GENE AMPLIFICATION--SCIARID DNA PUFFS

基因扩增--SCIARID DNA泡芙

• 批准号: 6385603
• 负责人: SUSAN A GERBI
• 金额: $ 29.46万
• 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 2002-12-04
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6385603
• 关键词: DNA replication origin; Diptera; chromatin; genetic mapping; genetic regulatory element; immunofluorescence technique; natural gene amplification; polymerase chain reaction; protein binding; yeast two hybrid system

We propose to analyze initiation of DNA replication in DNA puff II/9A of Sciara salivary gland polytene chromosomes because it is the only non-viral, metazoan eukaryotic origin unambiguously mapped to a small, well-defined region (less than 1 kb). We will address the following questions that have never been answered for any eukaryotic cellular origin: (1) What activates an origin to fire? (2) What control mechanism exists to ensure that an origin will fire not more than once per S phase? We will complete our studies on the structure of the II/9A origin: (a) definition of the boundaries of the II/9A origin and how it changes during development, (b) identification of replication initiation points (mapped to the nucleotide level), and (c) identification of cis-acting elements that regulate II/9A origin activity (by chromatin studies and P-element transformation). Our major thrust will be to identify proteins that associate with the II/9A origin and are important for control of initiation of DNA replication and prevention of rereplication. We will define the binding sites of the origin recognition complex (ORC) with the II/9a origin, having isolated the ORC2 polypeptide and raised antibodies against it. This will be the first case where ORC is demonstrated to bind to a specific DNA sequence in any metazoan. Also, we will investigate if ORC binds to a consensus sequence in the genome, which is currently unknown for any metazoan. Whether all ORC binding sites function as active ORIs in Sciara will be explored by PCR analysis of nascent strands and by double immunofluorescence of polytene chromosomes. Finally, we will identify by yeast one hybrid screens other proteins that bind the II/9A origin in a stage specific manner, differing between pre-amplification to amplification stages, suggesting a role for such proteins in rereplication control. Yeast two hybrid screens will identify interactions of these proteins with others, such as ORC. Cdc6 is an important component associated with ORC, and it appears to transmit cell cycle controls to ORC. We will clone Sciara Cdc6 and use it for a yeast two hybrid screen to identify interacting proteins that change from pre- amplification to amplification stage.

我们建议分析DNA Puff II/9A中DNA复制的启动 Sciara唾液腺聚腺染色体的染色体是唯一的 非病毒的，后生真核的起源，明确映射到一个小的， 定义明确的区域（小于1 kb）。 我们将解决以下内容 任何真核细胞从未回答过的问题 来源：（1）什么激活了起点？ （2）什么控制机制 存在以确保起源不会发射不超过一次 阶段？ 我们将完成有关II/9A起源结构的研究：（a） II/9A起源边界及其如何变化的定义 在开发过程中，（b）识别复制起始点 （映射到核苷酸水平）和（c）鉴定顺式作用 调节II/9A起源活性的元素（通过染色质研究和 P元素转换）。 我们的主要力量是确定与之相关的蛋白质 II/9A起源，对于控制DNA的启动很重要 复制和预防重复。 我们将定义绑定 具有II/9A起源的原点识别综合体（ORC）的位置， 分离出ORC2多肽并提出针对它的抗体。 这将是第一种证明兽人与A结合的情况 任何后生动物中的特定DNA序列。另外，我们将调查ORC是否 结合基因组中的共有序列，目前未知 对于任何后生动物。 所有兽人结合位点是否充当活性ORI 在Sciara中，将通过对新生链的PCR分析以及 聚单抗染色体的双重免疫荧光。 最后，我们会的 通过酵母筛选筛选其他结合II/9A的蛋白质 以特定阶段的方式起源，在放大前不同 放大阶段，表明此类蛋白质在 重复控制。 酵母两个混合动力屏幕将识别 这些蛋白质与其他蛋白质（例如ORC）的相互作用。 Cdc6是一个 与ORC相关的重要组件，并且似乎传输了单元格 循环控制到兽人。 我们将克隆sciara cdc6并将其用于酵母 两个混合屏幕，以识别从前变化的相互作用蛋白 放大到放大阶段。