Analysis of Environmental Endocrine Disrupting Activities in Wastewater Treatment Plant Effluents Using Recombinant Yeast Assays Incorporated with Exogenous Metabolic Activation System

Objective To measure the endocrine disrupting chemicals （EDCs） in wastewater and evaluate the EDCs removal efficiencies in the municipal wastewater treatment plants （WWTP）. Methods A battery of in vitro recombinant yeast bioassays incorporated with exogenous metabolic activation system （rat liver preparation, S9 mix） was conducted to assess the estrogen receptor （ER）, androgen receptor （AR）, progesterone receptor （PR）, and thyroid receptor （TR） ant/agonistic activities of effluents collected from Datansha WWTP. Results The indirect estrogenic, anti‐androgenic, anti‐progesteronic, and anti‐thyroidic activities were observed in the influent. The removal efficiencies of EDCs were above 74%, suggesting that the present wastewater treatment processes were good enough to remove most of these indirect endocrine disrupting chemicals. Conclusion The incorporation of exogenous metabolic capacity into the test system was valid for the study of indirect effects on ER, AR, PR, and TR.

Yeast One-hybrid System Used to Identify the Binding Proteins for Rat Glutathione S-transferase P Enhancer I

Objective To detect the trans-factors specifically binding to the strong enhancerelement (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. MethodsYeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library toidentify potential trans-factors that can interact with core sequence of GPEI(cGPEI).Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of trans-factors to cGPEI. Results cDNA fragments coding for the C-terminal part of thetranscription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. Thebinding of c-Jun and ANT to GPEI core sequence were confirmed. Conclusions Rat c-juntranscriptional factor and ANT may interact with cGPEI. They could play an important rolein the induced expression of GST-P gene.

In Search of Regulators of <i>LeSPL-CNR</i>by South-Western Blotting and Yeast One-Hybrid Library Screening System

LeSPL-CNR is a crucial transcription factor for fruit ripening of Solanum lycopersicum. The cnr (colorless non-ripening) epimutation resulted from hypermethylation in a 286 bp region of LeSPL-CNR promoter inhibits normal fruit ripening. In present study, potential regulators of LeSPL-CNR, which could bind to the specific 286 bp region, were screened via south-western blotting and yeast one-hybrid (Y1H) library screening system. Results indicated that a total of 13 and 19 candidate proteins were acquired respectively, and both ribulose-1,5-bisphosphate carboxylase/oxygenase and 40S ribosomal protein were identified by two methods. These would provide some information for revealing roles of DNA methylation and the regulatory mechanism for LeSPL-CNR.

Isolation of transcription factors binding auxin response elements using a yeast one-hybrid system

Plant hormones play an important role during higher plant embryogenesis. Auxin is central to the development of vascular tissues, formation of lateral and adventitious roots, control of apical dominance, and tropic responses. Auxin response element (AuxRE), present in the promoters of many auxin-induced genes, can confer auxin responsiveness. Using carrot somatic embryo under specific developmental phase, a cDNA expression library was constructed. Several plasmids were recombined containing the tetramer of AuxRE as a bait. After screening by a yeast one-hy- brid system, one positive clone was confirmed and characterized. Electrophoretic mobility shift assay showed that AxRF1 protein expressed in yeast cell could bind AuxRE in vitro. It suggests that AxRF1 participates in regulation of the expression of auxin responsive gene during carrot somatic embryogenesis.

Saccharomyces cerevisiae in neuroscience:how unicellular organism helps to better understand prion protein?

The baker's yeast Saccharomyces(S.)cerevisiae is a single-celled eukaryotic model organism widely used in research on life sciences.Being a unicellular organism,S.cerevisiae has some evident limitations in application to neuroscience.However,yeast prions are extensively studied and they are known to share some hallmarks with mammalian prion protein or other amyloidogenic proteins found in the pathogenesis of Alzheimer's,Parkinson's,or Huntington's diseases.Therefore,the yeast S.cerevisiae has been widely used for basic research on aggregation properties of proteins in cellulo and on their propagation.Recently,a yeast-based study revealed that some regions of mammalian prion protein and amyloidβ1–42 are capable of induction and propagation of yeast prions.It is one of the examples showing that evolutionarily distant organisms share common mechanisms underlying the structural conversion of prion proteins making yeast cells a useful system for studying mammalian prion protein.S.cerevisiae has also been used to design novel screening systems for anti-prion compounds from chemical libraries.Yeastbased assays are cheap in maintenance and safe for the researcher,making them a very good choice to perform preliminary screening before further characterization in systems engaging mammalian cells infected with prions.In this review,not only classical red/white colony assay but also yeast-based screening assays developed during last year are discussed.Computational analysis and research carried out using yeast prions force us to expect that prions are widely present in nature.Indeed,the last few years brought us several examples indicating that the mammalian prion protein is no more peculiar protein–it seems that a better understanding of prion proteins nature-wide may aid us with the treatment of prion diseases and other amyloid-related medical conditions.

