GENE AMPLIFICATION--SCIARID DNA PUFFS

基因扩增--SCIARID DNA泡芙

• 批准号: 3289380
• 负责人: SUSAN A GERBI
• 金额: $ 19.95万
• 依托单位: BROWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-03-01 至 1994-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3289380
• 关键词: DNA footprinting; DNA replication; DNA replication origin; Diptera; ecdysone; genetic mapping; genetic transcription; natural gene amplification; nucleic acid probes; nucleic acid sequence

Our main goal is to define the ORI (origin of DNA replication) sequences needed for DNA puffs II/9A and II/2B in the fungus fly Sciara coprophilia, and to understand the relationship between ecdysone induced initiation of amplification and transcriptional enhancers. DNA puff ORI will be mapped by chromosome walks across II/9A and II/2B coupled with quantitative Southern blots to map the region of maximum amplification for each puff. A newly developed 2-D gel method will be applied to each ORI domain for finer resolution mapping; this method will also show if the same ORI sites are used for prepuff replication (prior to amplification). The DNA sequence of ORI at both puffs will be determined and compared to one another Transcription units active during DNA puffing will be mapped by Northern blots with probes spanning the chromosome walks at both DNA puff loci, and their 5' start sites determined by primer extension. Transcriptional enhancers will be located by P element transformation in Drosophila using CAT assays. DNase I hypersensitivity and in vivo DMS footprinting of enhancer regions will locate binding sites for trans-acting proteins, and these areas will be sequenced. Do enhancers overlap ORI? Future studies will address whether the activated ecdysone receptor directly binds to upstream sites to initiate both amplification and transcription. Gene amplification is an important feature of some cancers, and DNA puffs provide an excellent model system to understand the mechanism of amplification and its hormonal control.

我们的主要目标是定义 ORI（DNA 复制起点）序列 真菌蝇 Sciara coprophilia 中 DNA 泡芙 II/9A 和 II/2B 所需， 并了解蜕皮激素诱导的启动之间的关系 扩增和转录增强子。 DNA 吹泡 ORI 将通过 II/9A 和 II/2B 上的染色体行走进行定位 结合定量 Southern 印迹来绘制最大区域 每次抽吸的放大。 新开发的二维凝胶方法将 应用于每个 ORI 域以实现更精细的分辨率映射；这个方法将 还显示是否使用相同的 ORI 站点进行预膨胀复制（在 放大）。 两次喷出的 ORI DNA 序列将被确定 并相互比较 DNA 膨化过程中活跃的转录单位将由 Northern 绘制 用跨越染色体游走的探针在两个 DNA 膨胀基因座上进行印迹，以及 它们的 5' 起始位点由引物延伸决定。 转录的 增强子将通过果蝇中的 P 元件转化来定位 CAT 检测。 DNase I 超敏反应和体内 DMS 足迹 增强子区域将定位反式作用蛋白的结合位点，并且 这些区域将被排序。 增强子是否与 ORI 重叠？ 未来的研究将解决是否 激活的蜕皮激素受体直接与上游位点结合以启动 扩增和转录。 基因扩增很重要 DNA 泡芙提供了一个极好的模型系统 了解放大机制及其激素控制。