Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912, USA.
Chromosome Res. 2013 Jul;21(4):345-60. doi: 10.1007/s10577-013-9360-1. Epub 2013 Jun 5.
The steroid hormone ecdysone induces DNA amplification and subsequent DNA puff formation in late fourth larval instar salivary gland polytene chromosomes of the fungus fly, Sciara coprophila. Previous in vitro studies on DNA puff II/9A in Sciara demonstrated that the ecdysone receptor (ScEcR-A) efficiently binds an ecdysone response element adjacent to the origin recognition complex binding site within the II/9A amplification origin, implying a role for ScEcR-A in amplification. Here, we extrapolate the molecular details from locus II/9A to the rest of the genome using immunofluorescence with a ScEcR-A-specific antibody. ScEcR-A binds all DNA puff sites just as amplification begins and persists throughout the processes of amplification, transcription, and puffing. Ecdysone injections into pre-amplification stage larvae prematurely induce both DNA amplification and ScEcR-A binding to DNA puff sites. These data are consistent with a direct role for ScEcR-A in DNA amplification.
蜕皮激素诱导真菌果蝇晚期第四龄幼虫唾液腺多线染色体的 DNA 扩增和随后的 DNA 膨泡形成。先前在 Sciara 上的 DNA 膨泡 II/9A 的体外研究表明，蜕皮激素受体 (ScEcR-A) 能够有效地结合位于 II/9A 扩增起始点内的起始识别复合物结合位点附近的蜕皮激素反应元件，这表明 ScEcR-A 在扩增中起作用。在这里，我们使用 ScEcR-A 特异性抗体的免疫荧光法，从 II/9A 基因座推断出基因组其余部分的分子细节。ScEcR-A 在 DNA 扩增开始时结合所有的 DNA 膨泡位点，并在扩增、转录和膨泡过程中持续存在。蜕皮激素注射到预扩增阶段的幼虫中会提前诱导 DNA 扩增和 ScEcR-A 与 DNA 膨泡位点的结合。这些数据与 ScEcR-A 在 DNA 扩增中的直接作用一致。
