GENE AMPLIFICATION--SCIARID DNA PUFFS

我们建议在DNA puff II/9A中分析DNA复制的起始 的Sciara唾液腺多线染色体，因为它是唯一的 非病毒，后生动物真核生物起源明确映射到一个小的， 定义明确的区域（小于1 kb）。 我们将解决以下问题 对于任何真核细胞来说， 起源：（1）是什么激活了一个起源火？(2)什么控制机制 存在的目的是为了确保源在每个S中不会触发一次以上 阶段？ 我们将完成对II/9A原点结构的研究： II/9A原点边界的定义及其变化 在发育过程中，（B）鉴定复制起始点 （映射到核苷酸水平），和（c）顺式作用的鉴定 调节II/9A起始活性的元件（通过染色质研究和 P元素变换）。 我们的主要目标将是鉴定与这些蛋白质相关的蛋白质。 II/9A起源，并对控制DNA起始起重要作用 复制和预防再复制。 我们将定义绑定 与II/9a起源的起源识别复合物（ORC）的位点， 分离了ORC 2多肽并产生了针对它的抗体。 这将是第一次证明ORC与一种 特定的DNA序列。另外，我们会调查 与基因组中的共有序列结合，这是目前未知的 对于任何后生动物来说。 是否所有ORC结合位点都起活性ORI的作用 将通过新生链的PCR分析和 多线染色体的双重免疫荧光。 最后我们将 通过酵母鉴定，一个杂交体筛选结合II/9A的其他蛋白质 以特定阶段的方式起源，与预扩增不同 到扩增阶段，这表明这些蛋白质在 再复制控制 酵母双杂交筛选将识别 这些蛋白质与其他蛋白质的相互作用，如ORC。cdc 6是一个 与ORC相关的重要组成部分，它似乎将细胞 循环控制到ORC。 我们将克隆Sciara Cdc 6并将其用于酵母 双杂交筛选，以确定相互作用的蛋白质， 放大到放大阶段。

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.