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)

Epstein-Barr virus (EBV)-transformed human B-cell lines, L-KT9 and DH3 cells express CD23 antigen, and grow in a mixture of single and aggregated cells. The CD23 molecule has high amino acid sequence homology with C-type lectin and recently we have shown that the solubilized CD23 molecule can really interact with galactose residues on glycoproteins. In this study, therefore, we tested whether CD23 antigen on the cell surface really acts as a galactose-binding lectin in the aggregation of these cells. The EBV-transformed cells (L-KT9) were separated into an aggregated-cell-rich fraction and a single-cell-rich fraction. Aggregated cells disaggregated after removal of galactose by beta-galactosidase treatment, whereas single cells made large aggregation on sialidase treatment, and this aggregation was inhibited in the presence of asialo-fetuin. On the other hand, naturally aggregated cells become single cells with anti-CD23 monoclonal antibody (mAB) as well as the soluble form of CD23, but not with anti-CD21 mAB. In addition, L-KT9 and DH3 cells bound to asialo-fetuin-coupled Sepharose (ASF-Sepharose) and this binding was significantly inhibited by pre-treatment of cells with anti-CD23, but not with anti-CD21 or other anti-adhesion molecules. From these results, we conclude that the naturally aggregated state of EBV-transformed cells occurs mainly through the interaction of CD23 as a lectin molecule and galactose residues as its ligand.
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PMID:CD23 molecule acts as a galactose-binding lectin in the cell aggregation of EBV-transformed human B-cell lines. 757 99

The genomic localization of two immunodominant genes encoding two proteins of the Epstein-Barr virus capsid antigen (VCA) complex, VCA-p18 and VCA-p40, has been identified. For that purpose, lambda gt11-based cDNA libraries were constructed from HH514.c16 cells induced for virus production. The libraries were screened with a monoclonal antibody, EBV.OT41A, directed against VCA-p40 or with affinity-purified human antibodies against VCA-p18. Sequencing of the inserts of positive plaques showed that VCA-p18 and VCA-p40 are encoded within open reading frames (ORFs) BFRF3 and BdRF1, respectively. Peptide scanning analysis of the predicted protein of ORF BdRF1 resulted in defining the epitope of monoclonal antibody EBV.OT41A at the C-terminal region. The dominant VCA-p18 reactivity of human sera can be completely inhibited by preadsorption with Escherichia coli-expressed BFRF3-beta-galactosidase. Serum of a rabbit immunized with BFRF3-beta galactosidase reacts with a VCA-specific protein of 18 kDa. In addition, BFRF3-beta-galactosidase affinity-purified antibodies react with VCA-p18 of virus-producing cells (HH514.c16). Complete inhibition of viral DNA polymerase activity by phosphonoacetic acid is associated with the absence of RNAs and protein products of both ORFs, indicating that VCA-p18 and VCA-p40 are true late antigens.
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PMID:Gene mapping and expression of two immunodominant Epstein-Barr virus capsid proteins. 768 3

Levels of expression of two reporter genes cloned into SV40 or Epstein-Barr virus (EBV) ori-containing plasmids were measured following transient transfection of cell lines constitutively expressing T-antigen or EBV nuclear antigen 1 (EBNA1). The TSA201 and COS7 cell lines stably produce T-antigen and support replication of the SV40 ori-containing constructs while the 293EBNA cell line produces EBNA1 and supports replication of EBV ori-containing plasmids. We found that 293EBNA cells express > 25-fold more beta-galactosidase (beta Gal) per mg protein than COS7 cells and 11-fold more beta Gal than TSA201 cells. We also demonstrate that 293EBNA cells are able to express 70-100-fold more angiotensin II type-1 receptor (AT1) per mg protein than COS7 or TSA201 cells. We examined the suitability of each cell line for use in expression cloning using a NaOH 'scrape' method as an improvement over emulsion autoradiography for detection. Measurable AT1 signals can be detected when reporter plasmids are diluted up to 1000-fold for COS7 and TSA201 cells, and up to 80,000-fold for 293EBNA cells. These data demonstrate that 293EBNA cells offer a significant improvement in expression cloning technology as compared to the conventionally used T-antigen-based cell lines.
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PMID:Improved expression cloning using reporter genes and Epstein-Barr virus ori-containing vectors. 775 62

