GENE AMPLIFICATION

我们建议继续我们对真核DNA复制的研究，使用 DNA从苍蝇的多线染色体中喷发出来。作为一种 模型系统。DNA喷发是已知的仅有的两个病例之一 染色体内DNA扩增是发育过程中的正常事件(The 另一个例子是果蝇毛囊细胞中的绒毛膜基因扩增)。 我们刚刚将放大的原点映射到 DNApuff 11/9A以L kb区为主。因此，它现在提供了 唯一已被绘制的非病毒、后生动物真核起源 毫不含糊地延伸到一个小的、定义明确的区域。 我们建议继续进行地图研究，以确定该地图的位置。 基于2-D凝胶，使用另一种方法。我们将完成我们的 初步实验表明，相同的区域被用作 扩增前正常染色体复制的起源。我们 建议开发技术以将复制的起源映射到 核苷酸水平；这项技术将使用定义明确的 在应用于Sciara DNA泡芙之前，在酵母中加入ARS。 我们将从另一个大的DNA中分离和鉴定基因组克隆 泡芙(11/2B)。并绘制出它的原点。DNA泡芙11/9A中的起始区 和1L/2B将被排序，以便在它们之间进行比较 鉴定可能具有功能的保守区 对复制的重要性。 DNase I超敏反应研究将完成，体内和体内 进行体外足迹研究，以确定结合蛋白质的区域 原产地区域。发挥DNA序列作用的DNA序列的重要性 原产地和调节原产地活动的顺式作用元件将是 通过使用P元素转换的功能分析进行检验。 癌症可能被认为是一种DNA复制失控的疾病。所以，一个 对DNA复制的基本机制的理解 真核生物及其正常复制是未来研究进展的关键 癌症，控制似乎出了问题。

We propose to continue our studies on eukaryotic DNA replication, using DNA puffs from polytene chromosomes of the fly. Sciara coprophila, as a model system. DNA puffs are one of only two cases known to undergo intrachromosomal DNA amplification as a normal event in development (the other case is chorion gene amplification In Drosophila follicle cells). We have just mapped the origin for amplification to predominantly a l kb region In DNA puff 11/9A. Thus, it now provides the only non-viral, metazoan eukaryotic origin that has been mapped unambiguously to a small, well-defined region. We propose to continue our mapping studies to confirm this map location based on 2-D gels by using another method. We will complete our preliminary experiments that suggest that the same region is used as an origin for normal chromosomaI replication prior to amplification. We propose to develop techniques to map the origIn of replication to the nucleotide level; this technique will be developed using a well-defined ARS in yeast before application to Sciara DNA puffs. We will isolate and characterize genomic clones from another large DNA puff (11/2B). and map its origin. The origin regions in DNA puffs 11/9A and 1l/2B will be sequenced to allow comparison between them for Identification of conserved regions that could be of functional importance for replication. DNase I hypersensitivity studies will be completed, and in vivo and in vitro footprinting carried out to define areas that bind proteins in the origin region. The Importance of DNA sequences that function as the origin and the cis-acting elements that regulate origin activity will be tested by functional assays using P-element transformation. Cancer may be thought of as a disease of runaway DNA replication. so an understanding of the fundamental mechanism of DNA replication in eukaryotes and Its normal replication is key to future progress in cancer where the controls have seemingly gone awry.