GENE AMPLIFICATION

我们建议继续我们对真核DNA复制的研究， 果蝇多线染色体的DNA泡沫。嗜粪黑翅菌作为一种 模型系统DNA泡沫是已知的仅有的两种情况之一 染色体内DNA扩增是发育中的正常事件（ 另一例是果蝇滤泡细胞中的绒毛膜基因扩增）。 我们刚刚绘制了扩增的起源， 主要是DNA puff 11/9A中的1 kb区域。因此，它现在提供了 只有非病毒，后生动物真核生物起源已被映射 一个很小的，明确的区域。 我们建议继续进行测绘研究，以确定这个地图位置 基于2-D gels凝胶通过使用另一种方法。我们将完成我们的 初步实验表明，同一区域被用作 扩增前正常染色体复制的起点。我们 建议开发技术，将复制的起源映射到 核苷酸水平;该技术将使用定义明确的 ARS在应用于Sciara DNA泡芙之前在酵母中。 我们将从另一个大的DNA中分离和鉴定基因组克隆， 抽吸（11/2B）。并绘制出它的起源11/9A的起源区域 和1 l/2B将被测序，以允许它们之间的比较， 鉴定可能具有功能性的保守区域 复制的重要性。 将完成DNA酶I超敏反应研究， 进行体外足迹法来确定结合蛋白质的区域， 原产地。DNA序列的重要性 起源和调节起源活性的顺式作用元件将被 通过使用P-元件转化的功能测定进行测试。 癌症可以被认为是一种DNA复制失控的疾病。所以一个 了解DNA复制的基本机制， 真核生物及其正常复制是未来研究进展的关键。 控制似乎出了差错的癌症。

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.