Drosophila DNA Replication Origins

果蝇 DNA 复制起源

• 批准号: 6727572
• 负责人: JOHN Gerard TOWER
• 金额: $ 20.31万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6727572
• 关键词: DNA binding protein; DNA replication origin; Drosophilidae; chorioallantoic membrane; double stranded RNA; eukaryote; functional /structural genomics; gel electrophoresis; gene expression; gene induction /repression; gene mutation; genetic regulatory element; genetic screening; genetically modified animals; graafian follicles; immunoprecipitation; nucleic acid amplification techniques; polymerase chain reaction; protein protein interaction; protooncogene; southern blotting; tetracyclines; transposon /insertion element; tumor suppressor genes; yeast two hybrid system

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.

描述（由申请人提供）：本研究的长期目标 是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，并调节这些复制的激发 以组织和时间特异性方式起源。模型系统为 利用的是绒毛膜基因的发育调控扩增 果蝇卵巢滤泡细胞簇。 DNA复制的控制 与人类癌症的研究尤其相关。多种原癌基因 肿瘤抑制基因参与 DNA 复制的调节。 我们已经鉴定出 DNA 中涉及的两个不同的顺式调控元件 复制：复制子和起源。这些将通过突变来研究 体外绒毛膜基因位点，将突变的构建体重新引入 转基因动物的染色体，并测定构建体的能力 使用简单的定量 Southern 印迹进行扩增。二维凝胶 从卵巢中分离的 DNA 复制中间体的电泳 滤泡细胞允许作为起源的特定序列 确定。起源可以与称为 以顺式方式发挥作用以调节起源的复制子。我们假设 独特的序列元件“X”是复制子和/或起点的一部分，并且 通过与一个或多个相互作用来标记绒毛膜基因座以进行扩增 “X因素。”反式扩增所需的两种蛋白质正在被 分析了 k43 (dmORC2) 和雪纺。两种蛋白质都可以相互作用或成为其中的一部分 最后，新的遗传方法将用于识别其他 放大的反式调节器，包括因子 X。第一种方法使用 工程转座元件产生显性、条件性 （四环素依赖性）突变频率较高。第二种方法 涉及四环素调节的双链 RNA 表达，进而 引起基因表达的序列特异性抑制。实验将 检验一些关于组织和监管的具体假设 绒毛膜基因座 DNA 复制起点。