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 an anti-receptor mAb that blocks the attachment of echovirus 7 and related viruses (echoviruses 13, 21, 29 and 33), we have isolated a complementary DNA clone that encodes the human decay-accelerating factor (CD55). Mouse cells transfected with the CD55 clone bind echovirus 7, and this binding is blocked by the anti-receptor mAb. The method used (CELICS) allows rapid and direct cloning of genes encoding cell surface receptors. It is based on episomal replication and high efficiency expression of complementary DNA clones in the vector pCDM8 in COS or WOP cells, in conjunction with a sensitive immuno-focal screen that uses antibody probes linked to beta-galactosidase. Receptor positive cells were identified by a colour change and isolated individually using a micromanipulator. DNA extracted from a small number of cells was then cloned directly in Escherichia coli.
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PMID:Decay-accelerating factor CD55 is identified as the receptor for echovirus 7 using CELICS, a rapid immuno-focal cloning method. 752 74

The human breast carcinoma cell line T47D is known to express high-affinity calcitonin receptors (CTRs). PCR amplification of the CTR cDNA from T47D mRNA resulted in the identification of two different cDNAs that encode distinct receptor isoforms, h alpha CTR and h beta CTR. The two cDNAs are identical except that the h alpha CTR cDNA contains a 48 bp insert sequence that encodes a 16 amino acid domain in the first cytosolic loop of the receptor. Stable transfection of each receptor cDNA into murine erythroleukaemia (MEL) cells resulted in the expression of receptors with high affinity for radiolabelled salmon calcitonin (h alpha CTR Kd 0.09 nM, h beta CTR Kd 0.12 nM). Ligand competition binding studies did not reveal any significant pharmacological difference between the receptor isoforms. In transfected MEL cells and COS-1 cells the h beta CTR isoform was expressed at tenfold higher levels than the h alpha CTR. A reporter gene assay that monitored the coupling of CTR to adenylate cyclase by increases in beta-galactosidase activity indicated that both receptors were able to stimulate cyclic AMP production in response to ligand binding.
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PMID:Identification of multiple human calcitonin receptor isoforms: heterologous expression and pharmacological characterization. 761 7

Efficient translation of the mRNA encoding the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A (PP2A) is prevented by an out of frame upstream AUG and a stable stem-loop structure (delta G = -55.9 kcal/mol) in the 5'-untranslated region (5'-UTR). Deletion of the 5'-UTR allows efficient translation of the PR65 alpha message in vitro and overexpression in COS-1 cells. Insertion of the 5'-UTR into the beta-galactosidase leader sequence dramatically inhibits translation of the beta-galactosidase message in vitro and in vivo, confirming that this sequence functions as a potent translation regulatory sequence. Cells transfected or microinjected with a PR65 alpha expression vector lacking the 5'-UTR, express high levels of PR65 alpha, accumulating in both nucleus and cytoplasm. PR65 alpha overexpressing rat embryo fibroblasts (REF-52 cells) become multinucleated. These data and previous results (Mayer-Jaekel, R. E., Ohkura, H., Gomes, R., Sunkel, C. E., Baumgartner, S., Hemmings, B. A., and Glover, D. M. (1993) Cell 72, 621-633) suggest that PP2A participates in the regulation of both mitosis and cytokinesis.
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PMID:Deregulation of translational control of the 65-kDa regulatory subunit (PR65 alpha) of protein phosphatase 2A leads to multinucleated cells. 767 73

Using an assay for recombination that measures deletion of a beta-galactosidase gene positioned between two directly repeated 350-bp sequences in plasmids transiently maintained in COS cells, we have found that replication from a simian virus 40 origin produces a high frequency of nonhomologous recombination. In contrast, plasmids replicating from a herpesvirus origin (oris) in COS cells superinfected with herpes simplex virus type 1 (HSV-1) show high levels of homologous recombination between the repeats and an enhanced recombinogenicity of the HSV-1 a sequence that is not seen during simian virus 40 replication. When the same assay was used to study recombination between 120- to 150-bp repeats in uninfected Vero cells, the level of recombination was extremely low or undetectable (< 0.03%), consistent with the fact that these repeats are smaller than the minimal efficient processing sequence for homologous recombination in mammalian cells. Recombination between these short repeats was easily measurable (0.5 to 0.8%) following HSV-1 infection, suggesting that there is an alteration of the recombination machinery. The frequency of recombination between repeats of the Uc-DR1 region, previously identified as the only segment of the HSV-1 a sequence indispensable for enhanced a-sequence recombination, was not significantly higher than that measured for other short sequences.
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PMID:Herpes simplex virus type 1 DNA replication is specifically required for high-frequency homologous recombination between repeated sequences. 770 36

