Yeast-2-hybrid assay

Figure 18.7 Results of the yeast-2-hybrid assay. PYRI and three different pyrabactin-insensitive mutants were constructed as binding domain (BD) fusion proteins and tested for their interaction with activation domain (AD)-fused HABl in the presence of...

Figure 18.9 PYR/PYL proteins have different selectivity for responses to different small-molecule ligands, (a) A panel of PYR/PYL genes were generated as BD fusion proteins and tested in yeast-2-hybrid assays for interactions with HAB1 in the presence of (-H)-ABA, (—)-ABA, pyrabactin, apyrabactin (all at a concentration of 10 mM) or dimethyl sulfoxide (DMSO) (carrier solvent, 1%).

In general, protein target identification often employs genetic techniques such as expression cloning, expression profiling, screening of yeast mutations, and yeast three-hybrid assays. None of these techniques works for every situation. 

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. 

As an example of the application of these new approaches, we will examine the yeast two-hybrid system for the identification of protein-protein interactions within a cell. This system consists of a yeast genetic assay in which the physical association of two proteins is measured by the reconstitution of a functional transcriptional activator to drive a reporter gene such as P-galactosidase or an auxotrophic marker for selection ° (Figure 5.2). In general. 

It would seem that this has little to do with SM-protein interaction profiling, and that would be true were it not for the amazing versatility of the approach. To look at two examples of how modified yeast two-hybrid approaches can potentially be used for SM screening, let us consider SM-induced protein-protein interaction inhibition (PPII) and SM-protein binding. To assess the first situation, a modified two-hybrid assay involves a lethal reporter gene and a protein pair that is known to interact (Fig. 2-3). Now, an array of SMs potentially causing PPII... 

A number of powerful techifiques can be employed to study multiprotein complex function in yeast (e.g., gene disruption, two-hybrid assays, and DNA micro-array technology). To explain how some of these techniques can be used to study a specific complex, the characterization of a subunit in the Mediator complex of S. cerevisiae will be reviewed. 

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

---