Mechanisms and Phenotypic Consequences of Structural Genomic Variation

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Argueso Lab – PA-20-272 – Administrative Supplement to Existing Grants and Cooperative Agreements NIH Project Summary/Abstract: One of the copy number variation (CNV) mechanisms that we are investigating is de novo recurrent CNVs. These result in duplications and deletions in offspring that are not seen in parents. Furthermore, the same genetic breakpoints are seen in unrelated individuals. Non-allelic homologous recombination (NAHR) during meiosis, mediated by low copy repeat (LCR) regions in the genome causes these rearrangements. This is an important mechanism to study because some forms of autism and other neurodevelopmental disorders in humans are associated with recurrent CNVs, and about 5-10% of the human genome is made up of low copy repeats. The potential for these genome rearrangements exists in normal, healthy people. We are interested in finding the factors that modulate the frequency of recurrent CNVs using Saccharomyces cerevisiae as our detection system. In our parent award, we described an assay system in yeast to study this phenomenon and to positively detect the CNVs in resulting haploid spores. We did this by placing a CNV reporter system, which uses the genes SFA1 and CUP1, and drug resistance markers in between engineered LCRs. We can identify CNVs with this original system via the standard method of tetrad dissections, which is a tried- and-true method to follow the segregation of markers after meiosis but is slow and laborious. We have modified our original assay system to also include spore autonomous fluorescence markers in between our engineered LCRs. With our new fluorescence system, we can assess hundreds of tetrads in a matter of hours, whereas with tetrad dissections we can assess 100-200 tetrads in about a week. To image our cells, we need to: 1) see them using brightfield microscopy, and 2) use fluorescence microscopy to determine which of the four spores are glowing red or green. We have tested our strains with a fluorescence microscope that belongs to a neighbor lab in our department, and the assay system works beautifully. This is a great microscope, but it poses a couple of problems for us: 1) It is in high demand and we do not have priority access to it, so it is challenging to get time on it. This will severely limit our ability to drive our investigation of recurrent CNVs forward. 2) This microscope is much more capable than we need and is not appropriately set up to image yeast cells because it lacks phase contrast. We are proposing to purchase a simpler and more affordable fluorescence microscope that has appropriate specifications to meet our needs. This new microscope will enable us to efficiently assess recurrent CNVs and to expand our experiments to study the architecture of LCRs and to include more mutants in our investigation of the genetic control of recurrent CNV formation.