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 fused full length and the carboxyl-half of human MDR1 cDNA with the E. coli lacZ gene via a collagen linker and allowed their expression in yeast Saccharomyces cerevisiae. Using antibodies against beta-galactosidase we partially purified the fusion proteins by immunoprecipitation and show here that the full length fusion protein has ATPase activity. By contrast, the fusion protein containing the carboxyl-half of P-glycoprotein did not show ATPase activity, indicating that both domains of P-glycoprotein are necessary. By treatment of the immunoprecipitated fusion protein with collagenase, P-glycoprotein was released from the beta-galactosidase moiety. The results shown here open the possibility for a large scale purification of P-glycoprotein using this site specifically cleavable fusion protein.
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PMID:Production of a site specifically cleavable P-glycoprotein-beta-galactosidase fusion protein. 136 54

The larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is expressed at a very high level in the fat body of third-instar larvae. Here we report that Lsp-2 transcription in adult flies produces a unique mRNA localized in the adult adipose tissue of the head in both sexes. To identify regulatory regions of this Drosophila gene, Lsp-2 5'-flanking DNA sequences were fused to the E. coli beta-galactosidase gene (lacZ). Transient expression of the hybrid gene in third-instar larvae indicates that 230 bp just upstream from the 'TATA box' of the Lsp-2 gene are sufficient for larval fat body-specific expression.
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PMID:Fat-body-specific expression of the Drosophila Lsp-2 gene. 136 81

Keratins comprise a multigene family of structural proteins that form the 10-nm filaments present in epithelial cells. Keratin filament formation requires the presence of stoichiometric quantities of type I and type II keratin peptides. Each keratin peptide contains an N-terminal "head" segment, a C-terminal "tail" segment, and a highly conserved, alpha-helical central rod domain. To investigate the importance of these domains in situ, we have altered the DNA coding sequence of human cytokeratin K19 and transiently expressed the mutants in PtK2 cells that contain an endogenous keratin filament system. Interestingly, K19 mutants containing 4, 8, 12, and 24 amino acid insertions in the non-alpha-helical L1 region of the central rod domain successfully integrate into the endogenous PtK2 keratin filaments. Another K19 mutant, K19-bGAL, that encodes bacterial beta-galactosidase (bGAL) fused in phase to the 3' end of the K19 central rod domain, also integrates into the endogenous PtK2 keratin filaments. Our results demonstrate 1) that the spacing between the highly conserved amino and carboxy terminal ends of the K19 central rod domain can be increased without significantly effecting K19's ability to interact with keratin filaments and 2) that addition of a highly soluble 66-kDa tail to K19 does not impede its interaction with the filament system.
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PMID:Central rod domain insertion and carboxy-terminal fusion mutants of human cytokeratin K19 are incorporated into endogenous keratin filaments. 137 Feb 30

We devised an indicator gene for retrotransposition, nlsLacZRT, which contains the Escherichia coli lacZ gene fused to a nuclear location signal (nlsLacZ), engineered in such a way that the gene is expressed only if the structure in which it has been inserted transposes itself through an RNA intermediate. A cloned murine leukemia retrovirus with an ecotropic host range (Moloney murine leukemia virus), rendered defective by a large deletion encompassing the three viral gag, pol, and env open reading frames, was marked with this indicator gene and introduced by transfection into heterologous feline cells. No beta-galactosidase activity could be detected among the clonal cell population, unless the defective provirus was complemented in trans by the gag-pol gene products. Under these conditions, cell variants which disclosed an easily detectable nuclear blue coloration upon in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining were observed. Fluorescence-activated cell sorting of the beta-galactosidase-positive cells, followed by Southern blot analysis, demonstrated an unambiguous correlation between nlsLacZRT activation and retrotransposition of the marked provirus. Transposition occurs at a high frequency (up to 10(-4) events per cell per generation), which is dependent on the level of expression of the gag-pol gene and is concomitant with the release of noninfectious retroviruslike particles which are the hallmarks, but not the intermediates, of the intracellular transposition process.
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PMID:High-frequency intracellular transposition of a defective mammalian provirus detected by an in situ colorimetric assay. 137 Nov 67

We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer-Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.
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PMID:Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum. 137 17