<i>Arabidopsis</i>Transcription Factor WRKY33 Is Involved in Drought by Directly Regulating the Expression of <i>CesA8</i>

Arabidopsis (Arabidopsis thaliana) WRKY33 is a key transcription factor in pathogen-induced defense signaling, but its function in abiotic stresses remains largely unclear. In this study, we report on the use of a reverse-genetic approach, as well as a yeast (Saccharomyces cerevisiae) expression system, to determine the role of WRKY33 in drought. A T-DNA insertion deletion mutant of WRKY33 is more sensitive to dehydration. Through genome-wide screening the target genes of WRKY33 in yeast, we identified 23 candidate genes including a drought tolerance gene CesA8. Further results revealed that WRKY33 repressed CesA8 expression through binding to the W-box elements of CesA8 distal promoter region and probably interacting with the transcriptional activator of CesA8, MYB46. These findings revealed the primary molecular mechanism underlying the function of WRKY33 in response to

Clony color assay coupled with 5FOA negative selection greatly improves yeast three-hybrid library screening efficiency

The recently developed yeast three-hybrid system is a powerful tool for analyzing RNA-protein interactions in vivo. However, large numbers of false positives are frequently met due to bait RNA-independent activation of the reporter gene in the library screening using this system. In this report, we coupled the colony color assay with the 5FOA (5-fluoroorotic acid) negative selection in the library screening, and found that this coupled method effectively eliminated bait RNA-independent false positives and hence greatly improved library screening efficiency. We used this method successfully in isolation of cDNA of an RNA-binding protein that might play important roles in certain cellular process. This improvement will facilitate the use of the yeast three-hybrid system in analyzing RNA-protein interaction.

Mutagenic and Estrogenic Effects of Organic Compounds in Water Treated by Different Processes: A Pilot Study

Objective In this study, a pilot-scale investigation was conducted to examine and compare the biotoxicity of the organic compounds in effluents from five treatment processes (P1-P5) where each process was combination of preoxidation (O3), coagulation, sedimentation, sand filtration, ozonation, granular activated carbon, biological activated carbon and chlorination (NaClO). Methods Organic compounds were extracted by XAD-2 resins and eluted with acetone and dichlormethane (DCM). The eluents were evaporated and redissolved with DMSO or DCM. The mutagenicity and estrogenicity of the extracts were assayed with the Ames test and yeast estrogen screen (YES assay), respectively. The organic compounds were detected by GC-MS. Results The results indicated that the mutation ratio (MR) of organic compounds in source water was higher than that for treated water. GC-MS showed that more than 48 organic compounds were identified in all samples and that treated water had significantly fewer types and concentrations of organic compounds than source water. Conclusion To different extents, all water treatment processes could reduce both the mutagenicity and estrogenicity, relative to source water. P2, P3, and P5 reduced mutagenicity more effectively, while P1 reduced estrogenicity, most effectively. Water treatment processes in this pilot plant had weak abilities to remove Di-n-butyl phthalate or 1, 2-Benzene dicarboxylic acid.

Comparison of Different MARs(Matrix Attachment Regions) Effect on Transgene Expression

Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respectively. Sequence analysis showed that all thefragments had fairly high A/T content (73, 62 and 75%, respectively),harbored differentnumber and different type of some characteristic motifs of MARs, such as A-box and T-box,etc. The results of in vitro binding assay showed that the three MARs fragments derivedfrom different organisms could bind specifically to the matrix extracted from the tobacconuclei with different strength, which also demonstrated that these MARs fragments arefunctionally conserved during evolution. By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-bar-MARs into tobacco through Agrobacterium mediated procedures, the effects of MARs sequenceson the expression of transgenes in tobacco were investigated and compared. The GUSactivity in individual transformants showed that, comparing to the controls withoutadditional MARs, the overall transgene expression level in transformants with MARs hadbeen greatly increased while the variations in transgene expression among transformantswere decreased in different degrees. In accordance with the results of sequence analysisand in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overallexpression level.

