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)

It is poorly understood why vaccines could not be developed for the control and prevention of African swine fever (ASF) virus infection. The aim of our study was to identify genes non-essential for ASF virus replication because there were indications that certain viral gene products, which apparently are non-essential for viral replication, conferred protection from death due to ASF. A cosmid library representing the genome of ASF virus strain France 64 was established and characterized. Then, in order to inactivate viral genes by insertion, the beta-galactosidase (beta-gal) gene was introduced either randomly or at specific locations of selected cloned DNA fragments. These constructions were transfected into cells which had been previously infected with a cell-culture-adapted viral strain in order to allow the generation of recombinant progeny virus. Viable recombinant progeny was identified by at least one of the following means: (1) expression of beta-gal; (2) detection of beta-gal specific DNA by plaque hybridization, and (3) absence of a functional product of the inactivated gene. Presently, we are characterizing a recombinant virus with an insertionally inactivated thymidine kinase gene.
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PMID:Approaches to the identification of non-essential genes of African swine fever virus. 148 51

Homologous recombination is shown to be specifically induced in Vero cells by infection with African swine fever (ASF) virus. The frequency of recombination induced by ASF virus infection between cotransfecting plasmids is comparable to that found after infection with the prototype poxvirus, vaccinia virus. The induction of recombination is accompanied by replication of the plasmid templates in the ASF virus-infected cells. An ASF virus insertion/expression plasmid vector containing the Escherichia coli reporter gene beta-galactosidase (beta-gal) fused to a viral promoter sequence was constructed. Recombination between homologous sequences present in both the plasmid vector and the virus genome led to the generation of recombinant viruses expressing the beta-gal gene. Visual screening of beta-gal+ plaques allowed the isolation and plaque purification of recombinant ASF viruses. The characterization of a beta-gal+ virus isolate showed that the beta-gal gene had been stably inserted into the thymidine kinase locus of the virus genome, thus demonstrating that controlled genetic manipulation of ASF virus can be achieved by homologous recombination in infected cells.
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PMID:Genetic manipulation of African swine fever virus: construction of recombinant viruses expressing the beta-galactosidase gene. 156 85

We have developed a ligand-specific method for the visualization, isolation, and biochemical characterization of cell surface and intracellular membranes mediating endocytic transport. Iron dextran particles (FeDex) bearing either covalently conjugated galactosyl bovine serum albumin (GalBSA/FeDex) or asialofetuin (ASF/FeDex) are bound by the asialoglycoprotein receptor (ASGP-R) of HepG2 cells and transported to lysosomes with kinetics indistinguishable from those of free GalBSA or ASF. FeDex particles, which have a 3 to 5 nm electron-dense colloidal iron core, can be visualized by electron microscopy. Following incubation of GalBSA/FeDex with HepG2 cells at 37 degrees C, FeDex particles are seen at the cell surface, in endosomes, and in lysosomes. Surface membrane and intracellular organelles bearing a sufficient number of FeDex particles can be efficiently isolated from disrupted cells by high gradient magnetic affinity chromatography (HIMAC). Plasma membranes and endosomal/lysosomal membranes isolated by HIMAC are 35 to 40-fold enriched for GalBSA/FeDex or ASF/FeDex relative to the postnuclear supernatant. Alkaline phosphodiesterase I (APDE) and galactosyltransferase are each enriched 8-fold in the plasma membrane fraction prepared by HIMAC whereas neither beta-galactosidase nor glucose-6-phosphatase are detected in this fraction. The intracellular membrane fraction, containing both endosomes and lysosomes, is enriched for galactosyltransferase and beta-galactosidase but not for APDE or glucose-6-phosphatase. Use of FeDex conjugates in conjunction with HIMAC provides an effective method for ligand-specific isolation of membranes and correlation of morphological and biochemical characteristics.
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PMID:Ligand-specific isolation of endosomes and lysosomes using superparamagnetic colloidal iron dextran glycoconjugates and high gradient magnetic affinity chromatography. 168 Jun 81

The gene encoding the African swine fever virus protein of M(r) 11,500, present in the virus particle, has been mapped and sequenced in the genome of the Vero cell-adapted virus strain BA71V. A serum raised against virion proteins of M(r) 12,000 to 13,000 isolated from polyacrylamide gels was used to screen a plasmid expression library, containing viral DNA random fragments, that expresses viral polypeptides fused to beta-galactosidase. Using this method, we have identified and sequenced the open reading frame (ORF) A137R, which initiates at the right end of the EcoRI A restriction fragment and extends into the EcoRI F fragment. Expression of the protein in Escherichia coli has confirmed that ORF A137R encodes a protein with an M(r) of about 12,000. A specific serum was raised against the E. coli-expressed protein, and has been used to identify the protein encoded by the ORF, which is translated at late times of infection and incorporated into the virus particle. Immunofluorescence experiments have shown that the protein localizes in virus factories.
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PMID:Mapping and sequence of the gene encoding the African swine fever virion protein of M(r) 11500. 824 48

