Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P51532 (transcriptional activator)
 6,546 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. The biosensor is based on a plasmid incorporating the transcriptional activator xylR from the TOL plasmid of Pseudomonas putida mt-2. The XylR protein binds a subset of toluene-like compounds and activates transcription at its promoter, Pu. A reporter plasmid was constructed by placing the luc gene for firefly luciferase under the control of XylR and Pu. When Escherichia coli cells were transformed with this plasmid vector, luminescence from the cells was induced in the presence of benzene, toluene, xylenes, and similar molecules. Accurate concentration dependencies of luminescence were obtained and exhibited K1/2 values ranging from 39.0 +/- 3.8 microM for 3-xylene to 2,690 +/- 160 microM for 3-methylbenzylalcohol (means +/- standard deviations). The luminescence response was specific for only toluene-like molecules that bind to and activate XylR. The biosensor cells were field tested on deep aquifer water, for which contaminant levels were known, and were able to accurately detect toluene derivative contamination in this water. The biosensor cells were also shown to detect BETX (benzene, toluene, and xylene) contamination in soil samples. These results demonstrate the capability of such a bacterial biosensor to accurately measure environmental contaminants and suggest a potential for its inexpensive application in field-ready assays.
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PMID:Development and testing of a bacterial biosensor for toluene-based environmental contaminants. 950 40

The expression of heat shock genes in Escherichia coli is regulated by the antagonistic action of the transcriptional activator, the sigma32 subunit of RNA polymerase, and negative modulators. Modulators are the DnaK chaperone system, which inactivates and destabilizes sigma32, and the FtsH protease, which is largely responsible for sigma32 degradation. A yet unproven hypothesis is that the degree of sequestration of the modulators through binding to misfolded proteins determines the level of heat shock gene transcription. This hypothesis was tested by altering the modulator concentration in cells expressing dnaK, dnaJ and ftsH from IPTG and arabinose-controlled promoters. Small increases in levels of DnaK and the DnaJ co-chaperone (< 1.5-fold of wild type) resulted in decreased level and activity of sigma32 at intermediate temperature and faster shut-off of the heat shock response. Small decreases in their levels caused inverse effects and, furthermore, reduced the refolding efficiency of heat-denatured protein and growth at heat shock temperatures. Fewer than 1500 molecules of a substrate of the DnaK system, structurally unstable firefly luciferase, resulted in elevated levels of heat shock proteins and a prolonged shut-off phase of the heat shock response. In contrast, a decrease in FtsH levels increased the sigma32 levels, but the accumulated sigma32 was inactive, indicating that sequestration of FtsH alone cannot induce the heat shock response efficiently. DnaK and DnaJ thus constitute the primary stress-sensing and transducing system of the E. coli heat shock response, which detects protein misfolding with high sensitivity.
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PMID:Levels of DnaK and DnaJ provide tight control of heat shock gene expression and protein repair in Escherichia coli. 982 22

Non-invasive assessment of transgenic animals using bioluminescence imaging offers a rapid means of evaluating disease progression in animal models of disease. One of the challenges in the field is to develop models with robust expression to image repetitively live intact animals through solid tissues. The prostate-specific antigen (PSA) promoter is an attractive model for studying gene regulation due to its hormonal response and tissue-specificity permitting us to measure signaling events that occur within the native tissues. The use of the GAL4-VP16 activator offers a powerful means to augment gene expression levels driven by a weak promoter. We have used a two-step transcriptional amplification (TSTA) system to develop a transgenic mouse model to investigate the tissue-specificity and developmental regulation of firefly luciferase (fl) gene expression in living mice using bioluminescence imaging. We employed an enhanced prostate-specific promoter to drive the yeast transcriptional activator, GAL4-VP16 (effector). The reporter construct carries five Gal4 binding sites upstream of the fl gene. We generated a transgenic mouse model using a single vector carrying the effector and reporter constructs. The transgenic mice show prostate-specific expression as early as three weeks of age. The bioluminescence signal in the prostate is significantly higher than in other organs. We also demonstrate that blocking androgen availability can downregulate the fl expression in the prostate. The transgenic mice display normal physical characteristics and developmental behavior, indicating that the high level of GAL4 driven expression is well tolerated. These findings suggest that the GAL4-VP16 transactivator can be used to amplify reporter gene expression from a relatively weak promoter in a transgenic mouse model. The transgenic TSTA model in conjunction with other transgenic cancer models should also help to detect and track malignancies. The strategies developed will be useful for transgenic research in general by allowing for amplified tissue specific gene expression.
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PMID:Non-invasive imaging of a transgenic mouse model using a prostate-specific two-step transcriptional amplification strategy. 1586 48

