Bouhin H, Charles J P, Quennedey B, Delachambre J
Laboratoire de Zoologie, UA CNRS 674, Université de Bourgogne, Dijon, France.
Dev Biol. 1992 Jan;149(1):112-22. doi: 10.1016/0012-1606(92)90268-l.
Changes in translatable mRNAs from the wing epidermis of the Coleoptera Tenebrio molitor have been investigated during metamorphosis by analysis of in vitro translated products. Striking differences between the patterns obtained from mRNAs extracted during pupal and adult cuticle secretion indicated that a drastic change in gene expression occurs during the pupal-adult transition. In addition to these stage-specific modifications, the mRNA patterns changed within each cuticular synthesis program (pupal or adult), especially at ecdysis. After tritiated leucine incorporation, some of the major radiolabeled cuticular proteins showed similar changes suggesting that the sequential appearance of mRNAs corresponds to sequential deposition of cuticular proteins. In supernumerary pupae obtained after juvenile hormone analogue (JHA) application on newly ecdysed pupae, translatable mRNA were very similar to those of pharate pupae. The JHA seemed, therefore, to prevent the expression of the adult program. By immunoblotting in vitro translated products with a monoclonal antibody recognizing an adult-specific cuticular protein, the developmental profile of the corresponding mRNA was studied. This mRNA was detected in anterior wing epidermis during the first 80 hr of the pharate adult stage. Using the same antibody, a cDNA clone was isolated from epidermal mRNA. The hybrid selected mRNA coded for only one protein with an apparent MW of 22 kDa which was, furthermore, recognized by the antibody. The Northern blot analysis performed with the clone confirmed the Western blot analysis of the in vitro translation products. JHA application at the beginning of the pupal-adult reprograming prevented the appearance of this mRNA; however, this transcript was present during the following molting cycle. This reversibility of the JHA action was confirmed by immunogold labeling of the cuticles formed in treated animals.
通过对体外翻译产物的分析，研究了鞘翅目昆虫黄粉虫蛹期变态过程中翅表皮可翻译mRNA的变化。从蛹期和成虫表皮分泌期提取的mRNA所获得的模式存在显著差异，表明在蛹-成虫转变过程中基因表达发生了剧烈变化。除了这些阶段特异性修饰外，mRNA模式在每个表皮合成程序（蛹期或成虫期）内也发生了变化，尤其是在蜕皮时。在掺入氚标记的亮氨酸后，一些主要的放射性标记表皮蛋白显示出类似的变化，这表明mRNA的顺序出现对应于表皮蛋白的顺序沉积。在新蜕皮的蛹上施用保幼激素类似物（JHA）后获得的额外蛹中，可翻译的mRNA与预蛹的非常相似。因此，JHA似乎阻止了成虫程序的表达。通过用识别成虫特异性表皮蛋白的单克隆抗体对体外翻译产物进行免疫印迹，研究了相应mRNA的发育谱。在预成虫阶段的前80小时内，在前翅表皮中检测到了这种mRNA。使用相同的抗体，从表皮mRNA中分离出一个cDNA克隆。杂交筛选的mRNA仅编码一种表观分子量为22 kDa的蛋白质，而且该蛋白质能被该抗体识别。用该克隆进行Northern印迹分析证实了对体外翻译产物的Western印迹分析。在蛹-成虫重新编程开始时施用JHA可阻止这种mRNA的出现；然而，在随后的蜕皮周期中存在这种转录本。通过对处理动物形成的表皮进行免疫金标记，证实了JHA作用的这种可逆性。