Antibody neutralization of African swine fever (ASF) virus measured by a plaque reduction assay presents frequent difficulties because of the absence or delay in plaque formation by many strains, especially low-passage viruses. To overcome this problem, a new ASF virus neutralization test has been developed. The new test consists of a conventional plaque reduction assay in which the viral plaques are detected by expression of marker genes. For the development of this neutralization assay 4 mutant viruses were generated by homologous recombination, containing beta-galactosidase or beta-glucuronidase reporter genes inserted into the thymidine kinase locus of the viral genome. These recombinant viruses have the following advantages with respect to parental viruses: (1) the neutralization assay takes less than a third of the time needed using non-recombinant viruses; (2) the small plaques can be detected more accurately by color contrast; and (3) the neutralization-resistant virus clones can be recovered easily post-plaque counting. Additionally, these recombinant viruses permit differentiation by chromogenic staining of individual infected pig macrophages, the natural host cell for ASF virus, facilitating neutralization assays in these primary cultures as described in cell lines.
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PMID:Improvement of African swine fever virus neutralization assay using recombinant viruses expressing chromogenic marker genes. 853 64

Protein p54, one of the most antigenic structural African swine fever virus (ASFV) proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the p54 gene from ASFV by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the p54 gene and beta-glucuronidase selection marker under the control of p54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of p54 and beta-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of p54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the p54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.
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PMID:The structural protein p54 is essential for African swine fever virus viability. 872 12

The African swine fever virus (ASFV) strain Malawi LIL20/1 open reading frame (ORF) j13L was expressed in vaccinia virus (VV) from a strong synthetic late promoter as either a complete ORF (vSJ1) or lacking codons 1-31 (vSJ2). Each recombinant VV produced a small plaque which rapidly reverted to a normal size upon passage. The yield of infectious virus from a single cycle infection with vSJ1 or vSJ2 was reduced 50- to 100-fold compared to wild-type (wt) and a revertant virus (vSJ5) in which the j13L ORF was removed and the VV thymidine kinase gene restored. PCR analysis of nine spontaneous large plaque revertant viruses, recovered after passage of vSJ1 in BSC-40 cells, showed that six had lost the j13L ORF and the co-inserted beta-galactosidase gene. Three viruses retained the j13L and beta-galactosidase genes, but in each case the j13L protein was not expressed due to a different single base deletion near the 5' end of the j13L coding region which introduced a stop codon a short distance downstream. The formation of intracellular mature virus (IMV) and extracellular enveloped virus was reduced 50- to 75-fold in cells infected with vSJ1 compared to wt VV and revertant vSJ5. Electron microscopy showed aberrant IMV precursor structures in vSJ1-infected cells, and immunoelectron microscopy demonstrated that these structures contained j13L protein. These results indicate that expression of the j13L protein is toxic for VV replication due to interference with VV morphogenesis prior to IMV formation.
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PMID:Expression of African swine fever virus envelope protein j13L inhibits vaccinia virus morphogenesis. 960 32

PCR analysis of the genomes of 18 different African swine fever virus (ASFV) isolates showed that the I14L open reading frame (ORF) was present as either a long form or short form in all of the isolates. Sequencing of the ORF from eight isolates confirmed that both forms of the ORF were well conserved. Antisera raised against the I14L protein identified the long form of the protein as a 21 kDa protein expressed late during ASFV infection. Immunofluorescent analysis of transiently expressed haemagglutinin-tagged forms of the I14L protein showed that the long form of the protein localized predominantly to the nucleus and within the nucleoli. In contrast, although the short form of the protein was also present predominantly in the nucleus, it did not localize to the nucleoli. Deletion of the N-terminal 14 amino acids from the long form of the I14L protein, which includes a high proportion of basic Arg/Lys residues, abolished the specific nucleolar localization of the protein, although the protein was still present in the nucleus. Addition of this 14 amino acid sequence to beta-galactosidase or replacement of the N-terminal 14 amino acids of the I14L short form with those from the long form directed both of these modified proteins to the nucleolus. This indicates that this 14 amino acid sequence contains all the signals required for nucleolar localization.
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PMID:Nuclear and nucleolar localization of an African swine fever virus protein, I14L, that is similar to the herpes simplex virus-encoded virulence factor ICP34.5. 1009 89