Akgül Bünyamin, Tu Chen-Pei D
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.
Methods Enzymol. 2008;448:285-97. doi: 10.1016/S0076-6879(08)02615-3.
We have established an in vivo system to investigate mechanisms by which pentobarbital (PB), a psychoactive drug with a sedative effect, changes the rate of decay of gstD21 mRNA (encoding a Drosophila glutathione S-transferase). Here we describe methods for the use of hsp70 promoter-based transgenes and transgenic lines to determine mRNA half-lives by RNase protection assays in Drosophila. We are able to identify and map putative decay intermediates by cRT-PCR and DNA sequencing of the resulting clones. Our results indicate that the 3'-UTR of gstD21 mRNA is responsive to PB by regulating mRNA decay and that the cis-acting element(s) responsible for the PB-mediated stabilization resides in a 59 nucleotide sequence in the 3'-UTR of the gstD21 mRNA (Akgül and Tu, 2007).
我们建立了一个体内系统，以研究具有镇静作用的精神活性药物戊巴比妥（PB）改变gstD21 mRNA（编码一种果蝇谷胱甘肽S-转移酶）衰变率的机制。在这里，我们描述了使用基于hsp70启动子的转基因和转基因品系，通过果蝇中的核糖核酸酶保护试验来确定mRNA半衰期的方法。我们能够通过cRT-PCR和对所得克隆进行DNA测序来鉴定和定位假定的衰变中间体。我们的结果表明，gstD21 mRNA的3'-UTR通过调节mRNA衰变对PB有反应，并且负责PB介导的稳定作用的顺式作用元件位于gstD21 mRNA 3'-UTR的59个核苷酸序列中（阿克居尔和图，2007年）。