Tracking stem cell localization, survival, differentiation, and proliferation after transplantation in living subjects is essential for understanding stem cell biology and physiology. In this study, we investigated the long-term stability of reporter gene expression in an embryonic rat cardiomyoblast cell line and the role of epigenetic modulation on reversing reporter gene silencing. Cells were stably transfected with plasmids carrying cytomegalovirus promoter driving firefly luciferase reporter gene (CMV-Fluc) and passaged repeatedly for 3-8 months. Within the highest expressor clone, the firefly luciferase activity decreased progressively from passage 1 (843+/-28) to passage 20 (250+/-10) to passage 40 (44+/-3) to passage 60 (3+/-1 RLU/microg; P<0.05 vs. passage 1). Firefly luciferase activity was maximally rescued by treatment with 5-azacytidine (DNA methyltransferase inhibitor) compared with trichostatin A (histone deacetylase inhibitor) and retinoic acid (transcriptional activator; P<0.05). Increasing dosages of 5-azacytidine treatment led to higher levels of firefly luciferase mRNA (RT-PCR) and protein (Western blots) and inversely lower levels of methylation in the CMV promoter (DNA nucleotide sequence). These in vitro results were extended to in vivo bioluminescence imaging (BLI) of cell transplant in living animals. Cells treated with 5-azacytidine were monitored for 2 wk compared with 1 wk for untreated cells (P<0.05). These findings should have important implications for reporter gene-based imaging of stem cell transplantation.
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PMID:Effects of epigenetic modulation on reporter gene expression: implications for stem cell imaging. 1624 67

Most persimmon (Diospyros kaki) cultivars are astringent and require post-harvest deastringency treatments such as 95% CO2 (high-CO2 treatment) to make them acceptable to consumers. High-CO2 treatment can, however, also induce excessive softening, which can be reduced by adding 1-methylcyclopropene (1-MCP). Previous studies have shown that genes encoding the ETHYLENE RESPONSE FACTORS (ERFs) DkERF8/16/19 can trans-activate xyloglucan endotransglycosylase/hydrolase (DkXTH9), which encodes the cell wall-degrading enzyme associated with persimmon fruit softening. In this study, RNA-seq data between three treatments were compared, namely high-CO2, high-CO2+1-MCP, and controls. A total of 227 differentially expressed genes, including 17 transcription factors, were predicted to be related to persimmon post-deastringency softening. Dual-luciferase assays indicated that DkNAC9 activated the DkEGase1 promoter 2.64-fold. Synergistic effects on transcription of DkEGase1 that involved DkNAC9 and the previously reported DkERF8/16 were identified. Electrophoretic mobility shift assay indicated that DkNAC9 could physically bind to the DkEGase1 promoter. Bimolecular fluorescence complementation and firefly luciferase complementation imaging assays indicated protein-protein interactions between DkNAC9 and DkERF8/16. Based on these findings, we conclude that DkNAC9 is a direct transcriptional activator of DkEGase1 that can co-operate with DkERF8/16 to enhance fruit post-deastringency softening.
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PMID:High CO2/hypoxia-induced softening of persimmon fruit is modulated by DkERF8/16 and DkNAC9 complexes. 3192 21