Drosophila DNA Replication Origins

描述（由申请人提供）：本研究的长期目标 是为了了解高等真核细胞如何指定某些区域， 染色体DNA作为复制起点，并调节这些 以组织和时间特异性的方式起源。该模型系统是 利用的是绒毛膜基因的发育调节扩增， 果蝇卵泡细胞中的簇。DNA复制的控制 与人类癌症的研究特别相关。几种原癌基因 肿瘤抑制基因与DNA复制的调节有关。 我们已经确定了两个不同的顺式调控元件参与DNA 复制：复制者和起源。这些将通过突变 绒毛膜基因位点，将突变的构建体重新引入到 转基因动物的染色体，并测定构建体的能力， 用简单的定量Southern印迹进行扩增2-双向电泳 从卵巢中分离的DNA复制中间体的电泳 毛囊细胞允许特定的序列作为起源， 鉴定其起源可以从基本序列中区分出来， 顺式作用以调节起源的复制因子。我们假设 独特序列元件“X”是复制子和/或起点的一部分，并且 通过与一个或多个标记基因座相互作用来标记用于扩增的绒毛膜基因座， “X因素。“扩增所需的两种反式蛋白质正在被 分析，k43（dmORC 2）和雪纺。这两种蛋白质可能相互作用或部分 X因子最后，新的遗传学方法将用于确定额外的 反式放大调节因子包括因子X。第一种方法使用 工程化转座因子以产生显性的、条件性的 （四环素依赖性）突变。第二种方法 包括四环素调控的双链RNA表达， 导致基因表达的序列特异性抑制。实验将 测试一些具体的假设，以组织和监管， 绒毛膜基因座DNA复制起点。

DESCRIPTION (provided by applicant): The long term objective of this research is to understand how the higher eukaryotic cell designates certain regions of chromosomal DNA as replication origins, and regulates the firing of these origins in a tissue- and temporal-specific manner. The model system being utilized is the developmentally regulated amplification of the chorion gene clusters in Drosophila ovarian follicle cells. The control of DNA replication is particularly relevant to the study of human cancers. Several proto-oncogenes and tumor-suppresser genes are implicated in the regulation of DNA replication. We have identified two distinct cis-regulatory elements involved in DNA replication: replicators and origins. These will be studied by mutating the chorion gene locus in vitro, reintroducing the mutated constructs into the chromosomes of transgenic animals, and assaying the ability of the constructs to amplify using simple quantitative Southern blots. 2-Dimensional gel electrophoresis of DNA replication intermediates isolated from the ovarian follicle cells allows the specific sequences acting as origins to be identified. The origins can be distinguished from essential sequences called replicators which act in cis to regulate the origins. We hypothesize that unique sequence element(s) "X" are part of the replicator and/or origin(s), and mark the chorion loci for amplification by interacting with one or more "factors X." Two proteins required in trans for amplification are being analyzed, k43 (dmORC2) and chiffon. Both proteins may interact with or be part of factor X. Finally, novel genetic methods will be used to identify additional trans regulators of amplification including factor X. The first method uses an engineered transposable element to generate dominant, conditional (tetracycline-dependant) mutations at high frequency. The second method involves tetracycline-regulated expression of double-strand RNA, which in turn causes sequence-specific inhibition of gene expression. The experiments will test a number of specific hypotheses as to the organization and regulation of the chorion locus DNA replication origins.