The Role of Drosophila SIR2 in gene silencing, DNA meta*

果蝇 SIR2 在基因沉默、DNA 元*中的作用

• 批准号: 6532579
• 负责人: Peter J. Harte
• 金额: $ 31万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-15 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6532579
• 关键词: Drosophilidae; alleles; amidohydrolases; arthropod genetics; chromatin; chromatography; enzyme activity; gene dosage; gene expression; gene induction /repression; gene interaction; gene mutation; histones; homeobox genes; immunoprecipitation; longevity; nicotinamide adenine dinucleotide; nucleic acid metabolism; transcription factor

The long term goal of the proposed research is to understand the role of the Drosophila SIR2 protein in gene silencing and investigate its possible role in lifespan-extension by caloric restriction (CR) in higher eukaryotes. In the budding yeast Saccharomyces cerevisiae, SIR2 has been demonstrated to be a determinant of lifespan, a single extra copy of the Sir2 gene extending lifespan by up to 50 percent. CR promotes a similar extension of yeast lifespan that is dependent on SIR2. It is of great interest to discover whether the SIR2 orthologs in higher eukaryotes are also involved in determining lifespan and lifespan extension by CR. We have identified the Drosophila SIR2 ortholog (dSIR2) by database searches and have identified mutations affecting it. We plan to use these mutations, as well as alterations in SIR2 gene dosage and directed overexpression of dSIR2 to investigate its role in gene silencing and longevity. We will characterize the biochemical activities of dSIR2, in particular determining whether it has an NAD+-dependent histone deacetylase activity that is conserved in yeast and mouse SIR2 proteins. We also plan to carry out biochemical purification of complexes containing dSIR2 by conventional chromatography and identify their polypeptide components by peptide sequencing. In preliminary studies we have made intriguing connections between dSIR2 and Polycomb Group proteins, which are involved in stable long-term silencing of the homeotic genes in Drosophila and are highly conserved in higher eukaryotes. We plan to explore this connection further through genetic and biochemical studies to determine its functional significance. We expect that these studies will provide new insights into the role of dSIR2 in gene silencing and reveal whether and how it plays a role in determining lifespan and lifespan extension by CR in higher eukaryotes.

拟议的研究的长期目标是了解果蝇SiR2蛋白在基因沉默中的作用，并研究其在较高真核生物中通过热量限制（CR）在寿命扩展中的可能作用。 在酿酒酵母的萌芽酵母菌中，SIR2已被证明是寿命的决定因素，SIR2基因的单个额外副本可延长寿命高达50％。 CR促进了依赖于SIR2的酵母寿命的类似扩展。 发现较高真核生物中的Sir2直系同源物是否还参与CR确定寿命和寿命延长。 我们已经通过数据库搜索确定了果蝇SiR2直系同源物（DSIR2），并确定了影响其突变的突变。 我们计划使用这些突变，以及SIR2基因剂量的改变，并指示DSIR2的过表达来研究其在基因沉默和寿命中的作用。我们将表征DSIR2的生化活性，特别是确定其是否具有NAD+依赖性组蛋白脱乙酰基酶活性，该活性在酵母和小鼠SIR2蛋白中保守。 我们还计划通过常规色谱对含有DSIR2的复合物进行生化纯化，并通过肽测序鉴定其多肽成分。 在初步研究中，我们在DSIR2和PolyComb蛋白之间建立了有趣的联系，这些蛋白与果蝇中同源基因的长期沉默有关，并且在较高的真核生物中得到了高度保守。 我们计划通过遗传和生化研究进一步探讨这种联系，以确定其功能意义。 我们希望这些研究将为DSIR2在基因沉默中的作用提供新的见解，并揭示其在确定CR在较高真核生物中的寿命和寿命延长中是否发挥作用。