Mechanisms and Phenotypic Consequences of Structural Genomic Variation

Argueso Lab - PA-20-272 -现有赠款和合作的行政补充 协定 NIH项目摘要/摘要： 我们正在研究的拷贝数变异（CNV）机制之一是从头复发 CNVs。这些导致后代中的重复和缺失，而在父母中看不到。此外，委员会认为， 相同的基因断裂点在不相关的个体中可见。非等位基因同源重组 在减数分裂期间，由基因组中的低拷贝重复（LCR）区域介导的NAHR引起这些 重新安排这是一个需要研究的重要机制，因为某些形式的自闭症和其他 人类的神经发育障碍与复发性CNV有关，约5-10%的CNV与复发性CNV有关。 人类基因组由低拷贝重复序列组成。这些基因组重排的可能性是存在的 在正常健康的人身上我们感兴趣的是找到调节频率的因素， 使用酿酒酵母作为我们的检测系统。 在我们的母公司奖中，我们描述了酵母中的测定系统来研究这种现象， 在所得的单倍体孢子中阳性检测CNV。我们通过放置CNV报告系统来做到这一点， 它使用基因SFA 1和CUP 1，以及工程LCR之间的耐药标记。我们可以 通过四分体解剖的标准方法，用这个原始系统鉴定CNV，这是一种尝试过的方法， 在减数分裂后跟踪标记分离的方法是一种既慢又费力的方法。我们有 修改了我们原来的分析系统，也包括孢子自主荧光标记之间 我们的工程LCR有了我们新的荧光系统，我们可以评估物质中的数百个四分体 而四分体解剖我们可以在大约一周内评估100-200个四分体。 为了给我们的细胞成像，我们需要：1）使用明场显微镜观察它们，2）使用荧光 显微镜下观察，以确定四个孢子中哪一个发出红光或绿色光。我们测试了我们的菌株 用的是我们系隔壁实验室的荧光显微镜， 效果很好这是一个伟大的显微镜，但它给我们带来了几个问题：1）它是在高 需求，我们没有优先访问它，所以它是具有挑战性的时间。这将严重 限制了我们对复发性CNVs的研究。2)这台显微镜 能力比我们需要的，并没有适当的设置，以图像酵母细胞，因为它缺乏相 对比度我们建议购买一种更简单、更实惠的荧光显微镜， 有合适的规格来满足我们的需求。这种新的显微镜将使我们能够有效地 评估复发性CNVs，并扩大我们的实验，研究LCR的结构，包括 在我们对复发性CNV形成的遗传控制的研究中，

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.