Comprhensive Biology: Exploiting the Yeast Genome

描述(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，但对微小遗传变异对蛋白质功能的影响的了解滞后。我们开发了一种方法，使用下一代DNA测序和将基因型与表型联系起来的蛋白质显示格式，分析多达100万种蛋白质变体的功能。到目前为止，我们的工作使用了小的蛋白质结构域(通常是100个氨基酸)和酵母或体外的功能分析。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分析，特别是在人类细胞中的分析。我们的总体目标是开发这项技术，以快速评估具有多种活动和相互作用的大型人类蛋白质的所有变体在人类细胞中的功能。这项技术将使用BRCA1蛋白来制作原型--在这种蛋白中，胚系突变会导致乳腺癌和卵巢癌风险的极大增加--然后扩展到其他与癌症风险有关的蛋白。我们将把我们的化验结果与已知变异的疾病风险和进展数据进行比较，以确定我们的高通量方法的实用性。我们的驱动生物医学项目将使用人类细胞中的DNA修复试验来分析BRCA1变体的活性。我们的具体目标是：1)产生BRCA1蛋白中所有可能的单一氨基酸变化，并评估它们在DNA修复(使用整个蛋白质)和E3连接酶活性(使用304个氨基酸结构域)方面的熟练程度；2)比较在我们的检测中获得的BRCA1变体与疾病等位基因数据库的定量适合性；3)将方法扩展到其他与癌症相关的人类基因，并适用于类似的分析，如BRCA2、BARD1和CHEK2。

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.