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 present a rapid assay for tRNA suppression in living Escherichia coli. An amber, ochre, or opal nonsense mutation in a cloned luxB gene from the bacterium Vibrio harveyi was suppressed. Because luciferase (Lux) activity depends completely on the appearance of the full-length luxB gene product, the amount of light produced was proportional to tRNA-mediated nonsense suppression in the cell. This luminometric assay was notably quicker, easier, and more sensitive than a traditional colorimetric assay employing beta-galactosidase. Assays required only one addition to a growing culture and were complete within 1 min. Light output was directly proportional to the amount of bacterial luciferase in a sample over a range of greater than or equal to 40,000-fold. Fewer than 100 cells were required for detection of Lux with ordinary instrumentation; assays were 80-fold more sensitive than simultaneous beta-galactosidase measurements. Assayed cells survived and could be recovered as colony formers. The beta-galactosidase colorimetric assay and the luciferase assay were similarly reproducible. Light from colonies expressing Lux was visible to the dark-adapted eye and useful for screening. A rapid assay that does not depend on the formation of permanent transformants can be based on electroporation followed by luminometry.
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PMID:A simple and sensitive in vivo luciferase assay for tRNA-mediated nonsense suppression. 210 99

We have constructed a series of broad-host-range plasmids which use "visual screens" to detect promoter activity. These plasmids contain the pMB1 and pRO1600 origins of replication and are capable of replicating in a wide range of gram-negative bacteria. The genes encoding beta-galactosidase and alkaline phosphatase from Escherichia coli and bacterial luciferase from Vibrio harveyi supply the promoterless indicator genes. The constructs were tested in E. coli and Pseudomonas aeruginosa.
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PMID:Construction of broad-host-range plasmid vectors for easy visible selection and analysis of promoters. 211 10

Previously, we identified a class of genes in Dictyostelium that are prespore cell-type specific in their expression in the multicellular aggregate and are inducible by cAMP acting through cell-surface cAMP receptors. In this paper, we report the cloning and analysis of the regulatory regions controlling the expression of one such gene that encodes a spore coat protein, SP60. By use of a fusion of the firefly luciferase gene and Escherichia coli lacZ [expresses beta-galactosidase (beta-gal)], we have identified cis-acting regions required for proper spatial and temporal expression in multicellular aggregates and for cAMP induction in shaking cell culture. Deletion analysis suggests that a CA-rich element (CAE) and surrounding sequences present three times within the 5'-flanking sequence are required for proper regulation. SP60-lacZ fusions that include all three of these regions express lacZ only in the posterior approximately 85% of migrating slugs (prespore zone). Studies show that SP60 is expressed during mid to late aggregation, and SP60-lacZ-positive cells are spatially localized as a doughnut-shaped ring within the forming aggregate. Cells within the skirt that surrounds the aggregate and that are still migrating into the aggregate do not stain. Sequential 5' deletions of CAEs and surrounding regions affect the expression level of SP60-luciferase in response to developmental signals and cAMP, as well as the spatial pattern of SP60-lacZ. Deletion of the first (most 5') of these regions restricts the spatial expression of SP60-lacZ fusions to the anterior of the prespore zone. When both the first and second regions are removed, the expression level drops, and the staining is restricted to the prespore/prestalk boundary. Furthermore, the staining pattern that is seen with these two deletions is present as a gradient from anterior to posterior within the prespore zone. Deletion of all three regions results in a loss of both cAMP and developmentally induced expression. These results suggest the presence of a gradient within the prespore zone that differentially affects the activity of promoters containing different numbers of response elements.
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PMID:A spatial gradient of expression of a cAMP-regulated prespore cell-type-specific gene in Dictyostelium. 216 44

A series of promoter-probe plasmid vectors has been constructed which allows for the selection of DNA sequences containing divergent control elements. Each vector contains a pair of promoterless genes [encoding beta-galactosidase (lacZ), alkaline phosphatase (phoA), and bacterial luciferase (luxAB)] arranged in an antiparallel fashion and separated by a large intervening multiple cloning site. The vectors permit direct detection of promoter activity on indicator plates after transformation. Cloned promoters are selected based on production of coloured products in the case of lacZ and phoA, and by the emission of light in the case of luxAB. These vectors have been tested using known divergent promoter elements from pBR322 and Pseudomonas phage D3.
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PMID:Construction of broad-host-range vectors for the selection of divergent promoters. 219 27

In an attempt to question the toxic effect of heat shock and related stress, we have studied the activity of reporter enzymes during stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase were synthesized in mouse and Drosophila cells after transfection of the corresponding genes. Both enzymes are rapidly inactivated during hyperthermia. The corresponding polypeptides are not degraded but become insoluble even in the presence of non-ionic detergents. The heat inactivation is more dramatic in vivo within the living cell than in vitro, in a detergent-free crude cell lysate. The extent of enzyme inactivation at a given temperature depends on the cell type in which the enzyme is expressed. Luciferase is inactivated at lower temperatures within Drosophila cells than within mouse cells, whereas beta-galactosidase is inactivated at higher temperatures in E. coli than in mouse cells. A "priming" heat shock confers a transient increased resistance (thermotolerance) of cells against a second "challenging" heat shock. Enzyme inactivation during heat shock or exposure of the cells to ethanol is attenuated in heat shock-primed cells. A comparable thermoprotection is raised by a priming heat shock for both luciferase activity and protein synthesis. Thus, the study of reporter enzyme inactivation is a promising tool for understanding the molecular basis of the toxicity of heat shock and related stress as well as the mechanisms leading to thermotolerance.
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PMID:Protein denaturation during heat shock and related stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase inactivation in mouse cells. 249 80

