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)

Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41. These clusters are designated BS I, BS II, and BS III, respectively. (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal). The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible. However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E. coli beta-gal fusion protein. Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins. Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.
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PMID:Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals. 177 81

Adenovirus type 12 transcriptional complexes were isolated from cells during the early phase of infection. Sedimentation analysis identified a fast sedimenting complex type I and a slow sedimenting complex type II. Both complexes made virus specific RNA complementary to all the early genes and both contained viral DNA, which in type II but not in type I had nucleosome like configuration. Analysis of the proteins of the complexes with antiserum against Ad 12 EIa-beta-galactosidase fusion protein expressed in E. coli demonstrated the following: (a) type I complex contained EIa 45 K protein, which co-precipitated with cellular proteins of mol. wt. 42, 58, and 60 K, (b) type II complex contained EIa 47 K protein, which co-precipitated with major cellular proteins of 35, 40-46 K and minor proteins of 58, 60, 68, 76, 86, and 120-150 K. Association of EIa specific and cellular proteins to transcriptional complexes was sensitive to both 1 M NaCl and DNAse I indicating the DNA binding nature of these proteins. Treatment of transcriptional complexes with 1 M NaCl or DNase I released EIa proteins, which still remained strongly bound to cellular proteins. These findings suggested that EIa proteins bind to viral DNA and that this binding is probably mediated by cellular proteins.
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PMID:Adenovirus transcriptional complexes contain EIa encoded tumour antigens physically bound to cellular proteins. 297 76

Adenovirus vectors are a promising vehicle to deliver cystic fibrosis transmembrane conductance regulator (CFTR) cDNA to airway epithelia. However, the value of adenovirus vectors will depend on the efficiency with which the vector can correct the defective fluid transport that is though to underlie the pathogenesis of the disease. To address the efficiency of gene transfer, we applied adenovirus vectors expressing CFTR (Ad2/CFTR-1) or beta-galactosidase to the mucosal surface of primary cultures of airway epithelial cells grown as polarized epithelial monolayers on permeable filter supports. These conditions provide a model that reproduces the physiology of the airways in vivo. We found that after adding 1 moi Ad2/CFTR-1 to the mucosal surface, cAMP agonists stimulated fluid secretion that was within the range observed in epithelia from normal subjects. When we measured electrolyte transport, we found that as little as 0.1 moi partially restored cAMP-stimulated Cl- secretion, and at 10 moi Cl- secretion was in the normal range. A related vector encoding beta-galactosidase generated activity in approximately 20% of cells at an moi of 1 and 90% of cells at an moi of 10. These data suggest that Ad2/CFTR-1 is very efficient at restoring normal fluid and electrolyte transport to CF airway epithelia. Thus, they suggest that relatively low input doses could be used for gene transfer to CF airway epithelia.
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PMID:Correction of cAMP-stimulated fluid secretion in cystic fibrosis airway epithelia: efficiency of adenovirus-mediated gene transfer in vitro. 751 84

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

Adenovirus serotype 5 vectors which contain the Excherichia coli beta-galactosidase gene driven by the cytomegalovirus immediate-early promoter as a screenable marker have been made and successfully used in the construction of recombinant adenoviruses. The beta-galactosidase gene has been introduced into viruses in which the E3 region is maintained or deleted and in which the cis-acting packaging sequence has been reiterated at the right end of the chromosome. A unique BstBI site has been introduced 3' of the beta-galactosidase gene. Cotransfection of BstBI-digested vector DNA and a plasmid containing the left end of the viral chromosome followed by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) results in clear plaques when overlap recombination has occurred and blue plaques when ligation of the viral arms has occurred within the host cell. The beta-galactosidase-expressing viruses grow to lower titers than do the parental viruses, leading to a relative growth advantage for viruses resulting from overlap recombination. Combined with color selection based on the beta-galactosidase gene, this system permits efficient production and selection of recombinant viruses after cotransfection of BstBI-digested viral DNA with a plasmid including left-end viral sequences and the gene of interest. The beta-galactosidase-expressing viral DNAs were used to construct viruses containing BstBI sites on either side of the cis-acting packaging element as a means of testing their utility.
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PMID:Efficient selection of recombinant adenoviruses by vectors that express beta-galactosidase. 774 47

Adenovirus (Ad) vectors have been used extensively to obtain high-level expression of foreign genes in mammalian cells and are currently being studied for use as live viral-vectored vaccines and as gene transfer vectors for gene therapy. Many Ad recombinants have been generated that express foreign genes inserted in early region 3 (E3); however, little has been done to study the importance for gene expression of regulatory sequences flanking the gene. We have generated a series of Ad5 helper-independent vectors that contain the firefly luciferase gene or the bacterial beta-galactosidase gene (LacZ) with or without simian virus 40 (SV40) regulatory sequences, combined with E3 deletions of 1.88 or 2.69 kb. The greatest levels of luciferase expression were obtained with a vector containing the luciferase gene under the control of the SV40 promoter and polyadenylation signal inserted in a 1.88-kb E3 deletion. In contrast, LacZ expression was highest with a vector containing the LacZ gene with just the SV40 polyadenylation sequence combined with a 1.88-kb E3 deletion. It was also observed that regardless of the SV40 sequences flanking the reporter gene or the E3 deletion used, expression from the luciferase recombinants was dependent on viral DNA replication, whereas expression from the LacZ recombinants was only partially reduced when DNA replication was blocked. Analyses of RNA by dot blot hybridizations revealed that the levels of reporter gene-specific mRNA for various vectors in each series did not vary significantly. These results indicate that the kinetics and efficiency of expression of genes inserted into the E3 region, in nonconditional helper-independent vectors, may be more strongly dependent on the sequences in the foreign gene insert itself than on flanking regulatory sequences such as those used here, derived from SV40.
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PMID:Foreign gene expression by human adenovirus type 5-based vectors studied using firefly luciferase and bacterial beta-galactosidase genes as reporters. 779 76

