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)

We have constructed a series of insertion mutations at 18 sites in the coding sequences of early region 1A (E1A) of human adenovirus type 5 (Ad5). At each site we have introduced three types of mutation: a 39-bp insertion specifying a 13-aa residue oligopeptide, a 39-bp insertion containing chain termination codons in all three reading frames, and a "collapsed" insert of 6-bp forming a conventional linker insertion mutation. All mutants were sequenced to determine the precise location, structure, and orientation of the inserts. The mutants were assayed for their abilities to trans-activate and to repress using transient expression assays in HeLa cells cotransfected with the E1A mutant plasmids and a reporter plasmid containing the bacterial beta-galactosidase (lac Z) gene under the control of Ad5 early promoters. The mutants were also tested for their ability to transform baby rat kidney cells in cooperation with either E1B or the ras oncogene. Each mutant was rescued into virus and infectivity was compared in HeLa and 293 cells. In addition, E1A protein synthesis was analyzed in cells infected with the mutant viruses and the insertions were found to have pronounced but unpredictable effects on electrophoretic mobility of E1A proteins in SDS-polyacrylamide gels. The results of functional assays indicated that only mutations mapping in, or deleting, the unique region of the 13 S mRNA product had any effect on ability to trans-activate and that a perfect correlation existed between ability of a mutant to trans-activate and to replicate efficiently in HeLa cells or to transform baby rat kidney cells in an E1A plus E1B mediated assay. In contrast, insertions near conserved region 2 of exon I and in the NH2-terminal portion of exon II significantly reduced repression activity but left transforming activity with E1B or with ras essentially unaffected suggesting that the repression function of E1A is separate from, or at least nonessential in, transformation.
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PMID:Isolation and characterization of insertion mutants in E1A of adenovirus type 5. 182 28

Sera from 26 rats bearing tumours induced by wild-type (wt) and mutant human adenovirus type 12 (Ad12), or by cells transformed with these viruses, were analysed for antibodies against the early region 1 (E1) transforming proteins. Six Ad12-beta-galactosidase fusion proteins encoding different regions of the Ad12 E1 proteins were constructed. The sera from the tumour-bearing animals reacted most strongly with the fusion protein encoding the N terminus of the E1A protein. Tumour-bearing rats exposed to the E1B 54K and 19K proteins showed strong reactions with the N terminus of the 54K protein and the C terminus of the 19K protein. Monospecific polyclonal antisera were raised against five of the fusion proteins by immunization of rats and rabbits; these sera cross-reacted with the purified native protein. No antibodies could be obtained which recognized a fusion protein containing amino acids 136 to 268 of the 54K protein. The fusion proteins were also used to purify monospecific antisera from tumour-bearer sera using affinity chromatography.
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PMID:The use of beta-galactosidase fusion proteins encoding the early region 1 transforming proteins of adenovirus type 12 to examine the humoral response in tumour-bearing animals. 184 78

The safety of replication-defective viruses used as vectors is based on the deletion of essential gene(s). Adenovirus vector safety relies on the deletion of the E1A/E1B region. This region encodes the immediate-early proteins that trans activate all other early regions, so DNA replication in these deletion mutants is dramatically reduced. We have previously shown that E1A deletion is efficient in vivo and significantly reduces the dissemination of adenovirus in mice and cotton rats. However, the pattern of dissemination of E1A-deleted and wild-type viruses showed that both could be localized in the same tissues, thus involving a theoretical risk of phenotypic complementation if a recipient of E1A-deleted adenovirus is infected after adenovirus-mediated gene therapy by a wild-type adenovirus. In this report, we show that complementation can be evidenced in vitro in Vero cells infected with E1A/E1B-defective adenovirus vectors expressing reporter genes (either beta-galactosidase or luciferase), passaged three times until no infectious virus can be recovered by plating on 293 cells, and then infected with wild-type adenovirus 5. A mixed virus population was maintained at a stable state for at least 10 passages. In contrast, no evidence of complementation was found in cotton rats inoculated intravenously or intramuscularly with Ad-beta-gal-nls and Ad-luc and infected 24 h later intranasally with wild-type adenovirus 5. No increase in the level of luciferase expression was found in these animals, compared with that in controls, nor was any viral population expressing beta-galactosidase or luciferase isolated from various organs or any animal excretion or secretion.
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PMID:Lack of evidence of phenotypic complementation of E1A/E1B-deleted adenovirus type 5 upon superinfection by wild-type virus in the cotton rat. 766 53

