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)

A mutant of Escherichia coli is described whose cells show a spherical or irregular morphology, associated with leakage of beta-galactosidase and other intracellular proteins. The expression of the morphologic abnormality is most marked when the mutant is grown in rich media and is suppressed by D-alamine, D-serine, D-glutamate, or glycine supplementation. D-Alanine is the most effective amino acid supplement, half maximally supressing this anomalous property at a concentration of 75 mug/ml, as measured by the reduction in beta-galactosidase released from the cells. The mutant is more sensitive to penicillin G, D-methionine, and D-valine and it is relatively resistant to lysozyme. These phenotypic abnormalities are likewise corrected by the above supplementations. The relative rates of peptidoglycan synthesis in mutant and parent, grown under restrictive conditions, were measured both in vivo and in vitro by rates of incorporation of L-[14-D]alanine and uridine-5'-diphosphate-N-acetyl-D-[1-15C-A1-glucosamine, respectively. There is not metabolic block in the biosynthesis of uridine-5'-diphosphate-N-acetyl-muramyl-pentapeptide as shown by enzymic analysis and the lack of accumulation of uridine-5'-diphosphate-N-acetylmuramyl-peptide precursors. These preliminary studies suggest that the mutant possesses a defect in the biosynthesis of peptidoglycan although the exact lesion has not yet been established.
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PMID:D-Alanine-requiring cell wall mutant of Escherichia coli. 109 98

Biotinylation of fusion proteins in E. coli was studied using a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. As the biotinylation sequence, we examined two sequences: one was of amino acid residues [84-123] of 1.3S, a partial sequence containing a region from a conserved tetrapeptide (Ala-Met-Bct-Met) around the biotinyl lysine (Bct) to the carboxyl terminal; the other was of an almost entire sequence [18-123]. We constructed recombinant plasmids for fusion proteins of beta-galactosidase, of chloramphenicol acetyltransferase, and of alkaline phosphatase. We found the biotinylation in the [18-123] sequence fused to alkaline phosphatase.
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PMID:In vivo biotinylation of fusion proteins expressed in Escherichia coli with a sequence of Propionibacterium freudenreichii transcarboxylase 1.3S biotin subunit. 136 26

The gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one. The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis. The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (Ala-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.
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PMID:Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor. 142 10

The control of RepFIB replication appears to rely on the interaction between an initiator protein (RepA) and two sets of DNA repeat elements located on either side of the repA gene. Limited N-terminal sequence information obtained from a RepA:beta-galactosidase fusion protein indicates that although the first residue of RepA is methionine, the initiation of translation of RepA occurs from a CTG codon rather than from the predicted GTG codon located further downstream. Overexpressed RepA in trans is capable of repressing a repA:lacZ fusion plasmid in which the expression of the fusion protein is under the control of the repA promoter. The repA promoter has been located functionally by testing a series of repA:lacZ fusion plasmids. Both in vivo genetic tests and in vitro DNA-binding studies indicate that repA autoregulation can be achieved by RepA binding to one or more repeat elements which overlap the repA promoter sequence.
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PMID:Expression and regulation of the RepA protein of the RepFIB replicon from plasmid P307. 144 26

A bacterial lacZ gene was inserted into an isolate of the Lymantria dispar nuclear polyhedrosis virus (LdMNPV). The transfer vector was constructed by site-directed mutagenesis of the translation start site of the LdMNPV polyhedrin gene, within the BglII E fragment of the viral genome. A multiple cloning sequence was inserted at this start site and used for the insertion of the lacZ gene into the transfer plasmid. Liposome transfection was used to cotransfect L. dispar tissue culture cells with viral DNA and the transfer plasmid. Recombinant LdMNPV isolates were purified by isolation of plaques producing beta-galactosidase but not polyhedra. Restriction enzyme fragment profiles were used to determine the site of the lacZ gene insertion, and DNA sequencing of the 5' and 3' ends of the lacZ gene insert and the adjoining polyhedrin promoter and coding regions was performed to identify its precise location. Expression of the lacZ gene was examined by studying virus-induced protein using [35S]methionine pulse-labelling, SDS-PAGE fractionation and autoradiography. Expression of beta-galactosidase was examined in tissue culture cells using colorimetric assays. The maximum rate of beta-galactosidase production was approximately 50 international units (IU)/10(6) tissue culture cells/day between 3 and 4 days post-infection (p.i), and the peak total expression was 158 IU/10(6) cells 5 days p.i. beta-Galactosidase activity was first detected 48 h p.i. in haemolymph samples from fourth instar L. dispar larvae injected with 10(6) p.f.u. of virus. The peak beta-galactosidase activity in larval haemolymph samples was 1931 IU/ml of haemolymph at 11 days p.i., just prior to death.
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PMID:Genetic engineering of a Lymantria dispar nuclear polyhedrosis virus for expression of foreign genes. 160 70