Apolipoprotein (apo) B-100 is the major protein component in low density lipoprotein (LDL); it contains the binding domain for the LDL receptor and the attachment site for apolipoprotein(a) in lipoprotein(a). ApoB-48 is colinear with the amino-terminal half of apoB-100 and misses the part of the molecule required for LDL receptor interaction and lipoprotein(a) formation. ApoB-48 mRNA is produced by the editing of apoB-100 mRNA, a process by which the codon CAA for Gln-2153 is changed to UAA, an in-frame stop codon. We used the cloned catalytic component of the rat apoB mRNA-editing enzyme (REPR) to construct a replication-defective recombinant adenoviral vector containing REPR cDNA (AvREPR) and a control vector (Av1LacZ4) containing a beta-galactosidase cDNA to investigate the effect of REPR gene delivery in C57BL/6 mice. Intravenous injection of AvREPR in mice resulted in efficient transduction of liver cells, where REPR mRNA and protein were overexpressed, reaching a peak at 7 and 12 days, returning toward control levels at 39 days after AvREPR administration. ApoB mRNA editing activity in liver extracts showed changes parallel to those of REPR mRNA expression; the proportion of edited apoB mRNA in the total hepatic apoB mRNA increased from approximately 60% to more than 90% at the peak of REPR expression. The proportion of plasma apoB-100 in AvREPR-transduced animals decreased from approximately 50% to < 10% of total plasma apoB concentration. Plasma very low density lipoproteins were polydisperse in control animals with an average diameter of 54.9 +/- 20.6 nm (uninjected control) and 54.7 +/- 16.8 nm (Av1LacZ4-treated), respectively. They became much smaller (average diameter 39.3 +/- 12.7 nm) and more uniform in size at day 12 following AvREPR administration. On the same day, the normal plasma LDL (26.2-25.5 nm) was almost completely eliminated in treated animals. Adenovirus-mediated transfer of the REPR cDNA is an efficient method to reduce plasma apoB-100 and normal LDL production.
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PMID:Adenovirus-mediated gene transfer of rat apolipoprotein B mRNA-editing protein in mice virtually eliminates apolipoprotein B-100 and normal low density lipoprotein production. 796 18

Adenovirus-mediated gene transfer to retinal cells was evaluated using the replication-defective recombinant adenovirus vector Ad2/CMVlacZ-1 (coding for beta-galactosidase) both in an in vitro murine culture model and in vivo in adult mice. In vitro, no difference in infectability of neuronal and glial cells was observed, and 50% of neurons expressed the exogenous gene at low viral concentration (10 pfu/cell). In vivo, intraocular injection of 3 x 10(6) pfu Ad2/CMVlacZ-1 resulted in expression of the transferred beta-galactosidase gene in retinal pigment epithelium and ganglion cells. These results demonstrate that Ad2/CMVlacZ-1 is an effective vector for gene transfer into retinal cells.
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PMID:Adenovirus-mediated gene transfer to murine retinal cells in vitro and in vivo. 803 87

Adenovirus vectors containing a marker gene (lacZ from Escherichia coli) are potent for transferring the gene to neurones after intraparenchymal injections. Expression of the marker gene may lead to the synthesis of an enormous amount of beta-galactosidase which diffuses throughout the entire neurone, providing a 'Golgi-like' staining. This suggested that the technique may be used to study the morphology of specific neuronal populations. We have validated this hypothesis by analysing the postnatal development of motoneurones in the rat cervical cord. Injections of the viral suspension into one ventral horn were performed at different ages after birth. Histochemical staining using X-Gal revealed morphological changes occurring within the first 3 weeks with enlargement of the perikaryon and increased dendritic complexity. Immunoreactivity for CGRP was visualized in double-staining experiments. In vivo transfer of a marker gene therefore provides a new way to analyse neuronal morphology which allows selection of the cells to be studied and double-labelling with immunohistochemical markers.
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PMID:In vivo transfer of a marker gene to study motoneuronal development. 808 Sep 60

Adenovirus type 5 was modified by coupling an asialoglycoprotein-polylysine conjugate to the virus by reactions that activate carbohydrate residues. Wild-type virus modified in this manner had greatly decreased infectivity toward normally susceptible HeLa S3 (asialoglycoprotein receptor (-)) and SK Hep1 (asialoglycoprotein receptor (-)) cells leaving 91 and 86% viable, respectively, after 48 h. However, with Huh 7 (asialoglycoprotein receptor (+)) cells, modified virus retained its infectivity leaving only 19% of cells viable under identical conditions. Modified virus was complexed to DNA in the form of a plasmid, pSVHBV surf, containing the gene for hepatitis B surface antigen as a marker of gene expression. Huh 7, receptor (+), cells treated with modified wild type, and modified replication-defective d1312 virus complexed to DNA raised antigen levels by approximately 13- and 30-fold, respectively, compared with asialoglycoprotein-polylysine DNA complex alone. Competition with a large excess of an asialoglycoprotein blocked the enhancement by more than 95%. Using a beta-galactosidase marker gene, the number of cells transfected by modified virus was found to be 200-fold higher than complex alone. Yet, specificity was retained exclusively for asialoglycoprotein receptor-bearing cells. These data indicate that adenovirus can be chemically modified by coupling ligands resulting in targeted gene expression dictated specifically by receptor recognition of the attached ligand.
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PMID:Incorporation of adenovirus into a ligand-based DNA carrier system results in retention of original receptor specificity and enhances targeted gene expression. 815 85


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