[The construction and the expression of V5 epitope fused human androgen receptor vector in the yeast cell].

When we try to establish the gene recombinant yeast cell to screen the androgenic endocrine disruptors, the key procedure is the androgen receptor (AR) expression in the yeast cell. For this purpose, we obtained the GPD (glyceraldehyde-3-phosphote dehydrogenase) promoter from the yeast genosome of W303-1A using PCR system and inserting it into Swa I and BamH I sites of pYestrp2. The new constructed vector was named pGPD.

[Establishment of recombinant Lac Z reporter gene-transformed yeast cells for bioassay of androgen-like compounds in environment].

Objective: To establish the yeast-based bioassay system for the androgenic endocrine disruptors in the environment.

Methods: The recombinant gene-transformed yeast cells were constructed based on two episomal vectors. In the expression vector, the expression of androgen receptor (AR) gene was driven by 3-glyceraldehydephosphate dehydrogenase (GPD) promoter and the gene was fused to V5 epitope; in the reporter vector, the expression of the Lac Z gene as a report gene was under the control of the androgen response element (ARE).

[The effects of TATA-box in CYC1 promoter on the reporter gene regulated by ERE in the recombinant yeast cell].

The complementary oligonucleotides, each with two consensus estrogen response element (ERE)-sequences and 5'-Hind III and 3'-Sph I sticky ends were artificially synthesized. A solution with both the complementary DNA sequences was heated to 95'C and cooled down to room temperature to form double strand DNA (dsDNA). The set was cloned into the corresponding sites of CYC1 promoter of the pERE-CYC-yEGFP to yield pERE-CYCalpha-yEGFP vector.

Development of a rapid screening and surveillance for estrogenic chemicals in environment based on recombinant yEGFP yeast cell.

A recombinant yEGFP gene yeast strain necessary for the routine screening of estrogen activity in the chemical product of the environment was described. Two plasmids, one containing human estrogen receptor (hER) cDNA fused to the GPD gene promoter and another yEGFP inserted under the control of ERE, were constructed. The use of hER cDNA and the yEGFP reporter in the yeast cell sensor resulted in estrogenic chemical product-dependent light emission of yEGFP without additions owing its advantages: a simple and reagent-free measurement of GFP, and a non-toxic protein characteristic.

[Bioassay of recombinant green fluorescent protein gene yeast cell for a high throughput to screen estrogenic compounds].

We developed the recombinant green fluorescent protein gene yeast cell to screen estrogenic compounds based on two episomal vectors. In the expression vector the expression of human estrogen receptor alpha(hERalpha) was driven by 3-glyceraldehydephosphate dehydrogenase (GPD) promoter; in the reporter vector the expression of the yeast enhanced green fluorescent protein (yEGFP) gene was under the control of the estrogen response element (ERE). The vectors were transformed into yeast cell (W303-1A) to construct GFP recombinant yeast cell.

Bioassay of estrogenic activity of effluent and influent in a farm wastewater treatment plant using an in vitro recombinant assay with yeast cells.

Objective: Environmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life. However, the majority of researches have been focused on industrial discharges, while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated. In order to investigate the contribution of environmental estrogens from livestock, the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method.