Identification and Characterization of Human Genomic Binding Sites for Retinoic Acid Receptor/Retinoid X Receptor Heterodimers

All-trans retinoic acid (ATRA) triggers a wide range of effects on vertebrate development by regulating cell proliferation, differentiation, and apoptosis. ATRA activates retinoic acid receptors (RARs) which heterodimerize with retinoid X receptors (RXRs). RAR/RXR heterodimers function as ATRA-dependent transcriptional regulators by binding to retinoic acid response elements (RAREs). To identify RAR/RXR heterodimer-binding sites in the human genome, we performed a modified yeast one-hybrid assays and identified 193 RAR/RXR heterodimer-binding fragments in the human genome. The putative target genes included genes involved in development process and cell differentiation. Gel mobility shift assays indicated that 160 putative RAREs could directly interact with the RAR/RXR heterodimer. Moreover, 19 functional regulatory single nucleotide polymorphisms (rSNPs) on the RAR/RXR-binding sequences were identified by analyzing the difference in the DNA-binding affinities. These results provide insights into the molecular mechanisms underlying the physiological and pathological actions of RAR/RXR heterodimers.

Assessment of source water contamination by estrogenic disrupting compounds in China

Detection of estrogenic disrupting compounds（EDCs） in drinking waters around China has led to rising concerns about health risks associated with these compounds.There is,however,a paucity of studies on the occurrence and identification of the main compounds responsible for this pollution in the source waters.To fill this void,we screened estrogenic activities of 23 source water samples from six main river systems in China,using a recombinant two-hybrid yeast assay.All sample extracts induced significant estrogenic activity,with E2 equivalents（EEQ） of raw water ranging from 0.08 to 2.40 ng/L.Additionally,16 samples were selected for chemical analysis by gas chromatography-mass spectrometry.The EDCs of most concern,including estrone（E1）,17β-estradiol（E2）,17α-ethinylestradiol（EE2）,estriol（E3）,diethylstilbestrol（DES）,estradiol valerate（EV）,4-t-octylphenol（4-t-OP）,4-nonylphenols（4-NP） and bisphenol A（BPA）,were determined at concentrations of up to 2.98,1.07,2.67,4.37,2.52,1.96,89.52,280.19 and 710.65 ng/L,respectively.Causality analysis,involving comparison of EEQ values from yeast assay and chemical analysis identified E2,EE2 and 4-NP as the main responsible compounds,accounting for the whole estrogenic activities（39.74% to 96.68%）.The proposed approach using both chemical analysis and yeast assay could be used for the identification and evaluation of EDCs in source waters of China.

Functional Characterization of the Promoter and Second Intron of CUM1 During Flower Development in Cucumber(Cucumis sativus L.)

The characterization of flower specific promoter is critical during flower development by cucumber transformation technology.AGAMOUS(AG)is an organ identity gene that is required for carpel and stamen development in Arabidopsis.The promoter and second intron of AG contain multiple regulatory elements that confer proper spatial and temporal expression.Cucumber is an important vegetable with unisexual flowers.Cucumber MADS-box 1(CUM1)is the AG homolog in cucumber,belonging to the eu AG lineage along with AG.In situ hybridization showed that CUM1 was specifically expressed in the stamens and carpels of cucumber.GUS staining indicated that the second intron of CUM1 confers stamen-specific expression,while the promoter of CUM1 drives both stamen-and carpel-specific expression during the early stages of flower development,but is restricted to carpel-and connectivum-specific expression during the late stages of flower development.Furthermore,a yeast one-hybrid assay demonstrated that two auxin response factors(Cs ARF13 and Cs ARF17)had bound directly to the second intron of CUM1.Our data suggest that different regulatory circuits operate in AG homologs in plant species with distinct sex types.

In vitro thyroid disrupting effects of organic extracts from WWTPs in Beijing

It is generally known that there are many endocrine disrupting compounds （EDCs） in the effluents from wastewater treatment plants （WWTPs）. Most research has focused on the occurrence of estrogenic or androgenic activities, while ignoring that there are environmental chemicals disrupting thyroid system, which is essential for growth and development in both humans and animals. In the present work, a two-hybrid yeast assay was conducted to evaluate the removal efficiencies of agonistic or antagonistic thyroid receptor （TR） mediated effects in different treatment processes of three WWTPs located in Beijing. We found no TR agonistic, but TR antagonistic activities in all processes from the WWTPs. The TR antagonistic activities in organic extracts of water samples were then calibrated regarding to a known TR-inhibitor, amiodarone hydrochloride （AH）. The observed concentration of TR disrupting substances ranged from 2.35 × 10 8 to 6.19 × 10 7 mol/L AH in Gaobeidian WWTP, 3.76 × 10 8 to 8.75 × 10 8 mol/L AH in Lugouqiao WWTP, and 4.80 × 10 9 to 2.55 × 10 8 mol/L AH in Beixiaohe WWTP. Of the three WWTPs, the removal rates were 92.7%, 42.2%, and 23.1% respectively. Industrial sewage may contain more TR disrupting substances compared with domestic sewage. The recipient waters were found to contain considerable concentrations of TR disrupting substances that may cause adverse effects on the exposed organisms.

