SEQUENCE ARRANGEMENT IN EUKARYOTIC DNA & CHROMOSOMES

拟议研究的总体目标是通过以下方式确定机制： 一种类固醇激素，蜕皮激素，刺激一组转录， 相关基因，果蝇的卵黄蛋白基因。 我们有 已经克隆了这些基因，测序， 与mRNA互补，表明它们的转录速率可能是 受激素浓度控制，并转录克隆的 基因与部分纯化的果蝇RNA聚合酶II。 我们现在 我建议使用体外转录来研究转录 通过蜕皮激素和辅助物质引发和控制引发 因素 我们的实验表明，在体内和体外， 转录起始于CAP位点的上游。 通过脉冲追踪， 体外诱变实验，我们将确定是否这些上游 起始事件通过加工事件导致成熟mRNA。 的 同样的方法将被用来确定是否转录和 处理是耦合的矢量过程，并确定和纯化 假定的加工活动。 本加工研究的目的是 以确定在哪里发生了磁控制的启动。 类似 方法将被用来确定所需的核苷酸和蛋白质， 观察到特异性的体外转录起始。 最后，我建议 为了研究体外引发和体内引发之间的关系， 转录的激素控制。 为了实现这一目标，我计划 使用两种测定系统（加入细胞提取物及其级分 对于细胞的pol II转录反应和DNA结合测定， 对基因具有特异性亲和力的蛋白质）来检测其它因子， 可能是蛋白质，这是必要的激素控制，但 在体外pol II转录系统中缺失。 当前知识 表明类固醇激素作用的一般机制 （受体介导的与染色质或DNA的相互作用）类似， 果蝇和哺乳动物 我相信我们目前对蛋黄的分析 蛋白质基因系统足够先进，进一步的分析是 可能会对理解该机制做出实质性贡献， 其中一个受体-受体复合物控制转录速率。 这是 这当然是一个必须在健康人身上发挥作用的基本过程。

The overall aim of the proposed research is to determine the mechanism by which a steroid hormone, ecdysone, stimulates transcription of a set of related genes, the yolk protein genes of Drosophila melanogaster. We have already cloned these genes, sequenced them, identified the sequences complementary to the mRNAs, shown that their transcription rate is likely to be controlled by the hormone concentration and transcribed the cloned genes with a partially purified Drosophila RNA polymerase II. We now propose to use in vitro transcription to investigate transcription initiation and control of that initiation by ecdysone and ancillary factors. Our experiments have demonstrated that in vivo and in vitro transcription initiates upstream of the CAP site. By pulse-chase and in vitro mutagenic experiments we will determine whether or not these upstream initiation events lead to mature mRNA by way of processing events. The same methodology will be used to determine whether or not transcription and processing are coupled vectoral processes and to identify and purify the presumed processing activities. The objective of this processing study is to determine where the hormonally controlled initiation occurs. Similar methods will be used to identify nucleotides and proteins required for the observed specific in vitro initiation of transcription. Finally, I propose to investigate the relation between in vitro initiation and in vivo, hormonal control of transcription. In order to accomplish this I plan to use two assay systems (addition of cellular extract and fractions thereof to the pol II transcription reaction and DNA binding assays for cellular proteins with specific affinity for the genes) to detect other factors, presumably proteins, that are necessary for hormonal control but are missing from the in vitro pol II transcription system. Current knowledge suggests that the apparent general mechanism of steroid hormone action (receptor mediated interaction with the chromatin or DNA) is similar in Drosophila and mammals. I believe that our current analysis of the yolk protein gene system is sufficiently advanced that further analysis is likely to make a substantial contribution to understanding the mechanism by which a hormone-receptor complex controls transcription rate. This is certainly a fundamental process that must work well in a healthy human.