To evaluate the feasibility of using a replication-deficient recombinant adenovirus to transfer human genes to the human endothelium, human umbilical vein endothelial cells were infected in vitro with adenovirus vectors containing the lacZ gene or a human alpha 1-antitrypsin (alpha 1AT) cDNA. After in vitro infection with the lacZ adenovirus vector, cultured endothelial cells expressed beta-galactosidase. In parallel studies with the alpha 1AT adenovirus vector, infected cells expressed human alpha 1AT transcripts, as evidenced by in situ hybridization and Northern analysis, and de novo synthesized and secreted glycosylated, functional alpha 1AT within 6 hr of infection, as shown by [35S]methionine labeling and immunoprecipitation. Quantification of the culture supernatants demonstrated 0.3-0.6 micrograms of human alpha 1AT secreted per 10(6) cells in 24 hr, for at least 14 days after adenovirus vector infection. To demonstrate the feasibility of direct transfer of genes into endothelial cells in human blood vessels, lacZ or alpha 1AT adenovirus vectors were placed in the lumen of intact human umbilical veins ex vivo. Histologic evaluation of the veins after 24 hr demonstrated transfer and expression of the lacZ gene specifically to the endothelium. alpha 1AT adenovirus infection resulted both in expression of alpha 1AT transcripts in the endothelium and in de novo synthesis and secretion of alpha 1AT. Quantification of alpha 1AT in the vein perfusates showed average levels of 13 micrograms/ml after 24 hr. These observations strongly support the feasibility of in vivo human gene transfer to the endothelium mediated by replication-deficient adenovirus vectors.
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PMID:Adenovirus-mediated transfer of a recombinant human alpha 1-antitrypsin cDNA to human endothelial cells. 163 Nov 46

The MET14 gene of Saccharomyces cerevisiae, encoding APS kinase (ATP:adenylylsulfate-3'-phosphotransferase, EC 2.7.1.25), has been cloned. The nucleotide sequence predicts a protein of 202 amino acids with a molecular mass of 23,060 dalton. Translational fusions of MET14 with the beta-galactosidase gene (lacZ) of Escherichia coli confirmed the results of primer extension and Northern blot analyses indicating that the ca. 0.7 kb mRNA is transcriptionally repressed by the presence of methionine in the growth medium. By primer extension the MET14 transcripts were found to start between positions -25 and -45 upstream of the initiator codon. Located upstream of the MET14 gene is a perfect match (positions -222 to -229) with the previously proposed methionine-specific upstream activating sequence (UASMet). This is the same as the consensus sequence of the Centromere DNA Element I (CDEI) that binds the Centromere Promoter Factor I (CPFI) and of two regulatory elements of the PHO5 gene to which the yeast protein PHO4 binds. The human oncogenic protein c-Myc also has the same recognition sequence. Furthermore, in the 270 bp upstream of the MET14 coding region there are several matches with a methionine-specific upstream negative (URSMet) control element. The significance of these sequences was investigated using different upstream deletion mutations of the MET14 gene which were fused to the lacZ gene of E. coli and chromosomally integrated. We find that the methionine-specific UASMet and one of the URSMet lie in regions necessary for strong activation and weak repression of MET14 transcription, respectively. We propose that both types of control are exerted on MET14.
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PMID:Cloning, nucleotide sequence, and regulation of MET14, the gene encoding the APS kinase of Saccharomyces cerevisiae. 165 9

