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 是为了理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 起源于特定于组织和时间的方式。该模型系统是 利用的是发育调节的绒毛膜基因的扩增 果蝇卵巢卵泡细胞内的簇状结构。DNA复制的控制 与人类癌症的研究特别相关。几个原癌基因 而肿瘤抑制基因与DNA复制的调控有关。 我们已经确定了dna中涉及的两个不同的顺式调控元件。 复制：复制者和起源。这些将通过突变 体外定位绒毛膜基因，将突变的构建物重新导入 转基因动物的染色体，并检测构建物的能力 利用简单的定量Southern杂交进行扩增。二维凝胶 卵巢DNA复制中间体的电泳法研究 滤泡细胞允许作为起源的特定序列被 确认身份。它们的起源可以区别于称为 在顺式结构中起作用以调节起始点的复制子。我们假设 唯一序列元素(S)“X”是复制子和/或原点(S)的一部分，以及 通过与一个或多个相互作用标记绒毛膜基因座以进行扩增 “因素X。”在反式中扩增所需的两种蛋白质 分析，k43(DmORC2)和雪纺。这两种蛋白质都可能与之相互作用或参与其中。 最后，新的遗传方法将被用来识别其他 包括因子X在内的扩增的反式调节因子。第一种方法使用 设计转座元件以生成主要的、有条件的 (四环素依赖)高频突变。第二种方法 涉及四环素调控的双链RNA的表达，进而 导致对基因表达的序列特异性抑制。这些实验将会 测试一些关于组织和监管的具体假设 绒毛膜DNA复制起源于绒毛膜。