Molecular mechanisms of herbicide-inducible gene expression of tobacco CYP71AH11 metabolizing the herbicide chlorotoluron.

Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5'-flanking region of CYP71AH11 was cloned, ligated to the reporter ß-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5'-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (-1281 to -770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75 kDa, respectively. Particularly, interaction between AAAAAG and <30 kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants.

Assays of PCB congeners and organochlorine insecticides with the transgenic Arabidopsis and tobacco plants carrying recombinant guinea pig AhR and GUS reporter genes.

Certain congeners of polychlorinated biphenyls (PCBs) and organochlorine insecticides are ligands of aryl hydrocarbon receptors (AhRs) in animals. A recombinant guinea pig (g) AhR, XgDV, was constructed by fusing the ligand-binding domain of gAhR, the DNA-binding domain of LexA, and the transactivating domain of VP16. Then, the expression unit of ß-glucuronidase (GUS) reporter gene regulated by XgDV was introduced into Arabidopsis and tobacco plants. When the transgenic Arabidopsis XgDV plants were cultured on Murashige-Skoog (MS) medium containing PCB congeners, the GUS activity in the plants increased toxic equivalent (TEQ)-dependently. The GUS activity in the transgenic Arabidopsis XgDV plants cultured on MS medium containing the organochlorine insecticide dieldrin was also induced. On the other hand, in the case of DDT, the GUS activity induced by 3-methylcholanthere in the plants decreased. The transgenic Arabidopsis XgDV plants detected 1000 ng g(-1) PCB126 in 1 g of soils. Thus the XgDV plants seemed to be useful for convenient assays of PCB congeners and organochlorine insecticides, without any extraction and purification steps.

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated ß-glucuronidase reporter gene expression system.

The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated ß-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorodibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of life and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

Assays of dioxins and dioxin-like compounds in actually contaminated soils using transgenic tobacco plants carrying a recombinant mouse aryl hydrocarbon receptor-mediated ß-glucuronidase reporter gene expression system.

The transgenic tobacco plant XD4V-26 carrying the recombinant mouse aryl hydrocarbon receptor XD4V-mediated ß-glucuronidase (GUS) reporter gene expression system was used for assay of dioxins and dioxin-like compounds consisting of polychlorinated dibenzeno-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls (Co-PCBs) in actually contaminated soils. The transgenic tobacco plant XD4V-26 showed a significant dose-dependent induced GUS activity when cultured on MS medium containing PCB126 [toxic equivalency factor (TEF) = 0.1]. In contrast, PCB169 and PCB180, which have 0.03 of TEF and unassigned TEF values, respectively, did not significantly induce GUS activity under the same conditions as with PCB126. When the tobacco plants were cultivated for up to 5 weeks on actually contaminated soils with dioxins and dioxin-like compounds collected from the periphery of an incinerator used for disposal of residential and industrial wastes, GUS activity in the leaves was dose-dependently increased. The plants clearly detected 360 pg-TEQ g(-1) of dioxins and dioxin-like compounds in this assay. There was a positive correlation between GUS activity and TEQ value of dioxins and dioxin-like compounds in the plants. This assay does not require any extraction and purification processes for the actually contaminated soil samples.

Congener specificity in the accumulation of dioxins and dioxin-like compounds in zucchini plants grown hydroponically.

Zucchini cultivars Cucurbita pepo subsp. ovifera cv. Patty Green and subsp. pepo cv. Gold Rush were cultivated hydroponically in a nutrient solution supplemented with a mixture of dioxins and dioxin-like compounds. Patty Green and Gold Rush showed low and high accumulation of these compounds in the aerial parts respectively. In both cultivars, the accumulation of each congener negatively depended on its hydrophobicity. This suggests that desorption and solubilization were partly responsible for congener specificity of accumulation, since this was not found in soil experiments. In contrast, no clear difference in accumulation in the roots was observed between the cultivars, whereas the translocation factors, which are indicators of efficient translocation from the roots to the aerial parts, differed among the congeners hydrophobicity-dependently. There were positive correlations between accumulation in the roots and the hydrophobicity of the polychlorinated biphenyl congeners in both cultivars. These results indicate that translocation was also partly responsible for the congener specificity and accumulation concentrations.

Designed recombinant transcription factor with antibody-variable regions.

Many recombinant transcription factors have been invented, but we cannot select a substance used as an inducer. In this study, we have created a novel expression control system in which we can select a substance as an inducer toward which a monoclonal antibody (mAb) is prepared. The variable region fragments (Fvs) of the heavy and light chains (V(H) and V(L)) of the bisphenol A (BPA)-specific mAb BBA-2187 were each fused to the DNA-binding domain (DBD) of LexA and the transactivation domain (AD) of VP16. The association between the two recombinant proteins in the presence of BPA constituted a functional transcription factor. The recombinant proteins in which the DBD was fused to the N-terminal side of the Fv and in which the nuclear localization signal (NLS) was fused to the N-termini of the construct including the AD highly induced beta-galactosidase (lacZ) expression in recombinant yeast cells grown with BPA. When the Fvs of the polychlorinated biphenyl (PCB)-specific mAb 4444 were used, DBD-NLS-V(H) and NLS-AD-V(L) showed significantly increased lacZ activity in response to a PCB derivative. The Fv transcription factor may be useful in many fields such as gene therapeutics.

Differential uptake for dioxin-like compounds by zucchini subspecies.

