Comprhensive Biology: Exploiting the Yeast Genome

描述（由申请人提供）：许多人类外显子组和基因组的DNA序列数据正在以不断增长的速度积累，但对小遗传变异对蛋白质功能的影响的理解落后。我们已经开发出一种方法，使用下一代DNA测序和将基因型与表型联系起来的蛋白质展示格式来分析多达100万种蛋白质变体的功能。迄今为止，我们的工作使用了小蛋白结构域（通常<100个氨基酸）和酵母或体外功能测定。然而，技术的进步表明，我们应该能够将我们的方法扩展到更大的蛋白质和更复杂的测定，特别是人类细胞中的测定。我们的总体目标是开发一种技术，在人类细胞中快速评估具有多种活性和相互作用的大型人类蛋白质的所有变体的功能。这项技术将使用BRCA 1蛋白质进行原型设计--其中种系突变会导致乳腺癌和卵巢癌的风险大大增加--然后扩展到与癌症风险有关的其他蛋白质。我们将比较我们的检测结果与已知变异的疾病风险和进展数据，以确定我们的高通量方法的实用性。我们的Driving Biomedical Project将在人类细胞中采用DNA修复试验来分析BRCA 1变体的活性。我们的具体目标是：1）在BRCA 1蛋白中产生所有可能的单个氨基酸变化，并评估变体在DNA修复中的能力（使用全蛋白）和E3连接酶活性（使用304个氨基酸的结构域）; 2）将在我们的测定中获得的BRCA 1变体的定量适合度与疾病等位基因的数据库进行比较; 3）将该方法扩展到与癌症相关的其他人类基因，并适用于类似的测定，如BRCA 2，BARD 1和CHEK 2。

DESCRIPTION (provided by applicant): DNA sequence data for many human exomes and genomes are accumulating at an ever-increasing pace, yet an understanding of the effect of small genetic variations on protein function lags behind. We have developed a method to analyze the function of up to ~1 million variants of a protein using next generation DNA sequencing and protein display formats that link genotype to phenotype. Our work to date has used small protein domains (typically <100 amino acids) and functional assays in yeast or in vitro. However, advances in technology suggest that we should be able to extend our approach to much larger proteins and more complex assays, in particular assays in human cells. Our overall goal is to develop the technology to rapidly assess the function, in human cells, of all th variants of a large human protein that has multiple activities and interactions. This technology will be prototyped using the BRCAl protein - in which germ-line mutations result in a vastly increased risk of breast and ovarian cancer-and then extended to other proteins implicated in cancer risk. We will compare the results from our assays to the data on disease risk and progression for known variants in order to establish the utility of our high throughput approach. Our Driving Biomedical Project will employ a DNA repair assay in human cells to analyze the activity of BRCAl variants. Our specific aims are: 1) To generate all possible single amino acid changes in the BRCAl protein and to assess the variants for their proficiency in DNA repair (using the whole protein) and in E3 ligase activity (using a 304 amino acid domain); 2) To compare the quantitative fitness of the BRCA1 variants obtained in our assays with a database of disease alleles; 3) To extend the approach to other human genes relevant to cancer and amenable to similar assays, such as BRCA2, BARD1 and CHEK2.