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)

Hybrid genes were constructed. One, ompA153-dfr, encoded the precursor of the 325 residue Escherichia coli outer membrane protein OmpA up to residue 153 which was fused to the complete 186-residue dihydrofolate reductase of the mouse. The other, ompA219-lacZ, coded for the same precursor up to residue 219 which was fused to 1017 COOH-terminal residues of the 1023-residue subunit of the beta-galactosidase of E. coli. Full expression of the ompA153-dfr gene caused accumulation of its precursor and of that of the chromosomally encoded OmpA protein. When the amount of product was reduced, no pro-OmpA and very little pro-hybrid protein accumulated. The precursor was processed and the mature protein was fully accessible to trypsin in permeabilized cells. Expression of the ompA219-lacZ gene led to the presence of the hybrid protein at only 20-30% of the amount expected. About 20% of it appeared to be incorporated in the outer membrane. All of the hybrid was quantitatively accessible to trypsin in permeabilized cells. When the hybrid gene was overexpressed, the protein was found associated with the plasma membrane in the cytosol. It is concluded that both beta-galactosidase and dihydrofolate reductase could quantitatively traverse the plasma membrane, provided the amounts synthesized were sufficiently small.
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PMID:Dihydrofolate reductase (mouse) and beta-galactosidase (Escherichia coli) can be translocated across the plasma membrane of E. coli. 314 14

The Escherichia coli glpT gene encodes a transport protein that mediates uptake of sn-glycerol-3-phosphate. This permease is a member of a class of bacterial organophosphate permeases which transport substrates by antiport with inorganic phosphate. The glpT gene product, probably an oligomer of a single polypeptide chain, is thought to span the cytoplasmic membrane several times, as predicted by the hydropathic profile. Protein fusions, in which varying lengths of the amino-terminal end of the permease is attached to alkaline phosphatase (phoA) and to beta-galactosidase (lacZ) were constructed. On the assumption that phoA fusions only exhibit high enzymatic activity when fused to extra-cytoplasmic regions of the target protein, whereas lacZ fusions will only be active when the beta-galactosidase portion is attached to cytoplasmic domains of the target protein, the activities of the fusions were used to test a two-dimensional model for the permease. The model proposes that GlpT contains 12 transmembrane segments divided by a larger cytoplasmic region. Despite some limitation caused by hot-spot sites of transpositions, the TnphoA approach was consistent with the model. In contrast, we feel that the enzymatic activity of lacZ fusions is only a limited parameter for studying the topology of a complex membrane protein.
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PMID:The transmembrane topology of the sn-glycerol-3-phosphate permease of Escherichia coli analysed by phoA and lacZ protein fusions. 314 44

MalF is an essential cytoplasmic membrane protein of the maltose transport system of Escherichia coli. We have developed a general approach for analysis of the mechanism of integration of membrane proteins and their membrane topology by characterizing a series of fusions of beta-galactosidase to MalF. The properties of the fusion proteins indicate the following. (1) The first two presumed transmembrane segments of MalF are sufficient to anchor beta-galactosidase firmly to the inner membrane. (2) Hybrid proteins with beta-galactosidase fused to a presumed cytoplasmic domain of MalF have high beta-galactosidase specific activity; fusions to periplasmic domains have low activity. We propose therefore, that periplasmic and cytoplasmic domains of integral membrane proteins can be distinguished by the enzymatic properties of such hybrid proteins. In general, it appears that cleaved or non-cleaved signal sequences when attached to beta-galactosidase cause it to become embedded in the membrane, and this results in the inability of the hybrid proteins to assemble into active enzyme. Additional properties of these fusion proteins contribute to our understanding of the regulation of MalF synthesis. The MalF protein, synthesized as part of the malEFG operon of E. coli, is approximately 30-fold less abundant in the cell than MalE protein (the maltose-binding protein). Differential amounts of the fusion proteins indicate that a regulatory signal occurs within the malF gene that is responsible for the step-down in expression from the malE gene to the malF gene.
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PMID:Genetic analysis of the membrane insertion and topology of MalF, a cytoplasmic membrane protein of Escherichia coli. 329 21

DNA obtained from Chlamydia trachomatis (serovar L2) was partially digested with DNase I and inserted into the beta-galactosidase gene of bacteriophage lambda gt11. Seven recombinants were selected that produced immunoreactive fusion proteins which were detected with anti-C. trachomatis rabbit serum. One recombinant, designated lambda gt11/L2/33, reacted with various monoclonal antibodies that recognize species-, subspecies-, and type-specific determinants on the chlamydial major outer membrane protein (MOMP). Immunoblot analysis of a lambda gt11/L2/33 lysogen revealed a fusion protein that expressed a approximately 15,000-dalton carboxyl-terminal peptide of the chlamydial MOMP. This moiety of the MOMP possesses epitopes responsible for each of the unique reactivities demonstrated by anti-MOMP monoclonal antibodies. The lambda gt11/L2/33 recombinant contained a 1.1-kilobase DNA insert which hybridized to DNA isolated from each of the 15 C. trachomatis serovars.
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PMID:Molecular cloning and expression of Chlamydia trachomatis major outer membrane protein antigens in Escherichia coli. 388 65