Annexin XI is a newly identified annexin which localizes mainly in the nucleus of rat embryonic fibroblasts. There are no typical nuclear localization signals (NLS) in the molecule. To define the region responsible for its nuclear localization, a series of mutants and chimeric cDNA were constructed. These were transiently expressed in COS-7 cells, and the subcellular distributions of the mutants and chimeric proteins were determined by indirect immunofluorescence microscopy. Wild-type annexin XI was located predominantly within the nucleus. Deletion of the N-terminal tail domain (residues 3-196) changed the distribution of the protein from the nucleus to the cytoplasm whereas deletion of the C-terminal core domain (residues 208-504) still kept the protein sorting to the nucleus. Three other mutants lacking 60-80 amino acids in the N-terminal region (residues 3-61, 61-115, and 115-197, respectively) no longer efficiently imported into the nucleus. Furthermore, Escherichia coli beta-galactosidase polypeptide was efficiently localized to the nucleus only when fused with the whole N-terminal region of annexin XI (residues 1-207), not with part of the N-terminal region. In primary cultured rat hepatocytes, annexin XI was distributed in the cytoplasm but not in the nucleus. These results suggest that the whole N-terminal tail domain of annexin XI is necessary and sufficient for its nuclear localization, and may function as NLS in a cell-type specific manner.
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PMID:The long amino-terminal tail domain of annexin XI is necessary for its nuclear localization. 772 57

The core protein of hepatitis C virus (HCV) is considered to be cleaved from the N terminus of the large precursor polyprotein by cellular signalase. The HCV cDNA encoding the core protein was expressed (i) in monkey COS cells by a plasmid expression vector driven by the SR alpha promoter, and (ii) in insect cells by a recombinant baculovirus. The expressed product had an M(r) of 22,000 and was located in the cytoplasm. When the C-terminal hydrophobic domains were deleted, however, the truncated core proteins were translocated into the nucleus. The truncated core proteins were located in the nucleus even when they were expressed as a fusion protein with E. coli beta-galactosidase, which is essentially localized in the cytoplasm. Plasmids containing HCV cDNAs with a deletion in one of the regions encoding clusters of basic amino acids were expressed in COS cells and the localization of the core protein was examined. The residues PRRGPR were suggested to play an important role in nuclear localization. HCV is an RNA virus and its life cycle was originally considered to be confined to the cytoplasm; the present study, however, suggests that the HCV core protein can translocate into the nucleus under certain circumstances.
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PMID:Nuclear localization of the truncated hepatitis C virus core protein with its hydrophobic C terminus deleted. 784 42

To produce antibodies for determination of the protein mass of human cholesterol 7 alpha-hydroxylase, a fusion protein was prepared from an in-frame fusion gene containing the cDNA for human cholesterol 7 alpha-hydroxylase near the 3' terminus of the lacZ gene of Escherichia coli. The fusion protein was purified by (NH4)2SO4 fractionation and gel-filtration chromatography on a Sephacryl column. Rabbits were immunized with this fusion protein and antisera were obtained. IgG was prepared by submitting antisera to chromatography on protein-A--Sepharose. Antibodies directed against bacterial proteins including beta-galactosidase were removed by affinity chromatography on a column to which bacterial proteins of E. coli containing beta-galactosidase had been immobilized. Evidence that the antibodies are indeed reactive against human liver cholesterol 7 alpha-hydroxylase was obtained by immunoblot analysis with human cholesterol 7 alpha-hydroxylase expressed in COS cells from the coding region of the human cholesterol 7 alpha-hydroxylase cDNA. The antiserum inhibited the activity of cholesterol 7 alpha-hydroxylase in human liver microsomes by approximately 70%. On immunoblotting of solubilized human liver microsomes, a positive band was obtained at a position corresponding to the protein mass for human cholesterol 7 alpha-hydroxylase. When calibration was performed using the fusion protein, a linear relationship was observed between the density and the amount of protein. Proportionality was also observed between the density and the amount of protein for microsomes of COS cells transfected with the coding region of the human cholesterol 7 alpha-hydroxylase cDNA. Liver microsomes from patients treated with cholestyramine (n = 3) were shown to contain levels of cholesterol 7 alpha-hydroxylase protein approximately twofold higher than those of liver microsomes from untreated patients (n = 6; P < 0.02), whereas cholesterol 7 alpha-hydroxylase activity was approximately six-fold higher in liver microsomes from the cholestyramine-treated patients than in the corresponding preparations from the untreated patients (P < 0.02). The higher activities observed in cholestyramine-treated patients, therefore, cannot be explained only by an increased amount of protein, suggesting a posttranslational mechanism to increase the activity of human cholesterol 7 alpha-hydroxylase in addition to the transcriptional control.
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PMID:Immunochemical determination of human cholesterol 7 alpha-hydroxylase. 788 95

