Replacement of Fluorescence Imaging System

项目总结 父母基金的中心目标是开发一种全基因组范围内鉴定配对的方法。 同源DNA片段之间的相互作用。同源DNA序列的并列是一个关键 染色体结构、基因组稳定性和基因表达调控的决定因素。在体细胞中， 姐妹染色单体之间的配对确保了准确的染色体断裂修复和染色体 种族隔离。在生殖系中，同源染色体之间的配对确保了基因组单倍化。 用于有性繁殖。在发育过程中，同源染色体片段之间的相互作用 是建立单等位基因表达所必需的。配对缺陷与 染色体错误分离、基因组重排和基因剂量异常，导致出生 缺陷、癌症和过早衰老。开发一种检测方法，以识别涉及到 同源配对是从机制上理解这一过程的关键一步。在父级中 格兰特，我们正在开发同源配对捕获试验，以定量检测同源DNA 互动。虽然适用于大量的发育阶段和生物体，但我们正在使用 芽殖酵母减数分裂作为检测发展的模式系统。同步性减数分裂可诱导 酵母在数百万个细胞中培养，促进在全基因组同源阶段捕获细胞 配对。我们的许多方法包括通过荧光监测细胞减数分裂周期进程 显微镜。在这份补充提案中，我们申请资金来替换我们老化的例行公事 荧光显微镜和集成了最新成像系统的灵敏相机。我们的 目前的系统已经变得不可靠，即将结束其生命周期。一种更新的荧光 显微镜将使我们能够保持并进一步增强一个高影响力的研究计划，以 同源染色体配对的机制。

PROJECT SUMMARY The central aim of the parent grant is to develop an assay for genome-wide identification of pairing interactions between homologous DNA segments. Juxtaposition of homologous DNA sequences is a key determinant of chromosome structure, genome stability and gene expression control. In somatic cells, pairing between sister chromatids ensures accurate chromosome break repair and chromosome segregation. In the germ line, pairing between homologous chromosomes ensures genome haploidization for sexual reproduction. During development, interactions between homologous chromosome segments are required for establishing monoallelic gene expression. Pairing defects are associated with chromosome missegregation, genome rearrangements and aberrant gene dosage, resulting in birth defects, cancer, and premature aging. Developing an assay that identifies genome segments involved in homologous pairing is an essential step for a mechanistic understanding of this process. In the parent grant, we are developing the Homolog Pairing Capture assay, to quantitatively detect homologous DNA interactions. While applicable to a large number of developmental stages and organisms, we are using budding yeast meiosis as a model system for assay development. Synchronous meiosis can be induced in yeast cultures in millions of cells, facilitating the capture of cells at the stage of genome-wide homologous pairing. Many of our approaches involve monitoring of meiotic cell cycle progression via fluorescence microscopy. In this supplement proposal, we are requesting funds for a replacement for our aging routine fluorescence microscope and a sensitive camera integrated with an up-to-date imaging system. Our current system has become unreliable and is near the end of its lifetime. An updated fluorescence microscope will enable us to maintain and further enhance a high-impact research program into mechanisms of homologous chromosome pairing.