Comprhensive Biology: Exploiting the Yeast Genome

综合生物学：利用酵母基因组

• 批准号: 8416531
• 负责人: Trisha N. Davis
• 金额: $ 35.41万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-10 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8416531
• 关键词: Affect; Alleles; Amino Acids; Automobile Driving; BARD1 gene; BRCA1 gene; BRCA2 gene; Biological Assay; Biology; CHEK2 gene; Cancer-Predisposing Gene; Cells; Complex; DNA Repair; DNA Sequence; Data; Databases; Disease; Disease Progression; Fungal Genome; Genes; Genetic Variation; Genome; Genotype; Germ-Line Mutation; Goals; Human; In Vitro; Individual; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Methodology; Methods; Phenotype; Proteins; Risk; Technology; Tertiary Protein Structure; Variant; Work; Yeasts; cancer risk; disorder risk; exome; fitness; malignant breast neoplasm; mutant; next generation; protein function; public health relevance; ubiquitin-protein ligase

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.

描述（由申请人提供）：许多人类外部和基因组的DNA序列数据正在以不断增长的速度积累，但了解小遗传变异对蛋白质功能的影响落后。我们已经开发了一种方法，可以使用下一代DNA测序和蛋白质显示格式分析蛋白质多达100万种变体的功能，这些格式将基因型与表型联系起来。迄今为止，我们的工作使用了酵母或体外的小蛋白质结构域（通常<100氨基酸）和功能测定。但是，技术的进步表明，我们应该能够将方法扩展到更大的蛋白质和更复杂的测定法，特别是在人类细胞中的测定法。我们的总体目标是开发该技术，以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第三种变体的功能。该技术将使用BRCAL蛋白进行原型 - 其中种系突变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。我们将将我们的测定结果与已知变体的疾病风险和进展数据进行比较，以确定我们高通量方法的实用性。我们的驱动生物医学项目将在人类细胞中采用DNA修复测定法，以分析BRCAL变体的活性。我们的具体目的是：1）在BRCAL蛋白中产生所有可能的单氨基酸变化，并评估其熟练程度（使用整个蛋白质）和E3连接酶活性（使用304氨基酸结构域）的变体； 2）比较在我们的测定中获得的BRCA1变体的定量适应性与疾病等位基因数据库相比； 3）将方法扩展到与癌症相关的其他人类基因，并适合类似测定法，例如BRCA2，BARD1和CHEK2。