We describe here a simple and efficient transfection method for transient expression of cloned genes in cell lines and primary cultured cells. The method involves the use of DEAE-dextran to target DNA to the cellular endocytotic pathway and the use of a human adenovirus to ensure efficient lysis of endosomal vesicles. The procedure allows effective delivery of DNA into the cytoplasm and, therefore, results in a higher fraction of cells expressing exogenous proteins. Using this method, we routinely obtain 60%-90% of COS cells or Chinese hamster ovary cells expressing beta-galactosidase, as determined by in situ staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). We have also obtained much improved levels of expression in cells that are difficult or impossible to use in transient expression assays, such as rat-1 fibroblasts or primary osteoblast cultures. We successfully used the method to express heteromeric proteins that require subunit assembly for proper function. The method also proved effective to express functions in which the exogenous protein needs to couple to the endogenous cellular machinery. Thus, this transient transfection method should prove valuable for many functional studies in a broad variety of cell lines and primary cultures.
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PMID:Adenovirus-mediated transfection of cultured cells. 798 Sep 40

We constructed two genes specific to melanogenesis, human tyrosinase (HT) and tyrosinase-related protein-1 (TRP-1) genes, into two separate expression vectors so that the cloned genes were under the control of a human cytomegalovirus promoter and enhancer. Monkey kidney COS-7 cells and human amelanotic and melanotic melanoma cells were then cotransfected by both HT and TRP-1 or transfected individually with each gene. The transfectants were examined for mRNA expression by reverse transcription-mediated RNA-PCR amplification. HT or TRP-1 mRNA was strongly expressed in HT or TRP-1 transfectants and cotransfectants of the two genes. Both light and electron microscopic observations indicated that degeneration and premature death of melanocytes occurred in HT transfectants, but not in TRP-1 transfectants or in HT and TRP-1 cotransfectants. Cotransfected cells from five cell lines revealed numerous granular reaction products with an anti-TRP-1 antibody and lysosomal granules with electron-dense material. Our melanin assay confirmed the new production of melanin pigments in these cells, indicating that the lysosomal granules would contain melanin pigments. The gene expression studies of lysosomal protein (beta-galactosidase, CD63, Lamp-1, and Lamp-2) revealed a dramatically elevated gene expression of Lamp-1, which is associated with the membrane receptor of lysosomal granules, in HT- and TRP-1-cotransfected cells. Conversely, the treatment of melanoma cells with antisense oligodeoxynucleotides against Lamp-1 resulted in a decreased expression of TRP-1 protein by immunoprecipitation, supporting the observations of the HT and TRP-1 cotransfection study regarding the up-regulation of Lamp-1 expression. We conclude that HT, TRP-1, and Lamp-1 gene products may function together, being expressed as a multiprotein complex within the melanosomal compartment. Specifically, HT and TRP-1 may function together via Lamp-1 by stabilizing the enzyme-protein complex within the melanosome and prevent the premature death of melanocytes due to tyrosinase-mediated cytotoxicity.
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PMID:Cotransfection of genes encoding human tyrosinase and tyrosinase-related protein-1 prevents melanocyte death and enhances melanin pigmentation and gene expression of Lamp-1. 802 May 95

A cDNA clone encoding a new type of GalNAc alpha 2,6-sialyltransferase (ST6GalNAc II) with a structure similar to that of a previously cloned GalNAc alpha 2,6-sialyltransferase (ST6GalNAc I; Kurosawa, N., Hamamoto, T., Lee, Y.-C., Nakaoka, T., Kojima, N., and Tsuji, S. (1994) J. Biol. Chem. 269, 1402-1409) was obtained from chicken testes. The predicted amino acid sequence of ST6GalNAc II encodes a protein with type II transmembrane topology, as found for other glycosyltransferases, and showed 32% identity with that of ST6GalNAc I. Transfection of the full length ST6GalNAc II gene into COS cells led to GalNAc alpha 2,6-sialyltransferase activity with a different substrate specificity from that of ST6GalNAc I. Moreover, asialofetuin after treatment with beta-galactosidase did not serve as an acceptor for this enzyme. 14C-Sialylated oligosaccharides obtained from resialylated asialobovine submaxillary mucin with this enzyme were identical to Gal beta 1,3([14C]NeuAc alpha 2,6)GalNAc-ol but not [14C]NeuAc alpha 2,6GalNAc-ol. These results clearly show that the expressed enzyme is a novel type of sialyltransferase that requires beta-galactoside residues linked to GalNAc residues, whereas sialic acid residues linked to galactose residues are not essential for the activity.
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PMID:Cloning and expression of Gal beta 1,3GalNAc-specific GalNAc alpha 2,6-sialyltransferase. 803 63


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