Determination of estrogens and estrogenic activities in water from three rivers in Tianjin, China

Studies on estrogenic disrupting compounds （EDCs） occurrence and identification of main responsible compounds in river water discharged into the sea are of significance. In the present research, we screened estrogenic activities of 10 river water samples from 3 main rivers discharged into Bohai Sea in Tianjin using a recombinant two-hybrid yeast assay and chemical analysis by gas chromatography-mass spectrometry. All sample extracts induced significant estrogenic activity, with 17^-estradiol equivalents （EEQ） of raw water ranging from 5.72 to 59.06 ng/L. Six most concerned EDCs in the fiver water samples including estrone, 17（5-estradiol, 17ct-ethinylestradiol, estriol, diethylstilbestrol and estradiol valerate were determined, with their concentrations up to 50.70, 31.40, 24.40, 37.20, 2.56, and 8.47 ng/L, respectively. Through causality analysis by comparing the EEQ values of yeast assay and chemical analysis, 17ct-ethinylestradiol and 17[3-estradiol were identified as the main contributors to the estrogenic effects of the river samples, accounting for the whole estrogenic activities （62.99% to 185.66%）, and estrogen antagonistic compounds might presented in the heavy polluted water samples. The proposed approach using both chemical analysis and bioassay could be used for identification and evaluation of the estrogenic activity of EDCs in river water.

ThERF1 regulates its target genes via binding to a novel cis-acting element in response to salt stress

Ethylene responsive factors (ERFs) are plant-specific transcription factors that are involved in a variety of biological processes. We previously demonstrated that an ERF gene from Tamarix hispida, ThERF1, encodes a protein binding to GCC-box and DRE motifs and negatively modulates abiotic stress tolerance. In the present study, microarray analysis was performed to study the genes regulated by ThERF1 on a genomic scale. There were 154 and 307 genes (respectively representing 134 and 260 unique genes) significantly up- and downregulated by ThERF1 under salt stress conditions, respectively. A novel motif, named TTG, was identified to be recognized by ThERF1, which commonly presents in the promoters of ThERF1-targeted genes. The TTG motif is also bound by other ERFs of a different subfamily from T. hispida and Arabidopsis, indicating that it is commonly recognized by ERF proteins. The binding affinities of ERFs to the TTG motif are significantly induced by salt stress. The TTG motif is more enriched than the GCC-box and DRE motifs in the promoters of ThERF1-targeted genes. Taken together, these studies suggested that the TTG motif plays an important role in the gene expression regulated by ERFs in response to salt stress.

In vitro agonistic and antagonistic endocrine disrupting effects of organic extracts from waste water of different treatment processes

Effluents from wastewater treatment plant （WWTP） have been reported to have a broad spectrum of endocrine disrupting compounds （EDCs）. The majority of studies have focused on the occurrence of estrogenic activity, while ignoring nuclear hormone receptors （NRs） pathways. In the present study, a battery of in vitro yeast bioassays and a cell bioassay, including antagonistic and agonistic effects on estrogen receptor （ER）, androgen receptor （AR）, progesterone receptor （PR）, estrogen- related receptor （ERR） and aryl hydrocarbon receptor （AHR）, were conducted to evaluate the removal efficien- cies of EDCs by different treatment processes of a WWTP located in Beijing. Estrogenic, anti-estrogenic, anti- androgenic, anti-progesteronic, anti-ERR and the activa- tion of AHR activities were detected in samples from all treatment processes and the receiving water. The concen- tration of estrogenic contaminants with estradiol （E2） equivalent concentrations ranged from 0.82 x 10-9 to 3.54 x 10 9g Ee_EQ.L-1. The concentration of anti-estrogenic contaminants with 4-hydroxytamoxifen （4-OHT） equiva- lent concentrations ranged from 1.24 × 10-6 to 2.36 x 10-6 g 4-OHT-EQ.L-1. The concentration of anti-androgenic contaminants ranged from 2.21 x 10-s to 3.52 × 10-6g flutamide-EQ. L-1. The concentration of anti-progesteronic contaminants ranged from 3.15 x 10^-5 to 2.71 x 10^-4g RU486-EQ. L-1. The concentration of anti-ERR contami- nants ranged from 7.09 x 10-5 to 6.50 x 104 g 4-OHT-EQ × L^-10. The concentration of AHR activators ranged from 1.7 × 10-10 to 3.4 × 10^-10g TCDD-EQ-L-1. These processes including secondary clarifier, coagulation, as well as coal and sand filtration could eliminated 67.2% of estrogenic contaminants, 47.0% of anti-estrogenic contaminants, 98.3% of anti-androgenic contaminants, 88.4% of anti- progesteronic contaminants, 65.4% of anti-ERR contami- nants and 46.9% of AHR activators. WWTP effluents contain multiple receptor disruptors may have very complex adverse effects on exposed organisms.