The rate of derepressed synthesis of a membrane protein required for lactose transport (M protein) by Escherichia coli is increased in response to increased gene dosage to the same extent as the rates of synthesis of beta-galactosidase and galactoside acetylase. However, elevated gene dosage does not increase beta-galactoside transport to the same extent that it increases synthesis of M protein and of the soluble proteins of the lac operon. Though the factor or factors other than M protein which limit induction of the transport system at high levels of lac operon expression have not been identified, studies with Escherichia coli mutants blocked in the synthesis of unsaturated fatty acids indicate that unsaturated fatty acids must be supplied during the course of induction of the lac operon to permit synthesis of a functional lactose transport system, but not of beta-galactosidase or galactoside acetylase.
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PMID:A lipid requirement for induction of lactose transport in Escherichia coli. 489 80

The nucleotide sequence of an Epstein-Barr virus gene expressed in latently infected growth-transformed cells is known to include a long open reading frame containing a 33-base-pair repeat element. A bacterial fusion protein constructed from a portion of the reading frame and Escherichia coli beta-galactosidase was used to produce sera in rabbits against the previously unidentified gene product. The viral protein detected with these sera in latently infected cells varies in size with the number of copies of the DNA repeat element. Translation of the RNA in vitro yields a protein of similar size. As expected from its primary sequence, the protein is a membrane protein. Immunofluorescence studies with the rabbit antisera suggest that the protein is in the plasma membrane. Thus, this protein could be the lymphocyte-determined membrane antigen (LYDMA) responsible for the generation of T-cell immunity to latently infected cells.
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PMID:A membrane protein encoded by Epstein-Barr virus in latent growth-transforming infection. 609 74

OmpA protein, a major outer membrane protein of Escherichia coli, is synthesized from a messenger RNA containing a 134-nucleotide 5' leader region. The role of this leader region in efficient ompA expression was investigated using a series of ompA-lacZ fusion plasmids. These plasmids differ in the amount of DNA encoding the ompA leader region which is fused to the lacZ structural gene. The fusion containing all but six nucleotides of the ompA leader produced the highest beta-galactosidase activity, while the fusion containing the shortest leader synthesized only 4% as much beta-galactosidase. Fusions with leaders intermediate in length produced between 6% and 24% of the activity found in the most efficient fusion. Quantitation of lacZ mRNA synthesis by DNA-RNA hybridization revealed differences in lacZ mRNA production reflecting the observed differences in beta-galactosidase activity. The primary effect of the ompA leader in maintaining high levels of mRNA is discussed in terms of the roles of mRNA secondary structure.
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PMID:Roles of the 5' leader region of the ompA mRNA. 620 57

The previously described hybrid plasmid pC7 which carries lacI+O+delta(Z)Y+A+ on a 12.3 X 10(6)-Mr DNA fragment [Teather et al. (1978) Mol. Gen. Genet. 159, 239-248] was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to d(A-A-T-T) and ligated to the vector pB322. lac Y-carrying inserts of various sized (Mr 1.5-4.7 X 10(6)) were obtained. Hybrid plasmid pTE18 (2300-base-pair insert) carries part of the I (repressor) gene, the promotor-operator region, part of the Z (beta-galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18-harbouring strains the Y-gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12-16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.
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PMID:Lactose carrier protein of Escherichia coli. Structure and expression of plasmids carrying the Y gene of the lac operon. 625 Aug 28

We previously obtained strains of Escherichia coli in which the beginning of gene lacZ, which codes for beta-galactosidase, is replaced by the beginning of gene lamB, which codes for a maltose-inducible outer membrane protein. In some of these strains the induction (with maltose) of lamB-lacZ hybrid protein synthesis was lethal because of membrane damage resulting from an incomplete export of this protein to the outer membrane. We describe here a class of maltose-resistant mutants obtained from one such strain. Mutants in this class fail to produce the lamB-lacZ hybrid protein but retain the ability to express lacY, which is located distal to the hybrid gene. Some of the mutants carry deletions within the hybrid gene. The others carry point mutations which most probably affect the initiation of translation at the beginning of the hybrid gene. One of these is located in the sequence that codes for the presumed ribosome interaction site on the mRNA. Three others, of which two are located in the coding region (sixth codon), are believed to result in an alteration of mRNA secondary structure such that the accessibility of the ribosome interaction site is reduced.
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PMID:Mutations that affect lamB gene expression at a posttranscriptional level. 626 27

We have developed an Escherichia coli plasmid vector for the identification and expression of foreign DNA segments that are open reading frames (ORFs). The 5' end of ompF, an E. coli gene encoding an abundant outer membrane protein, is used to provide a strong, regulated promoter, translation initiation site, and signal sequence for export from the cytoplasm. This sequence is coupled to the lacZ gene of E. coli so that expression of beta-galactosidase requires ompF transcription and translation signals. However, this hybrid gene is LacZ- because lacZ is out of frame with respect to ompF. Restriction enzyme recognition sites are located between ompF and lacZ to allow convenient insertion of DNA fragments. If an insert is an ORF of the correct length, ompF and lacZ become realigned in frame, resulting in a LacZ+ gene that produces a tribrid protein with the translation product of the insert sandwiched between OmpF and beta-galactosidase. The LacZ+ phenotype thus identifies clones containing an expressed ORF. To demonstrate the vector's utility we inserted a fragment from the herpes virus thymidine kinase gene and used the resulting tribrid protein to raise antibodies that precipitate thymidine kinase from herpes virus-infected cells. We also inserted a fragment from the E. coli lexA gene to produce a tribrid protein that is precipitated by antiserum raised with LexA protein. Thus, tribrid fusion proteins can be used to produce or detect antibodies and also to identify the product of a cloned gene.
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PMID:Open reading frame expression vectors: a general method for antigen production in Escherichia coli using protein fusions to beta-galactosidase. 630 25


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