GENE AMPLIFICATION--SCIARID DNA PUFFS

我们建议分析DNA Puff II/9A中DNA复制的启动 因为它是唯一的 非病毒，后生动物的真核起源明确地映射到一个小的， 清晰的区域(小于1kb)。我们将解决以下问题 对于任何真核细胞来说都没有得到解答的问题 起火点：(1)是什么激活了起火点？(2)什么控制机制 存在以确保一个原点不会在每个S身上触发一次 阶段？ 我们将完成对II/9A起源的结构的研究：(A) II/9A原点边界的定义及其变化 在开发期间，(B)确定复制起始点 (映射到核苷酸水平)，以及(C)识别顺式作用 调节II/9A起源活动的元件(通过染色质研究和 P元素变换)。 我们的主要任务将是确定与 II/9A起源，对DNA起始的控制很重要 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体(ORC)的位置， 分离了ORC2多肽并提高了抗体水平。 这将是第一个演示ORC绑定到 任何后生动物的特定DNA序列。此外，我们将调查ORC是否 与基因组中的一个共识序列结合，目前尚不清楚 对于任何后生动物来说。是否所有ORC结合位点都作为活动ORI运行 将通过对新生链的聚合酶链式反应分析和 多线染色体的双重免疫荧光。最后，我们会 酵母鉴定一种杂交物筛选与II/9A结合的其他蛋白质 以特定阶段的方式起源，不同于预扩增 到扩增阶段，这表明这些蛋白质在 复制控制。酵母双杂交筛选将鉴定 这些蛋白质与其他蛋白质的相互作用，如ORC。CdC6是一种 与ORC相关的重要组件，它似乎传递细胞 循环控制到兽人。我们将克隆Sciara CDC6并将其用于酵母 两个杂交筛选以确定相互作用的蛋白质从前 扩增到扩增阶段。

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.