Maize Gene Regulatory Network for Phenolic Metabolism

The translation of the genotype into phenotype, represented for example by the expression of genes encod- ing enzymes required for the biosynthesis of phytochemicals that are important for interaction of plants with the environment, is largely carried out by transcription factors （TFs） that recognize specific cis-regulatory elements in the genes that they control. TFs and their target genes are organized in gene regulatory net- works （GRNs）, and thus uncovering GRN architecture presents an important biological challenge necessary to explain gene regulation. Linking TFs to the genes they control, central to understanding GRNs, can be car- ried out using gene- or TF-centered approaches. In this study, we employed a gene-centered approach uti- lizing the yeast one-hybrid assay to generate a network of protein-DNA interactions that participate in the transcriptional control of genes involved in the biosynthesis of maize phenolic compounds including gen- eral phenylpropanoids, lignins, and flavonoids. We identified 1100 protein-DNA interactions involving 54 phenolic gene promoters and 568 TFs. A set of 11 TFs recognized 10 or more promoters, suggesting a role in coordinating pathway gene expression. The integration of the gene-centered network with informa- tion derived from TF-centered approaches provides a foundation for a phenolics GRN characterized by in- terlaced feed-forward loops that link developmental regulators with biosynthetic genes.

Osdof28: A New Member of the DOF Transcription Factor Family from Rice

DOF is a novel family of plant-specific proteins that share a unique and highly conserved DNA binding domain with one CX2CX21CX2C zinc finger motif. In this study, the Osdof28 gene, which codes a characteristic amino acid sequence of the DOF transcription factor family, was screened from rice （Oryza sativa japonica） using a yeast one-hybrid assay. Great amounts of the Osdof28 transcripts were found to accumulate in stems and leaves, with less in the roots, and no detectable transcription found in the germs. Osdof28 can be induced by salicylic acid and INA, which suggests that it may be related to the plant systemic acquired resistance （SAR）. The relationship was confirmed through biological induction of SAR using Xanthomonascampestrispv. oryzae, with more expression of Osdof28 observed in the systemic tissues after infection.

The interaction between ADAM22 and 14-3-3β

ADAM family consists of a number of transmembrane proteins that contain a disintegrin and metalloprotease domain. ADAMs are involved in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis and inflammatory response. The ADAM proteins have both cell adhesion and protease activities. Adam22 is highly expressed in human brain. The adam22-/- mice presented severe ataxia and died before weaning, but the function of ADAM22 is still unknown. 14-3-3β interacting with ADAM22 was detected by using yeast two-hybrid assay. The specificity of interaction between ADAM22 and 14-3-3β was proved by in vitro binding assay and immunoprecipitation. The major 14-3-3β binding site was located in the last 28 amino acid residues of ADAM22 cytoplasmic tail. Protein 14-3-3β is abundant and plays an important role in mediating cell diffusion, migration and cell cycle control. The interaction of ADAM22 and 14-3-3β suggests that the ADAM22 may play a crucial role in neural function and development.

Genotoxicity of gold nanoparticles functionalized with indolicidin towards Saccharomyces cerevisiae

The toxic effects of gold nanoparticles surface-functionalized with the antimicrobial peptide indolicidin（Au NPs-indolicidin） towards the yeast Saccharomyces cerevisiae, one of the major eukaryotic model organisms, have been evaluated. Growth and survival,genotoxicity, as measured by comet assay, and expression of the YCA1, an apoptosis indicating gene, following 72 hr exposure of yeast to Au NPs-indolicidin, and to Au NPs and indolicidin alone have been examined. The gold nanoparticles exerted toxicity with DNA damage, accompanied by reactive oxygen species production（ROS）, but they do not inhibit yeast growth and viability. Genotoxicity was less pronounced for surface-functionalized nanoparticles, showing that S. cerevisiae is quite resistant to the complex Au NPs-indolicidin.A progressive reduction of the genotoxic effect was observed along 72 hr exposure,presumably due to the activation of DNA repair mechanisms. These findings suggest the occurrence of a physiological protective response of S. cerevisiae towards nanoparticles,thereby providing useful information to the assessment of the environmental impact of metal nanoparticles.