Reducing genetic buffering to identify QTLs that affect cell shape and growth

描述(由申请人提供)：遗传缓冲是理解疾病分子基础的主要障碍。当一个基因的突变的影响被另一个基因的表达所掩盖时，缓冲就发生了。因此，导致疾病的遗传变异更难识别，因为在某些情况下，它们的表型是看不见的 遗传背景。为了克服这一障碍，我们将测试伴侣抑制是否减少了遗传缓冲，从而增加了定位研究的能力，以检测影响细胞形态和生长的数量性状基因座(QTL)。我们的研究旨在(1)确定影响这些疾病相关性状的QTL，(2)引入一种有助于发现疾病潜在基因的技术。我们将使用酿酒酵母进行QTL筛选，酿酒酵母是一种广泛应用于研究人类疾病分子基础的模式生物。我们确定的QTL可能包含与人类同源的酵母基因，因为基本过程，如细胞生长，在酵母和人类之间是保守的。我们将通过抑制热休克蛋白(HSPs)来减少这个作图家族的遗传缓冲，热休克蛋白是一种蛋白质折叠伴侣蛋白。热休克蛋白可以帮助突变蛋白正常折叠和功能，从而掩盖与这些突变相关的功能表型的丧失。我们将通过用格尔达霉素(GDA)处理细胞来限制HSP的功能，GDA是一种特定热休克蛋白HSP90的小分子抑制剂。对果蝇、植物和酵母的研究表明，HSP90的抑制揭示了以前被掩盖的表型变异，这种变异在遗传上不同的菌株之间有所不同。不足为奇的是，许多不同的菌株都含有对HSP90敏感的表型突变。首先，许多基因产物与HSP90相互作用。其次，大多数氨基酸替换破坏了蛋白质折叠的稳定性，产生了依赖HSP90实现功能构象的蛋白质。我们假设，在我们的QTL筛选中阻碍HSP功能将揭示一些不稳定突变的表型效应，揭示它们功能表型的丢失，并能够检测到额外的QTL。我们将通过在有和没有伴侣抑制的情况下进行相同的QTL筛选并比较结果来检验这一假设。如果成功，我们的技术可以用来提高在其他生物中的QTL检测，因为热休克蛋白在进化上是保守的。我们实现了几个额外的功能来增强对细胞形态和生长的QTL的检测。我们研究野生酵母菌株，以探索实验室菌株研究中尚未调查到的自然遗传变异。我们还使用高通量显微镜平台，允许跟踪每个细胞的220个形态表型，并测量每个单日实验约50,000个微集落的生长曲线。这种方法可以区分生长和形态上的微小表型差异，并可能发现以前无法检测到的QTL。识别影响细胞形态和生长的QTL是了解疾病，特别是癌症的遗传基础的重要目标。

DESCRIPTION (provided by applicant): Genetic buffering represents a major obstacle in understanding the molecular basis of disease. Buffering occurs when the effects of mutations in one gene are masked by expression of another gene. As a result, the genetic variants that contribute to disease are more difficult to identify because their phenotypes are invisible in some genetic backgrounds. To contend with this obstacle, we will test whether chaperone inhibition reduces genetic buffering thereby increasing the power of mapping studies to detect quantitative trait loci (QTL) contributing to cell morphology and growth. Our study stands to (1) identify QTLs contributing to these disease-relevant traits and (2) introduce a technique that will facilitate discovery of genes underlying disease. We will perform QTL screening using Saccharomyces cerevisiae, a widely used model organism for researching the molecular basis of human disease. The QTLs we identify are likely to contain yeast genes with human homologs because basic processes, like cell growth, are conserved between yeast and humans. We will reduce genetic buffering in this mapping family through inhibition of heat shock proteins (HSPs), a type of protein-folding chaperone. HSPs can help mutant proteins to fold and function normally, thereby masking loss of function phenotypes associated with these mutants. We will limit HSP function by treating cells with geldanamycin (GdA), a small-molecule inhibitor of a specific heat shock protein, HSP90. Studies in flies, plants, and yeast have shown that HSP90 inhibition reveals previously masked phenotypic variation that varies between genetically distinct strains. It is not surprising that many different strains harbor mutations with phenotypes that are HSP90 sensitive. Firstly, many gene products interact with HSP90. Secondly, the majority of amino acid substitutions destabilize protein folding, creating proteins that depend on HSP90 to achieve a functional conformation. We hypothesize that hindering HSP function in our QTL screen will reveal the phenotypic effects of some destabilizing mutations, uncovering their loss of function phenotypes and enabling detection of additional QTLs. We will test this hypothesis by performing identical QTL screens with and without chaperone inhibition and comparing the results. If successful, our technique can be utilized to enhance QTL detection in other organisms because HSPs are evolutionarily conserved. We implement several additional features to enhance detection of QTLs for cell morphology and growth. We study wild yeast strains in order to explore natural genetic variation that has not already been surveyed in studies of laboratory strains. We also use a high throughput microscopy platform that allows tracking of 220 morphological phenotypes per cell and measures growth curves for ~50,000 microcolonies per single-day experiment. This method can distinguish tiny phenotypic differences in growth and morphology, and may allow discovery of previously undetectable QTLs. Identifying QTLs contributing to cell morphology and growth is an important goal in understanding the genetic basis of diseases, cancers in particular.