GENE AMPLIFICATION--SCIARID DNA PUFFS

我们的主要目标是确定ORI（DNA复制起点）序列 在嗜粪粪黄帚菌中需要DNA泡II/9A和II/2B， 并了解蜕皮激素诱导的 扩增和转录增强子。 DNA puff ORI将通过II/9A和II/2B的染色体步移进行定位 再加上定量Southern印迹，以绘制最大区域 放大每一口。 一种新开发的二维凝胶方法将在 应用于每个ORI域以进行更精细的分辨率映射;此方法将 还显示是否使用相同的ORI位点进行预喷复制（在 放大）。 将测定两次抽吸时ORI的DNA序列 并且相互比较 在DNA膨化过程中活跃的转录单位将由北方 用跨越两个DNA puff基因座的染色体步移的探针进行印迹， 它们的5 ′起始位点由引物延伸确定。 转录 增强子将通过果蝇中的P元件转化定位， CAT测定。 DNA酶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 这些地区将按顺序排列。 增强子与ORI重叠吗？ 未来的研究将探讨 激活的蜕皮激素受体直接结合到上游位点， 扩增和转录。 基因扩增是重要的 一些癌症的特征，DNA泡沫提供了一个很好的模型系统， 了解放大的机制及其激素控制。

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.