ZEBRA has been shown to activate model reporter genes consisting of synthetic oligomerized ZEBRA response elements upstream of a minimal CYC1 promoter fused to beta-galactosidase in the yeast Saccharomyces cerevisiae. Here it is shown that in S. cerevisiae ZEBRA activates transcription of natural Epstein-Barr virus promoters. Two Epstein-Barr virus promoters were shown to be activated by ZEBRA in S. cerevisiae: Zp, the promoter that regulates expression of BZLF1, which encodes ZEBRA; and EAp, the promoter controlling expression of BMRF1, which encodes diffuse early antigen, EA-D. These observations indicate that neither mammalian-specific nor virally encoded coactivators are obligatory for ZEBRA to stimulate expression from these two promoters. Zp was also strongly activated by endogenous yeast factors. EAp was not activated by yeast factors. The results show that in S. cerevisiae and in B cells, ZEBRA dominates the response of EAp; ZEBRA plus endogenous cell factors activate Zp.
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PMID:Activation of the Epstein-Barr virus BMRF1 and BZLF1 promoters by ZEBRA in Saccharomyces cerevisiae. 793 54

To try epidermis as a target for somatic gene therapy we studied transfected primary human keratinocytes grown in culture and grafted onto athymic mice. We have developed a novel technique for grafting cultured epidermal sheets onto mice. First, the graft is placed on the dorsal muscle fascia underneath the mouse skin using the latter as a bandage. Secondly, the mouse skin above the graft is removed, which exposes the grafted skin to open air and thus stimulates terminal differentiation. A novel method for the discrimination between murine and human epidermal cells is also presented, employing in situ hybridization with human Alu repeated DNA sequences. During monolayer culture the keratinocytes were lipofected with the gene for human growth hormone in an Epstein-Barr virus-based expression vector. The cells were allowed to develop a multilayered tissue for 5 d, secreting human growth hormone into the medium at a daily rate of at least 50 ng/cm2 of tissue. The transfected tissues were then grafted onto mice. We detected human growth hormone at levels of up to 2.6 ng/ml in mouse serum for 4 d, but later no human growth hormone could be found, although the transplants survived for months. To investigate the fate of the transfected cells in the transplanted tissue, we labeled them with the beta-galactosidase reporter gene. The cells staining positive for X-gal were found exclusively in the most superficial differentiated layers at 7 d after transplantation. This may be the main reason why no human growth hormone is found in the mouse circulation at this time.
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PMID:Gene transfer into cultured human epidermis and its transplantation onto immunodeficient mice: an experimental model for somatic gene therapy. 807 6

This laboratory has previously reported that a tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate ("12-O-tetradecanoylphorbol 13-acetate"), induces BJAB cells, a Burkitt lymphoma cell line, to express cellular proteins that bind to the origin of plasmid DNA replication (oriP) of Epstein-Barr virus. These oriP-binding proteins interfere with the EBV-encoded nuclear antigen EBNA-1, which binds to oriP in Raji cells. To further characterize these proteins, a lambda phage expression cDNA library made from phorbol ester-induced BJAB cells was screened for fusion proteins which bind to oriP. Two recombinant phages containing sequences encoding beta-galactosidase fusion proteins and designated lambda-OBP-1 and lambda-OBP-2 were identified. lambda-OBP-1 and lambda-OBP-2 contained 0.43 kbp and 0.61 kbp of BJAB cell cDNA, respectively, of which 395 bp were shared. Using lambda-OBP-1 as probe, two cDNAs of 1.4 kbp and 1.2 kbp, designated OBP-1 and OBP-2, respectively, were isolated. These cDNAs also shared the 395-bp sequence at the 3' end. With these cDNAs as probes, Northern blot analyses of mRNA from BJAB cells gave 1.4, 2.4, and 3.4-kb bands, but a Southern blot of human genomic DNA revealed one band. It is likely that the oriP-binding proteins were derived from spliced mRNA(s) of a gene family.
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PMID:The cellular proteins that bind specifically to the Epstein-Barr virus origin of plasmid DNA replication belong to a gene family. 814 98