Marek's disease virus (MDV) gene clones, RA2 and GA8, constructed in E. coli bacteriophage lambda-gt11 (gt11) were identified by a monoclonal antibody (MAb), H19.47, against a putative transformation-related viral antigen consisting of a complex of three phosphorylated polypeptides, pp41, pp38, and pp24. Both recombinants have a MDV-DNA insert of about 0.5 kb and are mapped to the region of BamHI-H or EcoRI-X fragments of the MDV genome by Southern blot hybridization. Immunoblot and immunoprecipitation with H19.47 identified a recombinant beta-galactosidase-MDV 140-kD fusion protein for RA2 and a 127-kD fusion protein for GA8. Immunoprecipitation of 35S-methionine-labeled, MDV-infected chicken embryo fibroblasts (CEF) with antisera against RA2 and GA8 fusion proteins recognized five polypeptides, of which three (p41, p38, and p24) are specified by H19.47 and the remaining two, p135 and p20, have not been previously identified. Immunoprecipitation of 32P-phosphate-labeled or 3H-glucosamine-labeled, GA-MDV-infected CEF with the antiserum against RA2 fusion protein identified a phosphorylated polypeptide of 38 kD and two glycoproteins of 60 and 49 kD, respectively. The antisera against recombinant fusion proteins thus revealed the existence of epitopes common to the phosphorylated polypeptides and other MDV-specific polypeptides. Sera from chickens or mice hyperimmunized with the purified fusion proteins reacted with serotype 1, MDV-infected CEF in the fluorescent antibody (FA) test to significant titers. These immune sera did not react with either serotype II or III, indicating the serotype specificity of the phosphorylated polypeptides.
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PMID:Marek's disease virus gene clones encoding virus-specific phosphorylated polypeptides and serological characterization of fusion proteins. 169 56

A lambda gt11 expression library containing cDNA from total chick embryo was screened with S103L, a rat monoclonal antibody which reacts specifically with the core protein of the chick cartilage chondroitin sulfate proteoglycan. One clone was identified which produced a 220-kDa beta-galactosidase/S103L-binding fusion protein. Sequencing the entire 1.5-kilobase cDNA insert showed that it contained a single open reading frame, which encoded a portion of the proteoglycan core protein from the chondroitin sulfate domain. This was confirmed by comparison with amino acid sequence data from peptide CS-B, which was derived from the chondroitin sulfate domain (Krueger, R.C., Jr., Fields, T. A., Hildreth, J., IV, and Schwartz, N.B. (1990) J. Biol. Chem. 265, 12075-12087). Furthermore, the 3' end of the insert overlapped with 23 bases at the 5' end of the published sequence for the C-terminal globular domain (Sai, S., Tanaka, T., Kosher, R. A., and Tanzer, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 5081-5085), which oriented this clone, as well as the CS peptide, along the protein core. The cDNA insert hybridized with a 9-kilobase mRNA from sternal chondrocytes as well as a similar sized message in brain but did not hybridize to any message from rat chondrosarcoma or from undifferentiated limb bud mesenchyme. In further studies, the fusion protein as well as a cyanogen bromide fragment (70 kDa) derived from it were isolated and shown to react with S103L, indicating that cleavage at methionine residues does not disrupt the antibody recognition site. Purification and N-terminal sequencing of the antigenic CNBr fragment derived from the fusion protein revealed that its N terminus is preceded by a methionine in the fusion protein and overlaps with the N terminus of peptide CS-B. As peptide CS-B is not recognized by S103L and the C terminus of peptide CS-B lies beyond the proteoglycan portion of the antigenic CNBr fragment, the S103L epitope is either contained within the 11 amino acids preceding the N terminus of peptide CS-B or it spans the clostripain cleavage site at the origin of the N terminus of peptide CS-B.
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PMID:Chick cartilage chondroitin sulfate proteoglycan core protein. II. Nucleotide sequence of cDNA clone and localization of the S103L epitope. 169 53

The DNA sequence of the Salmonella typhimurium metA control region is presented. S1 nuclease mapping was used to determine the transcription initiation site. By measuring beta-galactosidase levels in Escherichia coli strains lysogenized with lambda phage carrying a metA-lacZ gene fusion, the MetR protein was shown to activate the metA gene. Homocysteine, an intermediate in methionine biosynthesis, plays a negative role in the MetR-mediated activation mechanism. Gel mobility shift assays and DNase I protection experiments showed that the MetR protein binds to a DNA fragment carrying the metA control region and protects a 26-bp region beginning 9 bp upstream of the -35 promoter sequence.
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PMID:Regulation of the Salmonella typhimurium metA gene by the metR protein and homocysteine. 172 33


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