Visualization of G4 Tetraplex DNA on Yeast Meiotic Chromosomes

酵母减数分裂染色体上 G4 四链体 DNA 的可视化

• 批准号: 9510732
• 负责人: Walter Gilbert
• 金额: $ 2.5万
• 依托单位: Harvard University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-01 至 1996-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9510732&HistoricalAwards=false
• 关键词: Visualization; G4; Tetraplex; DNA; Yeast

9510732 Gilbert The objective of this research is to obtain evidence for G4 tetraplex DNA in vivo; more specifically, on meiotically paired chromosomes. Biochemical and genetic evidence suggests that this novel form of DNA exists in vivo as a structural intermediate for several cellular processes including meiosis, telomere functioning, and G-rich sequence mediated recombination. The yeast gene KEMI encodes a G4-DNA dependent nuclease. A homozygous deletion of this gene blocks yeast cells at meiosis I, where homologous chromosomes are paired but fail to resolve, supporting the hypothesis that G4-DNA is involved in meiotic chromosome pairing. Naked chromosomes will be isolated from meiotically blocked (kem1() yeast cells and their DNA will be visualized and examined by a light microscopy, and by CHEF gel electrophoresis and Southern blotting in comparison with chromosomes from wild type cells. Chromosomes from keml( mutant cells blocked in meiosis I should exhibit higher-order structural changes including retarded mobility in CHEF gels, and tetrameric (4N) complexes of DNA from homologous chromosomes joined at multiple points. This research will shed new light on meiotic chromosome pairing by demonstrating DNA-DNA interaction consistent with G4-DNA pairing in vivo, and will establish the foundation to investigate other cellular processes in which this tetraplex DNA might be involved, such as telomere functioning and recombination. %%% The objective of this research is to obtain evidence for the novel G4 tetraplex DNA structure in meiotically paired chromosomes. Biochemical and genetic evidence suggests that this novel form of DNA exists in vivo as a structural intermediate for several cellular processes including meiosis, telomere functioning, and G-rich sequence mediated recombination. ***

9510732吉尔伯特这项研究的目标是获得G4四链DNA在体内存在的证据；更具体地说，是在减数分裂配对的染色体上。生化和遗传学证据表明，这种新形式的DNA在体内作为几个细胞过程的结构中间体存在，包括减数分裂、端粒功能和富含G的序列介导的重组。酵母基因Kemi编码一种依赖于G4 DNA的核酸酶。该基因的纯合缺失阻碍了减数分裂I的酵母细胞，在减数分裂I，同源染色体配对但无法分解，支持了G4-DNA参与减数分裂染色体配对的假设。裸露的染色体将从减数分裂阻断(Kem1)酵母细胞中分离出来，其DNA将通过光学显微镜、厨师凝胶电泳法和Southern blotting进行可视化和检查，并与野生型细胞的染色体进行比较。Keml(突变细胞在减数分裂I中受阻)的染色体应该表现出更高级别的结构变化，包括厨师凝胶中的移动迟缓，以及来自同源染色体的DNA四聚体(4N)复合体在多个点连接。这项研究将揭示减数分裂染色体配对的新线索，证明DNA-DNA相互作用与G4-DNA配对在体内是一致的，并将为研究这种四链DNA可能参与的其他细胞过程，如端粒功能和重组奠定基础。%本研究的目的是获得在减数分裂配对的染色体中存在新的G4四链DNA结构的证据。生化和遗传学证据表明，这种新形式的DNA在体内作为几个细胞过程的结构中间体存在，包括减数分裂、端粒功能和富含G的序列介导的重组。***