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 devised a general, one-step technique for isolation of strains in which the gene coding for an exported protein is fused to the gene for beta-galactosidase (lacZ). These fusions specify a hybrid protein comprised of an NH2-terminal portion of the exported protein and a large functional COOH-terminal portion of beta-galactosidase. The fusions are constructed with a derivative of the MudII (lac, Ap) phage. To overcome the lethality that is often associated with the expression of such a hybrid gene, we have recombined an early lacZ nonsense mutation onto this phage. With the use of strains that carry a temperature-sensitive nonsense suppressor, expression of the full-length hybrid protein can be controlled by varying the growth temperature. We demonstrated the utility of this technique by isolating a series of fusions to a gene, ompA, coding for a major outer membrane protein. As expected, strains containing these fusions are not viable under conditions that permit synthesis of a functional nonsense suppressor. Accordingly, this method should also be useful for direct selection of export-defective mutants.
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PMID:lacZ fusions to genes that specify exported proteins: a general technique. 633 Apr 98

Streptonigrin was used to select mutants impaired in the citrate-dependent iron transport system of Escherichia coli K-12. Mutants in fecA and fecB could not transport iron via citrate. fecA-lac and fecB-lac operon fusions were constructed with the aid of phage Mu dl(Ap lac). Strains deficient in ferric dicitrate transport which were mutated in fecB were as inducible as transport-active strains. They expressed the FecA outer membrane protein and beta-galactosidase of the fecB-lac operon fusions. In contrast, all fecA::lac mutants and fecA mutants induced with N-methyl-N'-nitro-N-nitrosoguanidine did not respond to ferric dicitrate supplied in the growth medium. tonB fecB mutants which were lacking all tonB-related functions were not inducible. We conclude that binding of iron in the presence of citrate to the outer membrane receptor protein is required for induction of the transport system. In addition, the tonB gene has to be active. However, iron and citrate must not be transported into the cytoplasm for the induction process. These data support our previous conclusion of an exogenous induction mechanism. Mutants in fur expressed the transport system nearly constitutively. In wild-type cells limiting the iron concentration in the medium enhanced the expression of the transport system. Thus, the citrate-dependent iron transport system shares regulatory devices with the other iron transport systems in E. coli and, in addition, requires ferric dicitrate for induction.
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PMID:Exogenous induction of the iron dicitrate transport system of Escherichia coli K-12. 637 72

We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this problem particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid proteins produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided evidence that the molecular mechanism of cellular protein localization is strinkingly similar in both bacteria and animal cells.
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PMID:A mechanism of protein localization: the signal hypothesis and bacteria. 644 3

The synthesis of a membrane-bound MalE beta-galactosidase hybrid protein, when induced by growth of Escherichia coli on maltose, leads to inhibition of cell division and eventually a reduced rate of mass increase. In addition, the relative rate of synthesis of outer membrane proteins, but not that of inner membrane proteins, was reduced by about 50%. Kinetic experiments demonstrated that this reduction coincided with the period of maximum synthesis of the hybrid protein (and another maltose-inducible protein, LamB). The accumulation of this abnormal protein in the envelope therefore appeared specifically to inhibit the synthesis, the assembly of outer membrane proteins, or both, indicating that the hybrid protein blocks some export site or causes the sequestration of some limiting factor(s) involved in the export process. Since the MalE protein is normally located in the periplasm, the results also suggest that the synthesis of periplasmic and outer membrane proteins may involve some steps in common. The reduced rate of synthesis of outer membrane proteins was also accompanied by the accumulation in the envelope of at least one outer membrane protein and at least two inner membrane proteins as higher-molecular-weight forms, indicating that processing (removal of the N-terminal signal sequence) was also disrupted by the presence of the hybrid protein. These results may indicate that the assembly of these membrane proteins is blocked at a relatively late step rather than at the level of primary recognition of some site by the signal sequence. In addition, the results suggest that some step common to the biogenesis of quite different kinds of envelope protein is blocked by the presence of the hybrid protein.
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PMID:Insertion of a MalE beta-galactosidase fusion protein into the envelope of Escherichia coli disrupts biogenesis of outer membrane proteins and processing of inner membrane proteins. 674 3

Escherichia coli strains have been constructed in which lacZ, the gene for the cytoplasmic enzyme beta-galactosidase, is fused to lamB, the gene for an outer membrane protein. One such strain produces a beta-galactosidase which remains cytoplasmic even though it possesses the complete signal sequence of the lamB protein precursor at the amino-terminal end.
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PMID:A signal sequence is not sufficient to lead beta-galactosidase out of the cytoplasm. 677 62

Strains of Escherichia coli K-12 deleted in the native lac operon and bearing both a wild-type glpT operon encoding for sn-glycerol 3-phosphate (G3P) transport and a hybrid operon in which glpT operator and promoter regions are fused to the lacZ gene were constructed. In strains with such a hybrid operon, beta-galactosidase and beta-galactoside permease become inducible by G3P. In these mutants the function and maturation of the glpT-coded proteins should be distinguishable from the level of gene expression, since the beta-galactosidase activity can serve as an index of the latter. With the aid of such mutants, it was shown that: (i) the expressions of the two neighboring operons, glpT and glpA (encoding anaerobic G3P dehydrogenase), are not coordinate; (ii) upon induction, the appearance of the cytoplasmic beta-galactosidase activity preceded that of methyl-beta-D-thiogalactoside transport activity (requiring only a cytoplasmic membrane protein) by about 4 min and that of G3P transport activity (requiring both a cytoplasmic membrane protein and a periplasmic protein) by about 9 min; and (iii) when cells grown at several temperatures from 24 to 42 degrees C were measured for G3P transport activity at 30 degrees C, the activity increased with the growth temperature, indicating that, within the range studied, the rate of transport increases with the fluidity of membrane phospholipids.
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PMID:Use of Escherichia coli operon-fusion strains for the study of glycerol 3-phosphate transport activity. 677 29

