SPECIFICITY OF RNA TRANSPORT IN VITRO

在真核细胞中控制基因表达的机制很复杂， 包括转录和转录后成分。由于 在他们的研究中，转录后机制有固有的困难 受到的关注相对较少；特别是RNA的运输 研究了快速标记的RNA在上清液中的出现 细胞核的体外孵化。这样的分析会更坚定地被接受 如果运输的RNA有更好的特性。这就是我们的目的 建议通过以下方式测试精心挑选的体外系统的保真度 检查特定序列的行为。使用微扰， 导致体内mRNA的形成和丰度有很大的差异， 急性时相反应物序列(α-酸性糖蛋白)的代谢， 荷尔蒙相关的球蛋白序列(α2Mu球蛋白)和标志物 成熟(白蛋白)和原始(甲胎蛋白)肝脏的序列 细胞将通过体外运输系统进行检测。一项额外的 Model将使用一种类似蛋白血症的大鼠品系，它转录白蛋白 序列进入核RNA，但将它们限制在细胞核内。已运输 核限制性RNA将被分离并在体外翻译 将对产品进行检查。RNA将按大小分开 电泳法：“Northern blotted”，与特定的 P32-cDNA探针和放射自显影将被开发出来。混合实验 将记录和量化细胞核和细胞质污染 运输的制剂。这些实验将确定在体外 传输检测可以支持处理/传输大小正确的 功能的信使核糖核酸，与体内环境中的调制平行。 可以建立提供特异性(如初步结果所示)， 体外转运试验的使用应有助于 了解基因表达的调控，特别是改变的基因 与癌变相关的转录后RNA转运。如果 无法确定具体情况，相当大的努力和资金 致力于将它们用于“体内特异性”的研究应该是 缩减了。

Mechanisms controlling gene expression in eukaryotic cells are complex, encompassing transcriptional and post-transcriptional components. Due to inherent difficulties in their study, post-transcriptional mechanims have received relatively less attention; in particular, RNA transport has been studied as the appearance of rapidly-labeled RNA in the supernate after incubation of nuclei in vitro. Such assays would be more firmly accepted if the transported RNA was better characterized. It is the intent of this proposal to test the fidelity of carefully selected in vitro system by examining the behavior of specific sequences. Using perturbations which result in great differences in mRNA formation and abundance in vivo, metabolism of an acute phase reactant sequence (alpha1-acid glycoprotein), a hormonally-responsible globulin sequence (alpha2Mu-globulin), and marker sequences for mature (albumin) and primitive (Alpha-fetoprotein) liver cells will be examined with an in vitro transport system. An additional model will employ an analbuminemic rat strain, which transcribes albumin sequences into nuclear RNA but restricts them to the nucleus. Transported and nucleus-restricted RNA will be isolated and in vitro translation products will be examined. RNA will be separated by size electrophoretically, "Northern blotted" and hybridized with specific P32-cDNA probes, and autoradiographs will be developed. Mixing experiments will document and quantitate cytoplasmic contamination of nuclear and transported preparation. These experiments will determine whether in vitro transport assays can support processing/transport of correctly-sized functional mRNA, with modulation paralleling in vivo circumstances. Providing specificity can be established (as preliminary results indicate), use of in vitro transport assays should contribute valuably to understanding controls of gene expression, especially the altered post-transcriptional RNA transport associated with carcinogenesis. If specificity cannot be established, the considerable effort and funding devoted to their use in studies of "in vivo specificity" should be curtailed.