GENE AMPLIFICATION

基因扩增

• 批准号: 2178136
• 负责人: SUSAN A GERBI
• 金额: $ 22.36万
• 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 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178136
• 关键词: DNA footprinting; DNA replication origin; Diptera; chromatin; eukaryote; gel electrophoresis; genetic mapping; genetic regulatory element; natural gene amplification; nucleic acid sequence; polymerase chain reaction; replicon; southern blotting; yeasts

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.

我们建议继续我们对真核 DNA 复制的研究，使用 来自果蝇多线染色体的DNA。 Sciara coprophila，作为 模型系统。 DNA 泡芙是已知的仅有的两起经历过的案例之一 染色体内 DNA 扩增是发育过程中的正常事件（ 另一种情况是果蝇滤泡细胞中绒毛膜基因扩增）。 我们刚刚将放大原点映射到 主要是 DNA 蓬松 11/9A 中的 l kb 区域。因此，它现在提供 唯一已绘制图谱的非病毒、后生动物真核起源 明确地指向一个小的、明确的区域。 我们建议继续进行地图研究以确认该地图位置 使用另一种方法基于二维凝胶。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 提议开发技术将复制起点映射到 核苷酸水平；该技术将使用明确定义的 在应用于 Sciara DNA 泡芙之前，在酵母中添加 ARS。 我们将从另一个大 DNA 中分离和表征基因组克隆 泡芙（11/2B）。并绘制其原点地图。 DNA 泡芙中的起源区域 11/9A 和 1l/2B 将被测序以允许在它们之间进行比较 识别可能具有功能的保守区域 对于复制的重要性。 DNase I 超敏性研究将完成，并且在体内和体内 进行体外足迹以确定结合蛋白质的区域 原产地地区。 DNA 序列的重要性 起源和调节起源活性的顺式作用元件将是 通过使用 P 元素转化的功能测定进行测试。 癌症可能被认为是一种 DNA 复制失控的疾病。所以 了解 DNA 复制的基本机制 真核生物及其正常复制是未来进展的关键 控制似乎出了问题的癌症。