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 复制的起始 Sciara 唾液腺多线染色体，因为它是唯一的 非病毒、后生动物真核起源明确映射到一个小的、 明确的区域（小于 1 kb）。 我们将解决以下问题 任何真核细胞都未曾回答过的问题 原点：(1) 是什么激活原点点火？ (2) 有哪些控制机制 存在是为了确保原点每 S 触发不超过一次 阶段？ 我们将完成II/9A起源结构的研究：(a) II/9A 原点边界的定义及其变化 在开发过程中，(b) 识别复制起始点 （映射到核苷酸水平），以及（c）顺式作用的鉴定 调节 II/9A 起源活性的元件（通过染色质研究和 P 元变换）。 我们的主要目标是识别与 II/9A 起源，对于控制 DNA 起始非常重要 复制和防止再复制。 我们将定义绑定 具有 II/9a 起源的起源识别复合物 (ORC) 位点， 分离了 ORC2 多肽并产生了针对它的抗体。 这将是 ORC 被证明可以与 任何后生动物中的特定 DNA 序列。另外，我们将调查 ORC 是否 与基因组中目前未知的共有序列结合 对于任何后生动物。 是否所有 ORC 结合位点都充当活性 ORI 在 Sciara 中，将通过新生链的 PCR 分析和通过 多线染色体的双重免疫荧光。 最后，我们将 通过酵母鉴定一种杂交筛选结合 II/9A 的其他蛋白质 以特定阶段的方式起源，预放大之间有所不同 到放大阶段，表明此类蛋白质在 再复制控制。 酵母两种杂交筛选将鉴定 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 相关的重要成分，它似乎可以传递细胞 循环控制到 ORC。 我们将克隆 Sciara Cdc6 并将其用于酵母 两个混合筛选来识别从预变化的相互作用蛋白质 放大到放大阶段。