The zucchini (Cucurbita pepo) cultivars 'Patty Green', 'Black Beauty', and 'Gold Rush' were cultivated on weathered dioxin-contaminated soil in pots, and concentrations of the 29 dioxin-like compounds that were assigned WHO-TEFs, three non-toxic polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs), and two non-dioxin-like polychlorinated biphenyls (PCBs) were analyzed. Toxic equivalent (TEQ) values accumulated in 'Black Beauty' and 'Gold Rush' were about 180 times higher than those in 'Patty Green'. The bioconcentration factor (BCF) based on total mass concentration of the twelve dioxin-like PCBs was higher than those of the seven PCDDs and ten PCDFs in all the cultivars. The BCFs for PCDD and PCDF congeners were negatively correlated with octanol-water partition coefficients in all the plants. No correlations were observed in PCB congeners in the high accumulators, although in 'Patty Green' the BCFs for PCB congeners were significantly correlated with octanol-water partition coefficients. Our findings suggest that the high accumulators had unknown, unique mechanisms for uptake of PCBs, whereas PCDDs and PCDFs were absorbed based on their physicochemical properties.

Xenobiotic response element binding enriched in both nuclear and microsomal fractions of rat cerebellum.

Xenobiotic response element (XRE) is a core nucleotide sequence at the upstream of inducible target genes for the transcription factor aryl hydrocarbon receptor (AhR) that is responsible for signal transduction of exogenous environmental pollutants in eukaryotic cells. Immunoblotting analysis revealed the constitutive expression of AhR-related proteins in rat liver and brain, while specific binding of a radiolabelled probe containing XRE was detected in nuclear preparations of both liver and brain on gel retardation electrophoresis. Among discrete rat brain structures examined, cerebellum exhibited the highest XRE binding with less potent binding in hypothalamus, midbrain, medulla-oblongata, hippocampus, cerebral cortex and striatum. In contrast to liver and hippocampus, cerebellum also contained unusually higher XRE binding in microsomal fractions than that in either nuclear or mitochondrial fractions. Limited proteolysis by V8 protease did not markedly affect XRE binding in cerebellar nuclear extracts, with concomitant diminution of that in hepatic and hippocampal nuclear extracts. In primary cultured cerebellar neurons, indigo was effective in significantly increasing XRE binding only when determined immediately after sustained exposure for 120 min in the presence of high potassium chloride. These results suggest the abundance of as-yet unidentified proteins with high affinity for XRE and responsiveness to indigo in both nuclear and microsomal fractions of rat cerebellum.

Modulation of DNA binding of nuclear transcription factors with leucine-zipper motifs by particular endogenous polyamines in murine central and peripheral excitable tissues.

Transcriptional regulation is one of the most important functions of polyamines in the nucleus of eukaryotic cells. The addition of the endogenous polyamines spermine and spermidine markedly increased DNA binding activity of the transcription factor activator protein-1 (AP1) in a concentration-dependent manner at a concentration range of 50 to 500 microM in nuclear extracts of murine whole brain when determined in the absence of added MgCl(2) on gel retardation electrophoresis. Similar but less potent potentiation was seen with DNA binding of cAMP responsive element binding protein (CREB), while both polyamines were ineffective in affecting that of c-Myc irrespective of the addition of MgCl(2). Unlabeled AP1 probe was invariably more potent in competing for AP1 binding than unlabeled CREB probe in either the presence or absence of spermine and spermidine. In addition to whole brain, both polyamines significantly increased AP1 binding in retina, adrenal and pituitary, without significantly affecting that in spleen. Moreover, ultraviolet and circular dichroism spectra analyses revealed that these two polyamines induced DNA topological transition of AP1 probe under the conditions favorable for the increase in AP1 binding. These results suggest that both spermine and spermidine may modulate gene transcription through cis- and trans-actions on AP1 binding in the nucleus in murine central and peripheral structures with high excitability.

Existence of xenobiotic response element binding in Dictyostelium.

Xenobiotic response element (XRE) is a core nucleotide sequence at the upstream of inducible target genes for the transcription factor aryl hydrocarbon receptor (AhR) that is responsible for recognition of exogenous environmental pollutants in eukaryotic cells. Gel retardation electrophoresis revealed the presence of binding of a radiolabeled probe containing XRE in both cytosolic and nuclear preparations of the slime mold Dictyostelium. Unlabeled XRE probe was more potent in competing for XRE binding in both fractions than unlabeled XRE probe with a point mutation at the core element. Limited proteolysis by V8 protease did not markedly affect XRE binding in both fractions, while XRE binding decreased during in vitro incubation at 30 degrees C for up to 24 h at decline rates proportional to increasing pHs at a range of 6.5-8.5 in cytosolic fractions in a manner different from those in nuclear fractions. Deprivation of nutrients induced aggregation of cells within 4-8 h later, followed by formation of first finger tips around 12 h later and subsequent development to mobile slugs within 16 h and then to fruit bodies between 20 and 24 h later. The starvation led to a marked decrement of XRE binding in cytosolic fractions 4-36 h later, followed by a robust but transient increment of that in nuclear extracts 12-20 h afterward. However, XRE binding was not affected by antibodies against AhR-related proteins known to date in both fractions irrespective of nutritional conditions. These results suggest the abundance of as-yet unidentified proteins with high affinity for XRE in the slime mold Dictyostelium. The possibility that those proteins may be translocated from the cytoplasm to the nucleus in response to cellular development during starvation is feasible.