| Autor: |
Lloyd D. Graham, Anna Pawlak-Skrzecz, Ronald J. Hill, Wynona M. Johnson, Garry N. Hannan, Donya Tohidi-Esfahani, Linda Howell, G.O. Lovrecz, Louis Lu, Marianne Bliese |
| Rok vydání: |
2009 |
| Předmět: |
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| Zdroj: |
Ecdysone: Structures and Functions ISBN: 9781402091117 |
| DOI: |
10.1007/978-1-4020-9112-4_19 |
| Popis: |
EcR- and USP-encoding cDNAs of four pest insects (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were cloned from high- quality lambda cDNA libraries and sequenced. Cognate EcR-USP cDNA pairs were shown to express functional ecdysone receptors in transfected cells. The amino acid sequences of the EcR ligand binding domains (LBDs) were employed in conjunc- tion with those known for other arthropods to construct a phylogenetic tree. Affinity tagged EcR-USP LBD heterodimers were co-expressed efficiently in insect cells using a baculovirus vector. The recombinant EcR and USP DE/F segments from each species associated spontaneously to form heterodimers that bound ecdysteroids with high affinity. An E/F segment pair (constructed only for H. armigera) also asso- ciated spontaneously to form a functional heterodimer, but one with ligand binding affinities several times lower than its DE/F counterpart. A fluorescein-inokosterone conjugate was synthesized and used to develop a novel ligand binding assay based on fluorescence polarization. This assay can be used in place of the classical ( 3 H)-ponasterone A binding assay, and is ideally suited to high-throughput screen- ing. The ligand binding data obtained in vitro using recombinant LBD heterodim- ers reflect the ability of agonists to induce ecdysone receptor controlled transgene expression in recombinant mammalian cells; in vitro binding data can also reflect the potency of ligands to act as insecticides. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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