The bovine adenovirus type 3 (BAV3) genome was sequenced from the left end to the HindIII site at 11%. This region comprises the entire E1 transcription unit including the open reading frames (ORF) for proteins homologous to the E1A, E1B proteins and protein IX of human adenovirus type 5 (Ad5). A portion of the BAV3 E1A protein showed significant homology with conserved region 3 (CR3), the principal transactivation region of Ad5 E1A. The BAV3 E1A protein also contains a consensus sequence known to be important for interaction with the cellular Rb protein but lacks most of the sequence corresponding to the second exon of Ad5 E1A. Promoter sequences for BAV3 E1B were not defined though the relevant region contains a 35-base pair repeat sequence. Two ORFs define the BAV3 E1B coding unit; one with regions homologous to sequences within the Ad5 E1B 19k protein, and an overlapping ORF with significant homology to the Ad5 E1B 55k protein. The encoded BAV3 E1B proteins of 157 and 420 amino acid residues (R) have predicted unmodified molecular weights of 17,393 and 46,734 respectively. Immediately following the E1B coding region there is a transcription unit containing an SP1 binding site and TATA box followed by an ORF which encodes a protein of 125R and predicted molecular weight of 13,706 with homology to protein IX of Ad5. Five concensus poly A addition sites are located in the 350 base pairs immediately following the protein IX coding region. The homology of sequences in the Ad5 E1A CR3 region and the corresponding BAV3 protein suggested that the BAV3 protein could transactivate certain Ad5 genes normally transactivated by the Ad5 E1A product. Evidence for this hypothesis was obtained in studies in which bovine cells in culture were coinfected with BAV3 and a human adenovirus type 5 (Ad5) recombinant viral vector lacking the E1A region and having a lacZ reporter gene within the E3 region dependent on E1A for its expression. Coinfection resulted in the induction of beta-galactosidase activity and the increased expression of other Ad5 early (E2A 72k) and late (hexon) proteins.
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PMID:The E1 sequence of bovine adenovirus type 3 and complementation of human adenovirus type 5 E1A function in bovine cells. 817 72

The 293 cell line that was generated by transforming human embryonic kidney cells with human adenovirus type 5 (HAV5) early region 1 (E1) sequences is an excellent host for generating and growing HAV5 recombinants with E1 deleted, but it does not support the replication of bovine adenovirus type 3 (BAV3). Madin-Darby bovine kidney (MDBK), an established bovine cell line, is an excellent host for growing and plaquing BAV3. For the purpose of combining the unique characteristics of these two cell lines (293 and MDBK), we generated a number of bovine x human hybrid (BHH) cell lines. Comparison of three BHH hybrid clones-BHH3, BHH8, and BHH2C-with 293-Puro (puromycin-resistant 293 cells) and MDBK-Neo (G418-resistant MDBK cells) cell lines for total cellular DNA content, species-specific surface markers, isoenzyme analysis, and karyotyping indicate that they are hybrid in nature. BHH clones constitutively expressed the E1 proteins (E1A, E1B-21kDa, and E1B-55kDa) of HAV5 and efficiently supported the replication of both wild-type and replication-incompetent bovine or human adenoviruses. Transient gene expression experiments with a plasmid encoding the bacterial beta-galactosidase gene demonstrated that BHH cell hybrids seem to have better transfection efficiencies than either of the parental cell lines. These cell lines will be useful for isolating and growing replication-competent human or bovine adenovirus recombinants with E1 deleted and for the study of cellular or viral factors important for viral replication. The development of somatic cell hybrids appears to be a simple way of combining some of the desirable characteristics present separately in two parental cell lines.
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PMID:Development and characterization of bovine x human hybrid cell lines that efficiently support the replication of both wild-type bovine and human adenoviruses and those with E1 deleted. 1202 21

Replication-competent oncolytic viruses are being developed for human cancer therapy. We previously reported that an attenuated adenovirus OBP-301 (Telomelysin), in which the human telomerase reverse transcriptase promoter element drives expression of E1A and E1B genes linked with an internal ribosome entry site, could replicate in and causes selective lysis of human cancer cells. Infection efficiency in target cancer cells is the most important factor that predicts the antitumor effects of OBP-301. The objectives of this study are to examine the effects of the histone deacetylase inhibitor FR901228 on the level of coxsackie and adenovirus receptor (CAR) expression and OBP-301-mediated oncolysis in human non-small cell lung cancer cell lines. Flow cytometric analysis revealed up-regulated CAR expression in A549 and H460 cells following treatment with 1 ng/ml of FR901228, which was associated with increased infection efficiency as confirmed by replication-deficient beta-galactosidase-expressing adenovirus vector. In contrast, neither CAR expression nor infection efficiency was affected by FR901228 in H1299 cells. To visualize and quantify viral replication in the presence of FR901228, we used OBP-401 (Telomelysin-GFP) that expresses the green fluorescent protein (GFP) reporter gene under the control of the cytomegalovirus promoter in the E3 region. Fluorescence microscopy and flow cytometry showed that FR901228 increased GFP expression in A549 and H460 cells following OBP-401 infection in a dose-dependent manner, but this effect did not occur in H1299 cells. In addition, OBP-301 and FR901228 demonstrated a synergistic antitumor effect in A549 cells in vitro, as confirmed by isobologram analysis. Our data indicate that FR901228 preferentially increases adenovirus infectivity via up-regulation of CAR expression, leading to a profound oncolytic effect, which may have a significant impact on the outcome of adenovirus-based oncolytic virotherapy.
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PMID:Histone deacetylase inhibitor FR901228 enhances the antitumor effect of telomerase-specific replication-selective adenoviral agent OBP-301 in human lung cancer cells. 1635 94

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