Epstein-Barr virus (EBV) is a herpesvirus that transforms B-cells (B-LCL) and has undergone intense scrutiny owing to its association with Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas. B-LCL have also proven useful in the study of human immunology. We describe a novel system for inducing efficient foreign gene expression in B-LCL using biotinylated adenovirus as an endosome-disrupting agent. Plasmid DNA is coupled to the exterior of viral particles by streptavidin-polylysine chimeric proteins. Up to 67% of B-LCL may be induced to express foreign genes in vitro in transient expression systems, and gene expression lasts for at least 17 days. We have expressed firefly luciferase, beta-galactosidase (beta-gal), chloramphenicol acetyltransferase, HIV gag, and env genes, as well as infectious HIV, and the EBV-specific BZLF gene in B-LCL with this system. In vivo delivery of a beta-gal reporter gene to B-LCL was documented in a SCID mouse model. Potential applications include study of genetic regulation of EBV infection and transformation events, study of potential gene therapies for EBV-related B-cell tumors, and production of antigen-presenting cells for use in immunologic assays. Because of the high percentage of cells transformed and the length of foreign gene expression, the possibility of examining foreign gene expression in transient assays, without selection for clonal populations, exists.
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PMID:Efficient foreign gene expression in Epstein-Barr virus-transformed human B-cells. 829 Dec 40

The Epstein-Barr virus BZLF1 gene product EB1 (also called ZEBRA and Zta), is a transcription factor belonging to the bZIP (basic domain leucine zipper) family of nuclear proteins. Translocation to the nucleus of EB1 (J. Becker, U. Leser, M. Marschall, A. Langford, W. Jilg, H. Gelderblom, P. Reichart, and H. Wolf, Proc. Natl. Acad. Sci. USA 88:8332-8336, 1991) and of two other bZIP proteins, c-Jun and c-Fos (P. Roux, J.-M. Blanchard, A. Fernandez, N. Lamb, P. Jeanteur, and M. Piechaczyk, Cell 63:341-351, 1990), has been shown to be subject to regulation. We show here that for both EB1 and Jun the nuclear targeting signals (NTS) in the proteins' primary sequences are two clusters of positively charged amino acids. These clusters, called BRA and BRB, are necessary and sufficient to direct beta-galactosidase to the nuclear compartment and act as a bipartite NTS. They are conserved among all the bZIP proteins, and although they are not identical, they probably share the same function. Site-directed mutagenesis studies made on these basic clusters suggest that they also act as a bipartite NTS in the EB1 protein. Our results also demonstrate that in EB1 and Jun, these bipartite NTS are superimposed with bipartite DNA-binding domains, since BRA and BRB are required in vitro for direct and specific contact between these proteins and their DNA-binding sites.
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PMID:The DNA-binding domain of two bZIP transcription factors, the Epstein-Barr virus switch gene product EB1 and Jun, is a bipartite nuclear targeting sequence. 838 Apr 64

The Epstein-Barr virus nuclear antigen 3A is expressed in the nuclei of cells latently infected by the Epstein-Barr virus. We have previously shown that a fragment of 265 amino acids was essential for the proper subcellular localization of the Epstein-Barr virus nuclear antigen 3A. As described in this paper, we have used deletion analysis to identify a decapeptide, RDRRRNPASR, which is essential for nuclear localization of this protein. Furthermore, this decapeptide is a functional nuclear localization signal as demonstrated by its ability to target expression of beta-galactosidase in the nuclei of transfected cells.
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PMID:Identification of a short amino acid sequence essential for efficient nuclear targeting of the Epstein-Barr virus nuclear antigen 3A. 838 17

The mannose transporter of the bacterial phosphotransferase system mediates carbohydrate transport across the cytoplasmic membrane concomitant with carbohydrate phosphorylation. It also functions as a receptor for bacterial chemotaxis [Adler.J. & Epstein, W. (1974) Proc. Natl Acad. Sci. USA 71. 2895-2899] and is required for infection of the cell by bacteriophage lambda where it most likely functions as a pore for penetration of phage DNA [Elliott, J. & Arber, W. (1978) Mol. & Gen. Genet. 161, 1-8]. The transporter consists of two transmembrane subunits (27-kDa IICMan and 31-kDa IIDMan) and a hydrophilic subunit (35-kDa IIABMan). Protein fusions of IICMan and IIDMan with beta-galactosidase (LacZ) and with alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the two proteins. Protein fusions were obtained by progressively deleting the manY and manZ genes from their 3' ends and ligating them to lacZ and 'phoA that lack promotor and leader sequences. Based on the analysis of 30 IICMan-PhoA. 10 IICMan-LacZ, 12 IIDMan-PhoA, and 30 IIDMan-LacZ fusions, it is predicted that IICMan has six membrane-spanning segments with the N- and C-termini on the cytoplasmic face of the membrane. IIDMan is anchored in the membrane by a single membrane-spanning segment at the end of the C-terminus, while most of the protein (250 residues) protrudes into the cytoplasm.
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PMID:Membrane topology of the mannose transporter of Escherichia coli K12. 877 30


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