Mechanisms and Phenotypic Consequences of Structural Genomic Variation

Argueso Lab-PA-20-272-对现有赠款和合作社的行政补充 协议 NIH项目摘要/摘要： 我们正在研究的拷贝数变异(CNV)机制之一是从头再发 CNV。这会导致后代的复制和缺失，这在父母身上是看不到的。此外， 在没有血缘关系的个体中也可以看到同样的基因断点。非等位基因同源重组 在减数分裂过程中，由基因组中的低拷贝重复(LCR)区域介导的(Nahr)导致这些 重新安排。这是一个重要的研究机制，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，大约5%-10%的CNV 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 在正常的、健康的人身上。我们感兴趣的是找到调节频率的因素 以酿酒酵母为检测系统的复发性CNV。 在我们的家长奖中，我们描述了一种在酵母中的检测系统来研究这一现象并 在产生的单倍体孢子中阳性检测CNV。我们通过放置CNV记者系统做到了这一点， 它使用基因SFA1和CUP1，以及工程LCR之间的耐药标记。我们可以的 通过四分体解剖的标准方法将CNV与这个原始系统进行鉴定，这是一种尝试过的方法- 减数分裂后遵循标记分离的正确方法，但速度慢且费力。我们有 修改了我们原来的检测系统，在两者之间加入了孢子自主荧光标记 我们设计的LCR。有了我们的新荧光系统，我们可以在一个物质中评估数百个四分体 几个小时，而使用四分体解剖，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 用显微镜来确定四个孢子中哪一个发出红色或绿色的光。我们已经测试了我们的菌株 使用属于我们部门邻居实验室的荧光显微镜，以及测试系统 效果很好。这是一个很棒的显微镜，但它给我们带来了几个问题：1)它太高了 需求，我们没有优先访问它，所以它是具有挑战性的时间。这将严重地 限制我们推进对复发CNV的调查的能力。2)这台显微镜要多得多 比我们所需要的更有能力，并且没有适当地设置为酵母细胞的图像，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 有适当的规格来满足我们的需求。这台新显微镜将使我们能够高效地 评估复发的CNV并扩展我们的实验以研究LCRs的结构并包括 在我们对复发CNV形成的遗传控制的研究中发现了更多的突变体。

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.