Studies on two ecdysone receptor isoforms of the spruce budworm, Choristoneura fumiferana.
A full-length cDNA clone corresponding to the Choristoneura fumiferana ecdysone receptor-A isoform (CfEcR-A) was isolated. The deduced amino acid sequence of CfEcR-A differed from CfEcR-B in the NH2-terminal region of the A/B domain. The CfEcR-A-specific region showed high amino acid identity with EcR-A isoforms of Manduca sexta, Bombyx mori, Drosophila melanogaster and Tenebrio molitor. Isoform-specific probes were used to study the expression of EcR-A and EcR-B mRNAs. Both probes detected 6 kb mRNAs that were present in second-sixth larval instars and in the pupae. Both EcR-A and EcR-B mRNA levels increased during the molting periods. In the sixth instar larvae, the increase in EcR-A and EcR-B mRNA levels were more pronounced in the midgut than in epidermis and fat body. Both EcR-A and EcR-B mRNAs were induced in CF-203 cells (a cell line developed from C. fumiferana midgut) grown in the presence of 4 x 10(-6) M 20E. EcR-B specific mRNAs were induced within 1 h of exposure to 20E, but EcR-A specific mRNAs were induced only after 3 h of exposure to 20E. Induction of mRNAs for both isoforms was unaffected by the presence of a protein synthesis inhibitor, cyclohexamide, in the culture medium. RH-5992, a stable ecdysone agonist, caused a similar induction pattern of EcR-A and EcR-B mRNAs in the midgut, epidermis and fat body of sixth instar larvae. In vitro translated CfEcR-A, CfEcR-B and CfUSP proteins were used to study the DNA binding and ligand binding properties of EcR-A/USP and EcR-B/USP protein complexes. The Kd values indicated that both complexes have similar binding affinities for ecdysone response elements and ponasterone A.
Perera
S C
SC
Great Lakes Forestry Centre, Canadian Forest Service, Sault Ste. Marie, Ont.
Ladd
T R
TR
Dhadialla
T S
TS
Krell
P J
PJ
Sohi
S S
SS
Retnakaran
A
A
Palli
S R
SR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D011987:Receptors, Steroid / Q000235:genetics* / Q000378:metabolism; D017421:Sequence Analysis
D018076:DNA, Complementary; D008024:Ligands; D020033:Protein Isoforms; D011987:Receptors, Steroid; C055469:ecdysone receptor