GENE AMPLIFICATION--SCIARID DNA PUFFS

我们的主要目标是定义ORI(DNA复制起点)序列 真菌蝇Sciara的DNA泡芙II/9A和II/2B所需的， 以及了解蜕皮激素诱导的 扩增和转录增强剂。 DNA泡芙ORI将通过跨越II/9A和II/2B的染色体走行来定位 结合定量Southern印迹绘制最大值区域 每一次喷雾的扩音器。一种新开发的二维凝胶方法将是 应用于每个ORI域以实现更精细的分辨率映射；此方法将 还显示是否将相同的ORI站点用于预膨胀复制(在此之前 放大)。将确定在两次喷雾中ORI的DNA序列 并相互比较 在DNA膨胀过程中活跃的转录单位将由Northern绘制 在两个DNA喷雾基因座上用跨越染色体的探针进行斑点扫描，以及 它们的5‘端起始点由引物延伸确定。转录的 利用P元件转化果蝇体内的增强子将被定位 CAT化验结果。DNase I超敏反应与体内DMS足迹 增强子区域将定位反式作用蛋白的结合部位，以及 这些地区将被排序。 增强剂是否与ORI重叠？未来的研究将解决是否 激活的蜕皮激素受体直接结合上游部位启动 包括扩增和转录。基因扩增是一个重要的 一些癌症的特征，DNA喷雾提供了一个很好的模型系统 了解扩增的机制及其荷尔蒙控制。

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.