In the last few years, several laboratories have demonstrated that many proteins (both from eukaryotic and prokaryotic organisms) that are destined to be localized in noncytoplasmic locations initially are synthesized as a precursor with a 15-30 amino acid extension at the NH2-terminal end of the molecule. This extra peptide has been termed the signal sequence, and it has been proposed that this signal plays a role in the localization of the extracytoplasmic protein. We are studying the process by which proteins are exported to the envelope region of Escherichia coli. Our work deals primarily with the outer membrane proteins, lambda receptor, the product of the lamB gene, and the major outer membrane (porin) proteins 1a and 1b, products of the ompF and ompC genes. Using techniques of gene fusion, we have demonstrated that information specifying the cellular location of the lambda receptor is contained within the lamB gene. Furthermore, we have shown that this information is capable of directing even a normally cytoplasmic protein, beta-galactosidase, to the outer membrane. Some of this information is contained within the signal sequence. Mutations that alter this sequence prevent export of the lambda receptor protein. Again using techniques of gene fusion, we have shown that the signal sequence alone is not sufficient to cause export of beta-galactosidase from the cytoplasm. Other information within the lamB gene is required. Selection procedures have been developed to isolate mutations that exhibit a general alteration in the export process. Genetic analysis of these mutations has provided evidence for the involvement of the ribosome in the process of protein localization. The structural genes for the porin proteins, 1a and 1b, are regulated at the transcriptional level by the ompB locus. This has permitted us to extend our studies on outer membrane protein localization to protein 1. With this genetic system, it should be possible to determine if E coli employs more than a single mechanism for the export of proteins to the outer membrane.
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PMID:Genetic studies on mechanisms of protein localization in Escherichia coli K-12. 701 77

We are studying the molecular mechanism of cellular protein localization. The availability of genetic techniques, such as gene fusion in Escherichia coli, has made this subject particularly amenable to study in this prokaryote. We have constructed a variety of strains in which the gene coding for an outer membrane protein is fused to the gene coding for a normally cytoplasmic enzyme, beta-galactosidase. The hybrid protein produced by such strains retain beta-galactosidase activity; this activity serves as a simple biochemical tag for studying the localization of the outer membrane protein. In addition, we have exploited phenotypes exhibited by certain fusion strains to isolate mutants that are altered in the process of protein export. Genetic and biochemical analyses of such mutants have provided important insights into the mechanism of protein localization.
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PMID:Genetic approaches to study export of LamB to the outer membrane. 704 34

The mtr gene of Escherichia coli K-12 encodes an inner membrane protein which is responsible for the active transport of trypotophan into the cell. It has been proposed that the Mtr permease has a novel structure consisting of 11 hydrophobic transmembrane spans, with a cytoplasmically disposed amino terminus and a carboxyl terminus located in the periplasmic space (J.P. Sarsero, P. J. Wookey, P. Gollnick, C. Yanofsky, and A.J. Pittard, J. Bacteriol. 173:3231-3234, 1991). The validity of this model was examined by the construction of fusion proteins between the Mtr permease and alkaline phosphatase or beta-galactosidase. In addition to the conventional methods, in which the reporter enzyme replaces a carboxyl-terminal portion of the membrane protein, the recently developed alkaline phosphatase sandwich fusion technique was utilized, in which alkaline phosphatase is inserted into an otherwise intact membrane protein. A cluster of alkaline phosphatase fusions to the carboxyl-terminal end of the Mtr permease exhibited high levels of alkaline phosphatase activity, giving support to the proposition of a periplasmically located carboxyl terminus. The majority of fusion proteins produced enzymatic activities which were in agreement with the positions of the fusion sites on the proposed topological model of the permease. The synthesis of a small cluster of hybrid proteins, whose enzymatic activity did not agree with the location of their fusion sites within putative transmembrane span VIII or the preceding periplasmic loop, was not detected by immunological techniques and did not necessitate modification of the proposed model in this region. Slight alterations may need to be made in the positioning of the carboxyl-terminal end of transmembrane span X.
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PMID:Membrane topology analysis of Escherichia coli K-12 Mtr permease by alkaline phosphatase and beta-galactosidase fusions. 781 18

HSV-1 derived amplicons expressing cytoplasmic beta-galactosidase (pA-SF1), or plasma membrane targeted TIMP-Thy1 (pA-TT1), were used to transduce glial cells in vitro. By monitoring the expression of reporter genes from both amplicons and helper virus, we determined that many cells were infected by both particles. In glial cells infected only by pA-SF1 beta-galactosidase immunoreactivity was restricted to the cytoplasm; co-infection with helper HSV-1 (wild type), resulted in additional nuclear beta-galactosidase immunoreactivity. Co-infection of cells with amplicon pA-TT1 and helper virus did not affect the plasma membrane localization of TIMP/Thy1. Thus, co-infection with wild type helper virus altered the localization of an amplicon encoded cytoplasmic, but not plasma membrane protein.
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PMID:Herpes simplex virus 1 (HSV-1) helper co-infection affects the distribution of an amplicon encoded protein in glia. 781 34


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