Mutagenesis with transposon mini-Mulac was used to identify loci containing genes for bioluminescence (lux) in the marine bacterium Vibrio harveyi. Transposon insertions which resulted in a Lux- phenotype were mapped to two unlinked regions of the genome. Region I contained the luxCDABE operon which was previously shown to encode the enzymes luciferase and fatty acid reductase, which are required for light production. The other locus, region II, which was identified for the first time in this study, appeared to have a regulatory function. In Northern blot analysis of mRNA from mutants with defects in this region, no transcription from the luxCDABE operon could be detected. Strains with transposon-generated lux::lacZ gene fusions were used to analyze control of the transcription of these regions. Expression of luminescence in the wild type was strongly influenced by the density of the culture, and in strains with the lacZ indicator gene coupled to the luxCDABE operon, beta-galactosidase synthesis was density dependent. So, transcription of this operon is responsive to a density-sensing mechanism. However, beta-galactosidase synthesis in strains with lacZ fused to the region II transcriptional unit did not respond to cell density. The organization and regulation of the lux genes of V. harveyi are discussed, particularly with regard to the contrasts observed with the lux system of the fish light-organ symbiont Vibrio fischeri.
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PMID:Identification of a locus controlling expression of luminescence genes in Vibrio harveyi. 254 Jan 49

Expression vectors have been constructed for a region of the human retinoic acid receptor-alpha (hRAR-alpha) and transferred into F9 embryonal carcinoma (EC) cells. When the vectors are overexpressed in F9 cells, clones can be selected for resistance to retinoic acid-induced differentiation. This effect is obtained even when the hRAR-alpha region is expressed as a beta-galactosidase fusion protein. Using the beta-galactosidase component of the fusion protein as a marker, overexpression of the fusion protein has been correlated with the retinoic acid-resistance effect. The clones resistant to retinoic acid no longer exhibit the normal retinoic acid induction of endo B cytokeratin, laminin B-1, and tissue plasminogen activator mRNAs observed with normal F9 cells. Retinoic acid induction of type IV alpha-1 collagen and Hox-1.3 RNAs is observed with these clones. When transfected with a thyroid receptor DNA-binding sequence (TRE)/thymidine kinase promoter/luciferase construct, the retinoic acid-resistant clones do not yield the same retinoic acid-induced level of luciferase obtained with F9 cells. It is hypothesized that the RAR vectors are interfering with endogenous RAR(s) in a dominant-negative manner to inhibit retinoic acid-induced differentiation of F9 EC cells.
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PMID:Retinoic acid receptor expression vector inhibits differentiation of F9 embryonal carcinoma cells. 255 44

Under certain conditions glucose represses the autoinducible synthesis of luminescence enzymes in Vibrio fischeri. To examine the genetic regulation of luminescence more closely, Escherichia coli catabolite repression mutants were transformed with a plasmid (pJE202) that contains V. fischeri genes specifying the luminescence enzymes and encoding regulatory functions for luminescence (the lux genes) or with plasmids (pJE413 and pJE455) containing transcriptional fusions between the lacZ gene on transposon mini-Mu and specific genes in each of the two lux operons. Unless cyclic AMP (cAMP) was added to the growth medium, an adenylate cyclase deletion mutant containing pJE202 produced very little light and low levels of the light-emitting enzyme luciferase. When grown in the presence or absence of cAMP, a cAMP receptor protein (CRP) deletion mutant produced low levels of light and luciferase. A mutant that does not make cAMP but does make an altered CRP which does not require cAMP for activity produced induced levels of luminescence after transformation with pJE202. To test the effects of cAMP and CRP on each of the two lux operons separately rather than on both together, the E. coli catabolite repression mutants were transformed with pJE413 and pJE455. From measurements of beta-galactosidase and luciferase activities it appeared that cAMP and CRP affected transcription of both lux operons. In the presence of autoinducer and its receptor, transcription of the operon encoding all of the luminescence genes except the receptor gene appeared to be activated by cAMP and CRP, whereas in the absence of the receptor, cAMP and CRP appeared to decrease transcription of this operon. Transcription of the operon encoding the autoinducer receptor appeared to be stimulated by cAMP and CRP in the absence of the receptor itself. These results demonstrate that cAMP and CRP are required for proper control of the V. fischeri luminescence system and suggest that lux gene transcription is required by a complex mechanism.
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PMID:Control of Vibrio fischeri luminescence gene expression in Escherichia coli by cyclic AMP and cyclic AMP receptor protein. 299 19

We discuss the utility of visual assays for the expression of genes introduced into plant cells. Such assays are valuable for both transient and stable gene expression studies. We review the properties of three visual assays that are already in use or under development phase for maize and other cereal crops. These assays depend on the expression of beta-galactosidase, luciferase, or structural genes required for anthocyanin pigment biosynthesis.
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PMID:Visual assays of transformation in plant cells. 310 71

We have introduced the firefly luciferase gene of Photinus pyralis into the vaccinia virus genome. This gene is expressed in a coordinate fashion during virus infection. Luminescence produced by the action of luciferase [Photinus-luciferin:oxygen 4-oxidoreductase(decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was easily detectable in infected cells in culture as well as in cells of tissues of infected mice. The limits of detection were about one infected cell in a background of a million noninfected cells. The luciferase assay was about 1000-fold more sensitive than that of beta-galactosidase. Our findings show that the luciferase assay can be conveniently used to follow viral gene expression and virus dissemination both in cell cultures and in tissues of infected animals.
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PMID:Expression of the firefly luciferase gene in vaccinia virus: a highly sensitive gene marker to follow virus dissemination in tissues of infected animals. 342 54


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