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