Palli S R, Hiruma K, Riddiford L M
Department of Zoology, University of Washington, Seattle 98195.
Dev Biol. 1992 Apr;150(2):306-18. doi: 10.1016/0012-1606(92)90244-b.
Using cDNAs for the human retinoic acid receptor alpha (hRAR alpha) and Drosophila hormone receptor 3 (DHR3), we isolated a cDNA encoding a member of the steroid hormone receptor superfamily from the tobacco hornworm, Manduca sexta. Sequencing showed that this cDNA is most closely related to DHR3 (97 and 68% amino acid identity in the DNA and ligand binding regions, respectively) followed by hRAR alpha (65 and 20% identity, respectively) and therefore is named MHR3. The cDNA hybridized to two mRNAs (3.8 and 4.5 kb) found in the epidermis during the ecdysteroid rises for the embryonic, larval, and pupal molts. Culture of fourth instar larval epidermis with 4 microM 20-hydroxyecdysone (2 micrograms/ml 20HE) caused the appearance of MHR3 mRNA within 3 hr and maximal expression by 6 hr; after 12 hr continuous exposure to 20HE, the mRNA level declined. The 4.5-kb mRNA appeared first, both were present in equal amounts by 12 hr, and by 20 hr the predominant transcript was 3.8 kb. Similar 20HE-induced expression was seen in epidermis explanted 1 day after the onset of wandering, although with a slower time course. The induction was largely independent of protein synthesis, but the subsequent decline required protein synthesis as is typical of the "early" puffs in Drosophila. Continuous exposure to 20HE was necessary for MHR3 expression; in its absence, the mRNA declined with a half-life of 2 hr. Thus, MHR3 is an ecdysteroid-inducible DNA binding protein that likely is a transcription factor involved in the cascade of gene activation and inactivation caused by ecdysteroids during the insect molt.
利用人类视黄酸受体α（hRARα）和果蝇激素受体3（DHR3）的cDNA，我们从烟草天蛾（Manduca sexta）中分离出一个编码类固醇激素受体超家族成员的cDNA。测序表明，该cDNA与DHR3关系最为密切（在DNA和配体结合区域的氨基酸同一性分别为97%和68%），其次是hRARα（分别为65%和20%同一性），因此被命名为MHR3。该cDNA与在胚胎、幼虫和蛹蜕皮的蜕皮甾类激素升高期间表皮中发现的两种mRNA（3.8和4.5 kb）杂交。用4微摩尔20-羟基蜕皮甾酮（2微克/毫升20HE）培养四龄幼虫表皮，3小时内出现MHR3 mRNA，6小时达到最大表达；连续暴露于20HE 12小时后，mRNA水平下降。4.5 kb的mRNA首先出现，12小时时两者含量相等，到20小时时主要转录本为3.8 kb。在化蛹前1天取出的表皮中也观察到类似的20HE诱导表达，尽管时间进程较慢。诱导在很大程度上不依赖于蛋白质合成，但随后的下降需要蛋白质合成，这是果蝇中“早期”胀泡的典型特征。持续暴露于20HE对MHR3表达是必要的；在没有20HE的情况下，mRNA以2小时的半衰期下降。因此，MHR3是一种蜕皮甾类激素诱导的DNA结合蛋白，可能是一种转录因子，参与昆虫蜕皮过程中由蜕皮甾类激素引起的基因激活和失活级联反应。
