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 其中一种类固醇激素，蜕皮激素，刺激一组 相关基因，果蝇卵黄蛋白基因。我们有 已经克隆了这些基因，对它们进行了测序，鉴定了序列 与mRNAs互补，表明它们的转录速度很可能 受激素浓度控制并转录克隆的 部分纯化的果蝇RNA聚合酶II的基因。我们现在 建议使用体外转录来研究转录 蜕皮激素及其辅助物引发和调控 各种因素。我们的实验已经证明，在体内和体外 转录起始于CAP位点的上游。通过脉冲追逐和输入 体外诱变实验我们将确定这些上游是否 启动事件通过处理事件的方式导致成熟的mRNA。这个 将使用相同的方法来确定转录和 处理是耦合的向量过程，并识别和提纯 推定的处理活动。这项处理研究的目标是 以确定荷尔蒙控制的启动发生在哪里。类似 方法将被用来鉴定所需的核苷酸和蛋白质 观察到体外转录的特异性启动。最后，我建议 为了探讨体外启动与体内启动的关系， 转录的荷尔蒙控制。为了实现这一目标，我计划 使用两种分析系统(添加细胞提取物及其部分 对PolII转录反应和细胞DNA结合试验的影响 对基因具有特定亲和力的蛋白质)以检测其他因素， 可能是荷尔蒙控制所必需的蛋白质，但 在体外PolII转录系统中缺失。当前知识 表明类固醇激素作用的表面一般机制 (受体介导的与染色质或DNA的相互作用)在 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 可能会对理解这一机制做出重大贡献 荷尔蒙-受体复合体控制转录速率。这是 当然，这是一个基本的过程，必须在一个健康的人身上很好地工作。

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.