Detection of juvenile hormone agonists by a new reporter gene assay using yeast expressing Drosophila methoprene‐tolerant

Abstract

Juvenile hormones (JHs) are sesquiterpenoids that play important roles in the regulation of growth, metamorphosis, and reproduction in insects. Synthetic JH agonists (JHAs) have been used as insecticides and are categorized as a class of insect growth regulators (IGRs). Natural JHs and synthetic JHAs bind to the JH receptor methoprene‐tolerant (Met), which forms a functional JH‐receptor complex with steroid receptor coactivators, such as Drosophila melanogaster Taiman (Tai). The ligand‐bound Met–Tai complex induces the transcription of JH response genes by binding to specific DNA elements referred to as JH response elements (JHREs). In the present study, we established a reporter gene assay (RGA) for detecting natural JHs and synthetic JHAs in a yeast strain expressing D. melanogaster Met and Tai. The yeast RGA system detected various juvenoid ligands in a dose‐dependent manner. The rank order of the ligand potencies of the juvenoids examined in the yeast RGA linearly correlated with those of RGAs for Met–Tai established in mammalian and insect cells. Our new yeast RGA is rapid, easy to handle, cost‐effective, and valuable for screening novel JHAs.

Development of a yeast reporter gene assay to detect ligands of freshwater cladoceran Daphnia magna ultraspiracle, a homolog of vertebrate retinoid X receptors

“…As alternative and heterologous host systems, we have developed RGAs in the yeast S. cerevisiae . S. cerevisiae is the simplest eukaryote with highly conserved gene expression systems to those of animals (Ito‐Harashima & Yagi, 2017), which enables the reconstitution of the mechanisms of action and ligand responses of the ligand‐dependent transcription factors of various animal species (Chu et al, 2009; Ito‐Harashima et al, 2015, 2017, 2020, 2021; Kawanishi et al, 2003, 2006; Matsui et al, 2016; Shiizaki et al, 2010, 2014).…”

“…The relative cell growth rate (% control) was also calculated for estimating cytotoxicity of the test compounds using the formula [OD 595 (18 h) − OD 595 (0 h) in exposed wells]/[OD 595 (18 h) − OD 595 (0 h) in control wells] × 100 (Ito‐Harashima et al, 2021). The 50% inhibitory concentration (IC 50 ) and 50% lethal dose (LD 50 ) values in antagonist assay and cytotoxicity test, respectively, were calculated for each compound.…”

“…As simple and convenient procedures for the detection of EDCs, we have established yeast‐based reporter gene assay (RGA) systems for NRs from humans, rodents, amphibians, and insects (Chu et al, 2009; Ito‐Harashima et al, 2015, 2017, 2020, 2021; Kawanishi et al, 2003, 2006; Matsui et al, 2016; Shiizaki et al, 2010, 2014). In the present study, we established a new RGA to detect ligand substances of Dapma‐USP by co‐expressing the steroid receptor coactivator (SRC) of D. melanogaster Taiman (DmTai) in wild‐type yeast cells carrying the lacZ reporter plasmid with an appropriate response element (RE) (Figure 1).…”