Screening systems for ecdysteroid mimetic or antiecdysteroid substances in plant extracts or libraries of synthetic compounds are commonly based on the observation of morphological and/or growth responses in insect cell lines. Because these responses are slow and require careful monitoring, existing screening systems are considered limited regarding their applicability to analysis in high-throughput (HT) formats. Here we describe the generation of transformed silkmoth (Bombyx mori) cell lines that respond to the addition of ecdysone-like substances through the expression of the green fluorescent protein (GFP) and the appearance of green fluorescence. Because tests consist of three simple steps, i.e., 1) distribution of transformed cells in microtiter plates; 2) addition of compounds/extracts at different concentrations; and 3) quantification of fluorescence intensity by a fluorescence plate reader, they can be performed quickly and be easily adapted to a HT format. The generated reporter cell lines are used for the screening of extracts from available plant collections for the presence of compounds with ecdysone mimetic or antagonistic activities as well as for monitoring subsequent activity during enrichment and purification steps. The same cell lines are also used here for the determination of structure-activity relationships among available synthetic dibenzoylhydrazine derivatives. Finally, for the identified agonists, we show that their activity as determined by the cell-based screening assays parallels their bioactivity in growth inhibition and toxicity assays carried out on live insects.
A comprehensive sequence analysis of three early chorion genes (6F6.1, 6F6.2, 6F6.3) which form a small subfamily is presented. Two main features characterize this subfamily: (1) the 6F6 gene copies are beta-branch genes and, unlike typical chorion genes which are organized in divergent gene pairs, they are unpaired, and (2) they are not clustered in genetic locus Ch3 but are dispersed in Ch1-2, which is about 3 to 4 centiMorgans away and contains middle and late chorion genes. Sequence comparisons show that members of this subfamily exhibit high identity values in their major coding region (94-96%) and that similarities also extend, but to a lesser degree, into their noncoding regions. The putative 6F6 promoter regions have no significant similarities with the corresponding regions of other early beta-genes but quite surprisingly share common elements with middle and late genes. The main difference among the 6F6 gene introns is the presence of inserted sequences: the insert into 6F6.2 ("IR"; 248 bp) is flanked by a 102-103-bp inverted repeat, while those into 6F6.1 ("FIB"; 184 bp) and 6F6.3 ("HOPE"; 951 bp) are carried by a partial Bm1 element. HOPE has features of a non-LTR retrotransposable element. Preliminary experiments indicate that the copy number of IR and HOPE in the Bombyx mori genome is about 5,000 and 20,000, respectively. The great similarity of 6F6 genes cannot be accounted for by selective pressure but rather appears to be the result of gene-conversion-like events, which are supposed to operate frequently in middle and late chorion genes but not in other known early beta-genes. Using the relative position and orientation of the 6F6 gene copies, it is possible to propose an evolutionary scheme for the formation of chorion locus Ch1-2.
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