Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A fragment of diphtheria toxin (tox) gene from beta 45 phage DNA was cloned on pUC19 plasmid in E. coli cells. The fragment is coding for toxA fragment of the toxin and contains the control region of the tox gene. The tox gene promoter is active in E. coli. The toxA protein is found mainly in periplasm of E. coli cells. The protein is enzymatically active in ADP-ribosilation of elongation factor 2 from eucaryotic cells. An in frame toxA-lacZ' fusion was constructed on pUC8 plasmid. The hybrid protein expresses both toxA and lacZ' activities. Two or seven base pairs were deleted from the central part of toxA gene by means of S1 nuclease digestion. Translation of hybrid toxA-lacZ' mRNA should be terminated downward the delections due to the frameshifts caused by them. Nevertheless, a functionally active alpha-peptide of beta-galactosidase is expressed by both the deletion fusions. The existence of another translational start site functioning in E. coli and located inside 3'-end region of toxA mRNA is suggested.
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PMID:[Construction of derivatives of the diphtheria toxin gene and their expression in Escherichia coli cells]. 283 93

The ribosome translocation step that occurs during protein synthesis is a highly conserved, essential activity of all cells. The precise movement of one codon that occurs following peptide bond formation is regulated by elongation factor G (EF-G) in eubacteria or elongation factor 2 (EF-2) in eukaryotes. To begin to understand molecular interactions that regulate this process, a genetic selection was developed with the aim of obtaining conditional-lethal alleles of the gene (fusA) that encodes EF-G in Escherichia coli. The genetic selection depends on the observation that resistant strains arose spontaneously in the presence of sublethal concentrations of the antibiotic kanamycin. Replica plating was performed to obtain mutant isolates from this collection that were restrictive for growth at 42 degrees C. Two tightly temperature-sensitive strains were characterized in detail and shown to harbor single-site missense mutations within fusA. The fusA100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fusA101 allele encoded a glutamine-to-proline alteration at position 495. Induction kinetics of beta-galactosidase activity suggested that both mutations resulted in slower elongation rates in vivo. These missense mutations were very near a small group of conserved amino acid residues (positions 483 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded that these sequences encode a specific domain that is essential for efficient translocase function.
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PMID:In vivo selection of conditional-lethal mutations in the gene encoding elongation factor G of Escherichia coli. 828 87

The enzymatically active A-fragment of diphtheria toxin enters the cytosol of sensitive cells where it inhibits protein synthesis by inactivating elongation factor 2 (EF-2). We have constructed a number of diphtheria toxin mutants that are degraded by the N-end rule pathway in Vero cells, and that display a wide range of intracellular stabilities. The degradation could be inhibited by the proteasome inhibitor lactacystin, indicating that the proteasome is responsible for N-end rule-mediated degradation in mammalian cells. Previously, the N-end rule has been investigated by studying the co-translational degradation of intracellularly expressed beta-galactosidase. Our work shows that a mature protein entering the cytosol from the exterior can also be degraded by the N-end rule pathway with a similar, but not identical specificity to that previously found. We found a correlation between the intracellular stability of the mutants and their toxic effect on cells, thus demonstrating a novel manner of modulating the toxicity of a protein toxin. The data also indicate that the inactivation of EF-2 is the rate-limiting step in the intoxication process.
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PMID:Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway. 943 Jun 52

Pseudomonas exotoxin A (PE) inhibits protein synthesis by NAD-dependent ADP-ribosylation of eukaryotic elongation factor 2. Traditionally, toxin activity has been characterized, either in living cells or cell-free systems, using radioactive compounds for quantification. The increased costs of radioactive waste disposal together with heightened security concerns have made the use of radioactive isotopes less attractive for routine laboratory assays. We therefore adapted a cell-free rabbit reticulocyte in vitro transcription-translation system that utilizes a reporter (beta-galactosidase) to measure toxin activity. The assay for PE is rapid, scalable, log-linear, NAD dependent and can be used to assess the neutralizing activity of anti-PE antibody preparations.
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PMID:A nonradioactive, cell-free method for measuring protein synthesis inhibition by Pseudomonas exotoxin. 1664 35

Cryo-electron microscopy (cryo-EM) single-particle analysis has come a long way in achieving atomic-level resolution when imaging biomolecules. To obtain the best possible three-dimensional (3D) structure in cryo-EM, many parameters have to be carefully considered. Here we address the often-overlooked parameter of the pixel size, which describes the magnification of the image produced by the experiment. While efforts are made to refine and validate this parameter in the analysis of cryo-EM experimental data, there is no systematic protocol in place. Since the pixel size parameter can have an impact on the resolution and accuracy of a cryo-EM map, and the atomic resolution 3D structure models derived from it, we propose a computational protocol to estimate the appropriate pixel size parameter. In our protocol, we fit and refine atomic structures against cryo-EM maps at multiple pixel sizes. The resulting fitted and refined structures are evaluated using the GOAP (generalized orientation-dependent, all-atom statistical potential) score, which we found to perform better than other commonly used functions, such as Molprobity and the correlation coefficient from refinement. Finally, we describe the efficacy of this protocol in retrieving appropriate pixel sizes for several examples; simulated data based on yeast elongation factor 2 and experimental data from Gro-EL chaperone, beta-galactosidase, and the TRPV1 ion channel.
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PMID:Computational Protocol for Assessing the Optimal Pixel Size to Improve the Accuracy of Single-particle Cryo-electron Microscopy Maps. 3200 95