Replacement of Fluorescence Imaging System

Cleveland State University, Cleveland OH

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Abstract

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.