We have analyzed the cis-regulatory elements in the 5' flanking region of the Drosophila choline acetyltransferase gene (ChAT, E.C.2.3.1.6). DNA fragments were fused to the Escherichia coli lacZ reporter gene and introduced into the Drosophila germ line by P-element-mediated transformation. A 7.4 kb 5' flanking sequence directed beta-galactosidase expression in the adult optic lobes and other well-defined CNS structures with a pattern very similar to the distribution of endogenous ChAT protein. In contrast, the proximal 3.3 kb and 1.2 kb of 5' flanking DNA directed lacZ expression in only selected subsets of the structures seen with the 7.4 kb lacZ construct. Our results indicate that both qualitative and quantitative regulatory elements are present in the 5' flanking DNA and that these elements distinguish various subsets of cholinergic neurons. We have also fused the same 5' flanking DNA sequences to wild-type ChAT cDNA and used these constructs to transform Chatsl mutant flies. Not only the 7.4 kb cDNA construct, but also the 3.3 and 1.2 kb constructs, rescued Chatsl from temperature-dependent paralysis and adult lethality, indicating that the regulatory information in any of these genomic fragments can drive sufficient wild-type ChAT expression to overcome these mutant phenotypes.
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PMID:Analysis of cis-regulatory elements in the 5' flanking region of the Drosophila melanogaster choline acetyltransferase gene. 137 60

The din23 fusion encodes a B. subtilis SOS-inducible regulatory region fused to the E. coli lacZ gene (Love et al., 1985). A strain encoding the din23 fusion and a recM13 allele showed low-level constitutive beta-galactosidase expression, was induced for beta-galactosidase production by DNA gyrase inhibitors but not by DNA-damaging agents, and was slightly induced by a variety of agents which do not normally induce the SOS regulon. The din23 fusion itself resulted in high levels of spontaneous prophage induction in wild-type, recM- and recA-hosts, despite the fact that the din23recM13 strain was not induced for beta-galactosidase production by DNA-damaging agents. The results suggest that the recM gene may be involved with the regulation of the RecA protease-mediated SOS response, while the din23 gene may be involved with the regulation of an alternative function which results in the cleavage of prophage repressor.
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PMID:Phenotypes conferred by the Bacillus subtilis recM13 mutation and the din23 fusion. 137 9

The analytical applicability of electrospray ionization mass spectrometry (ESIMS) to large glycoproteins in the molecular weight (MW) range of 150,000-200,000 was demonstrated. Multiply charged ions (charge state as high as 150+) of several typical macrosized glycoproteins of immunological significance were generated by pneumatically-assisted electrospray (ionspray) and their masses measured on a quadrupole mass spectrometer having a mass-to-charge (m/z) range of 2400. The resolution of the quadrupole instrument was insufficient to resolve the glycocomposition microheterogeneities in the MW range studied. Nevertheless, the average MWs of three immunoglobulin G (IgG) class murine monoclonal antibodies, anti-(human alpha 1-antitrypsin) (148,484 +/- 4), anti-(human alpha 1-acid glycoprotein) (149,599 +/- 12) and anti-(beta-galactosidase) (component I, 150,544 +/- 10, and component II, 151,496 +/- 17), and human alpha 2-macroglobulin monomer (186,100 +/- 100), and human complement component C4 (196,863 +/- 29) were still determined from the fused peak profiles of their constituent glyco components (the errors given reflect the measurement precisions of the simultaneous multichannel MW determinations). The difference between the measured average MW and the unmodified sequence MW was used to assess the degree of post-transitional modification in human alpha 2-macroglobulin (13.6%) and human complement component C4 (5.3%). For the large glycoproteins studied here, glycosylation did not appear to seriously affect the effectiveness of the electrospray ionization; up to 70% of their full charge-retaining capacities were fulfilled under the usual experimental conditions. These results show that ESIMS is capable of providing analytically useful information for macrosized proteins.
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PMID:Analysis of antibodies and other large glycoproteins in the mass range of 150,000-200,000 Da by electrospray ionization mass spectrometry. 138 90

The expression of the genes encoding ribonucleotide reductase in Escherichia coli was investigated in cultures synchronized by obtaining the smallest cells in a population after sucrose gradient centrifugation. Specific activity of ribonucleotide reductase and DNA initiation were found to increase in parallel, periodically as a function of the cell cycle. The expression of nrd was also determined in cells synchronized by periodic repeated doubling in a phosphate limited medium. Antibodies directed against the B2 subunit of ribonucleotide reductase were raised in a rabbit and purified. Immunoprecipitation of the B2 subunit and RNA-DNA dot blot hybridization assays were developed and employed to determine the expression of ribonucleotide reductase translational and transcriptional products during the cell cycle. Both of nrd-mRNA and B2 subunit expression were found to increase each generation at approximately the same time DNA synthesis was initiated and then to decrease back to the basal level shortly after DNA initiation. These results provided evidence of cell cycle dependent regulation of ribonucleotide reductase in E. coli. When the upstream regulatory region of nrd was fused to a promoterless lacZ gene on a single copy plasmid, lac-mRNA and beta-galactosidase were found to be synthesized in parallel to nrd expression from the chromosomal operon. When nrd sequences surrounding the promoter were removed from this construct, lac-mRNA and beta-galactosidase synthesis were no longer cell cycle regulated.
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PMID:Escherichia coli ribonucleotide reductase expression is cell cycle regulated. 138 14

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.
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PMID:On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli. 138 56


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