Two-hybrid assay yeast

While finding the system that works best for a particular experiment is still largely a matter of trial and error, the availability of several different systems makes it likely that a suitable system for any given experiment has already been developed. 

More recently additional systems have been developed that rely on other DBDs. Golemis and co-workers developed a system that uses the DBD from the bacteriophage X repressor cl protein [25]. Interestingly, the plasmid encoding the cl fusion protein carries the zeoR gene, which makes both yeast and E. coli containing the plasmids resistant to zeocin. Also, a system has been developed that uses the DBD from the human estrogen receptor [26]. 

The UAS provides the first level of control. The UAS is the region of DNA recognized by the DBD, and binding of a DBD-AD complex to the UAS is responsible for transcription activation. It has been established that the levels of transcription activation correlate with the number of DBD binding sites [27]. Thus, it is common to pre- 

L40 LexA/B42 Two-Hybrid Strains HIS3, lacZ MATa ade2 his3 leu2 trpl LYS2 lexAop(4x)-HIS3 URA3 lexAop (8x) -lacZ 12 

One key to minimizing false positives is to choose the optimal Y2FI format before beginning a selection. For example, if one wanted to evolve a peptide aptamer that 

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Kawamura, Y., Ogawa, Y., Nishimura, T., Kikuchi, Y., Nishikawa, J., Nishihara, T., and Tanamoto, K., 2003, Estrogenic activities of UV stabilizers used in food contact plastics and benzophenone derivatives tested by the yeast two-hybrid assay, J. Health Sci. 49 205-212. 

Another important consideration in the selection of a test set is to ensure that the chemicals in the data set relate to the real problem in question. It should be emphasized that the QSAR models developed in our project are used primarily to predict the activity of environmental chemicals, mostly pesticides and industrial chemicals. A data set reported by Nishihara et al. (Nishihara et al., 2000) was also selected as a test set. This data set contains 517 chemicals tested with the yeast two-hybrid assay, of which over 86% are pesticides and industrial chemicals. Only 463 chemicals were used for this validation study after structure processing. Only 62 chemicals were categorized as active on the basis of having on activity greater than 10% of 10 7M H2, as defined in the original paper (Nishihara et al., 2000). The majority of the chemicals were inactive, which is similar to the real-world situation where inactive chemicals are expected from a large proportion of those in the environment. 

We compared the assay results for 80 common chemicals from both the Nishihara et al. and NCTR data sets inconsistent assay results were observed for 12 chemicals. Specifically, of 30 active chemicals in the Nishihara et al. data set, one chemical was found inactive in the NCTR data set of 50 inactive chemicals in the Nishihara et al. data set, 11 chemicals were found active in the NCTR data set. These observations show that even using the experimental data from the ER binding assay (the NCTR data set) to predict the experimental results from the yeast two-hybrid assay (the Nishihara et al. data set), there may be about a 15% (12/80) discrepancy, or 3.3% (1/30) false negative rate and 22% (11/50) false positive rate. Care should be taken in interpreting the QSAR validation results using this data set (Hong et al., 2002). 

JEPA examined the literature on SDs and STs to determine whether risk assessment was really necessary. Except for the estrogenic effects, it was decided that the assessment was not necessary. Additional examinations were performed on ER binding assay, proliferation of MCF-7 human breast cancer cells (E-screen assay), yeast two hybrid assay and yeast estrogen selective (YES) assay [12]. From the results of these examinations, it was judged that estimating the risk of SDs and STs is not necessary at the present time. JEPA officially announced a revised edition of SPEED 98 in 2000, in which SDs and STs had been deleted from the list of EDs along with n-butylbenzene. 

Ogawa Y, Kawamra U, Wakui C, et al. Estrogenic activities of chemicals related to food contact plastics and rubbers tested by yeast two-hybrid assay. FoodAddit Contam 2006 23(4) 422 30. 

Yeast Two-Hybrid Assay for Protein-Protein Interactions... 

Widespread use of the yeast two-hybrid system led several groups to develop alternate transcription-based assays. While the yeast two-hybrid assay is quite powerful, a bacterial equivalent would increase by several orders of magnitude the number of proteins that could be tested, as the transformation efficiency and doubling rate of E. coli are significantly greater than those of S. cerevisiae. There may also be applications where it is advantageous to test a eukaryotic protein in a prokaryotic environment, in which many pathways are not conserved. The yeast two-hybrid assay cannot, however, be transferred directly to bacteria since the components of the transcription machinery and the mechanism of transcriptional activation differ significantly between bacteria and yeast. 

The yeast two-hybrid assay no doubt will continue to be a mainstay technique for the discovery of new protein-protein interactions. As biological pathways... 

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