and its metabolites. 250 9

We have isolated two cDNAs encoding human lysosomal beta-galactosidase, the enzyme deficient in GM1-gangliosidosis and Morquio B syndrome, and a beta-galactosidase-related protein. In total RNA from normal fibroblasts a major mRNA of about 2.5 kilobases (kb) is recognized by cDNA probes. A minor transcript of about 2.0 kb is visible only in immunoselected polysomal RNA. A heterogeneous pattern of expression of the 2.5-kb beta-galactosidase transcript is observed in fibroblasts from different GM1-gangliosidosis patients. The nucleotide sequences of the two cDNAs are extensively colinear. However, the short cDNA misses two noncontiguous protein-encoding regions (1 and 2) present in the long cDNA. The exclusion of region 1 in the short molecule introduces a frameshift in its 3'-flanking sequence, which is restored by the exclusion of region 2. These findings imply the existence of two mRNA templates, which are read in a different frame only in the nucleotide stretch between regions 1 and 2. Sequence analysis of genomic exons of the beta-galactosidase gene shows that the short mRNA is generated by alternative splicing. The long and short cDNAs direct the synthesis in COS-1 cells of beta-galactosidase polypeptides of 85 and 68 kDa, respectively. Only the long protein is catalytically active under the assay conditions used, and it is capable of correcting beta-galactosidase activity after endocytosis by GM1-gangliosidosis fibroblasts. The subcellular localization of cDNA-encoded beta-galactosidase and beta-galactosidase-related proteins is different.
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PMID:Alternative splicing of beta-galactosidase mRNA generates the classic lysosomal enzyme and a beta-galactosidase-related protein. 251 Dec 8

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

The BamHI Nhet fragment of the B958 strain of Epstein-Barr virus (EBV) encodes a membrane protein (BNLF-1) that is present in cells transformed by EBV. We made a hybrid protein in which a polypeptide sequence from the carboxyl-terminal part of BNLF-1 is fused to Escherichia coli beta-galactosidase. This hybrid protein was used to immunize rabbits, and the resulting antiserum was purified by immunoaffinity chromatography. The antiserum was able to immunoprecipitate BNLF-1 from cell lysates. We found that BNLF-1 is phosphorylated at serines in EBV genome-positive B-cell lines. Pulse-chase analyses with [35S]methionine indicated that BNLF-1 is turned over in lymphoblasts with a half-life of approximately 5 h. Protein immunoblots of EBV genome-positive B-cell lines revealed both a 62,000-molecular-mass band corresponding to BNLF-1 and a myriad of lower-molecular-mass bands. We postulate that these lower-molecular-mass bands are degradation products resulting from the turnover of BNLF-1 in cells. The BNLF-1 gene was expressed in COS cells, and the protein was both phosphorylated and turned over in these cells.
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PMID:Posttranslational processing of an Epstein-Barr virus-encoded membrane protein expressed in cells transformed by Epstein-Barr virus. 302 13

We cloned and sequenced the full-length cDNA for human placental beta-galactosidase. The 2379-nucleotide sequence contains 2031 nucleotides which encode a protein of 677 amino acids. The amino acid sequence includes a putative signal sequence of 23 amino acids and 7 potential asparagine-linked glycosylation sites. The cDNA in the expression vector pSVL was used to transfect COS cells. Expression of the cDNA in transfected COS cells produced immunoprecipitable proteins and led to an increase in beta-galactosidase activity.
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PMID:Cloning, sequencing, and expression of cDNA for human beta-galactosidase. 314 62

A hybrid gene in which the expression of an Escherichia coli beta-galactosidase gene was placed under the control of a Drosophila melanogaster 70,000-dalton heat shock protein (hsp70) gene promoter was constructed. Mutant derivatives of this hybrid gene which contained promoter sequences of different lengths were prepared, and their heat-induced expression was examined in D. melanogaster and COS-1 (African green monkey kidney) cells. Mutants with 5' nontranscribed sequences of at least 90 and up to 1,140 base pairs were expressed strongly in both cell types. Mutants with shorter 5' extensions (of at least 63 base pairs) were transcribed and translated efficiently in COS-1 but not at all in D. melanogaster cells. Thus, in contrast to the situation in COS-1 cells, the previously defined heat shock consensus sequence which is located between nucleotides 62 and 48 of the hsp70 gene 5' nontranscribed DNA segment is not sufficient for the expression of the D. melanogaster gene in homologous cells. A second consensus-like element 69 to 85 nucleotides upstream from the cap site is postulated to be also involved in the heat-induced expression of the hsp70 gene in D. melanogaster cells.
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PMID:The heat shock consensus sequence is not sufficient for hsp70 gene expression in Drosophila melanogaster. 392 May 9

As an approach to the study of mammalian gene expression, the promoters and translation initiation regions of the rat preproinsulin II and the simian virus 40 early genes were fused to the structural gene of Escherichia coli beta-galactosidase, a sensitive probe for gene expression. These fusions were introduced into COS-7 cells, a simian virus 40 large tumor-antigen-producing monkey kidney cell line, where they directed the synthesis of enzymatically active hybrid beta-galactosidase proteins. Conditions for transfection were varied to optimize the expression of beta-galactosidase activity in the transfected cells. The pH optimum of this activity was found to be 7.0, the same as that of native E. coli beta-galactosidase and distinct from the major lysosomal "acid" beta-galactosidase. The fused preproinsulin-beta-galactosidase was further characterized by gel electrophoresis of nondenatured cell extracts stained by a fluorogenic substrate and by immunoprecipitation and gel electrophoresis of 3H-labeled cell proteins. These results all indicate that fully active tetrameric beta-galactosidase hybrids can be produced in mammalian cells. The expression of preproinsulin-beta-galactosidase activity was measured in the presence of high glucose, insulin, dexamethasone, or epidermal growth factor but no regulatory changes were observed.
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PMID:Expression of a preproinsulin-beta-galactosidase gene fusion in mammalian cells. 631 May 64


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