SPECIFICITY OF RNA TRANSPORT IN VITRO

真核细胞中控制基因表达的机制是复杂的， 包括转录和转录后组分。 由于 转录后机制在其研究中存在固有困难， 受到的关注相对较少;特别是，RNA运输已经被 研究了快速标记的RNA在上清液中的出现， 体外培养细胞核。 这样的分析会被更坚定地接受 如果转运的RNA被更好地表征。 这样做的目的是 建议通过以下方式测试精心选择的体外系统的保真度： 检查特定序列的行为。 使用扰动， 导致体内mRNA形成和丰度的巨大差异， 急性期反应物序列（α 1-酸性糖蛋白）的代谢， 免疫相关球蛋白序列（α 2 μ-球蛋白）和标记物 成熟（白蛋白）和原始（甲胎蛋白）肝脏的序列 将用体外转运系统检查细胞。 额外 模型将采用转录白蛋白的无白蛋白血症大鼠品系 序列进入核RNA，但将它们限制在细胞核内。 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将按大小分离 在遗传学上，“北方印迹”并与特定的 将开发P32-cDNA探针和放射自显影。 混合实验 将记录和定量细胞核的细胞质污染， 运输准备。 这些实验将决定是否在体外 转运测定可支持正确大小的 功能性mRNA，具有与体内环境平行的调节。 提供特异性可以建立（如初步结果所示）， 体外转运试验的使用应有助于 了解基因表达的控制，特别是改变的 转录后RNA转运与癌发生相关。 如果 具体性无法确定，大量的努力和资金 专门用于“体内特异性”研究的， 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.