Hurban P, Thummel C S
Department of Human Genetics, Howard Hughes Medical Institute, University of Utah, Salt Lake City 84112.
Mol Cell Biol. 1993 Nov;13(11):7101-11. doi: 10.1128/mcb.13.11.7101-7111.1993.
Our insights into the regulatory mechanisms by which the steroid hormone ecdysone triggers Drosophila melanogaster metamorphosis have largely depended on puffs in the larval salivary gland polytene chromosomes as a means of identifying genes of interest. Here, we describe an approach that provides access to ecdysone-inducible genes that are expressed in most larval and imaginal tissues, regardless of their ability to form puffs in the polytene chromosomes. Several hundred cDNAs were picked at random from subtracted cDNA libraries and subjected to a rapid and sensitive screen for their ability to detect mRNAs induced by ecdysone in the presence of cycloheximide. Of the 15 genes identified in this manner, 2 correspond to early puffs in the salivary gland polytene chromosomes, at 63F and 75B, confirming that this screen functions at the desired level of sensitivity and is capable of identifying novel primary-response genes. Three of the genes, Eig45-1, Eig58, and Eig87, are expressed coordinately with the salivary gland early genes; one of them, Eig58, maps to the 58BC puff that is active when the 74EF and 75B early puffs are at their maximal size. Another gene identified in this screen, Eig17-1, encodes a novel cytochrome P-450. On the basis of its sequence identity and temporal profile of expression, this gene may play a role in steroid hormone metabolism and thus could provide a mechanism for feedback regulation of ecdysone production. Although all 15 genes have patterns of transcription that are consistent with ecdysone regulation in vivo, 5 genes do not appear to be induced by the late larval ecdysone pulse. This indicates that ecdysone induction in larval organs cultured with cycloheximide is not always indicative of a primary response to the hormone.
我们对于类固醇激素蜕皮激素触发黑腹果蝇变态的调控机制的深入了解，很大程度上依赖于幼虫唾液腺多线染色体中的胀泡，以此作为鉴定感兴趣基因的一种手段。在此，我们描述了一种方法，该方法能够获取在大多数幼虫和成虫组织中表达的蜕皮激素诱导基因，而不论它们在多线染色体中形成胀泡的能力如何。从消减cDNA文库中随机挑选了数百个cDNA，并对其在放线菌酮存在的情况下检测由蜕皮激素诱导的mRNA的能力进行了快速且灵敏的筛选。通过这种方式鉴定出的15个基因中，有2个对应于唾液腺多线染色体上63F和75B处的早期胀泡，这证实了该筛选在所需的灵敏度水平下起作用，并且能够鉴定出新的初级反应基因。其中三个基因，即Eig45 - 1、Eig58和Eig87与唾液腺早期基因协同表达；其中一个基因Eig58定位于58BC胀泡，当74EF和75B早期胀泡处于最大尺寸时该胀泡活跃。在此筛选中鉴定出的另一个基因Eig17 - 1编码一种新型细胞色素P - 450。基于其序列同一性和表达的时间模式，该基因可能在类固醇激素代谢中发挥作用，因此可能为蜕皮激素产生的反馈调节提供一种机制。尽管所有15个基因的转录模式在体内与蜕皮激素调节一致，但有5个基因似乎不受幼虫晚期蜕皮激素脉冲的诱导。这表明在用放线菌酮培养的幼虫器官中，蜕皮激素诱导并不总是表明对该激素的初级反应。
