Human variation in retrotransposon activity

描述（由申请方提供）：本提案的总体假设是，人类亚群正在获得突变负荷，因此可能以不同的速率进化。为了验证这一假设，人们需要研究具有全基因组效应的过程，而不是那些只改变单个基因表达的过程。这种通用的全基因组因子是L1反转录转座子。在这个提议中，我们确定了人类群体中L1反转录转座能力的变化，作为全基因组进化驱动的潜在替代标记。长散布核元件-1（LINE-1或L1）是一种丰富的反转录转座子，占人类DNA的约17%。绝大多数L1都是化石遗迹，不能再移动（即，逆转录转座），因为失活突变。我们已经确定，由人类基因组工作草案（HGWD）表示的个体人类基因组包含大约80-100个活性L1反转录转座子，但仅约6个占观察到的反转录转座活性的84%。另一方面，我们发现“私人”或基因组特异性L1并不罕见，这使我们假设在整个人类基因库中有非常大量的，也许是数百万的高活性L1，并且人类个体之间L1活性的变化是巨大的。这项资助代表了人类和哺乳动物反转录转座子领域两个经验丰富的小组工作的逻辑延伸，以回答人类生物学中的一个重要问题。这些小组带来了重要的技术专长领域，即1）在变性聚丙烯酰胺凝胶中显示任何个体的人类特异性L1的技术，2）组织培养中任何L1的相对反转录转座频率的高通量快速测定，以及3）确定不同L1多态性存在/不存在的高通量方法。使用这些方法，我们将通过确定各种L1的存在/不存在和他们的反转录转座能力的等位基因变异来测量个体之间L1反转录转座能力的总变异。我们还将确定在体内转录的组织培养试验中有活性的L1的分数。利用这些数据，我们将确定不同个体和地理群体之间存在的L1反转录转座能力的变化程度。

DESCRIPTION (provided by applicant): The overarching hypothesis of this proposal is that human subpopulations are acquiring mutational loads and thereby potentially are evolving at different rates. To test this hypothesis, one needs to study processes that have genome wide effects as opposed to those that only alter the expression of a single gene. Such a general, genomewide agent is the L1 retrotransposon. In this proposal, we determine the variation in L1 retrotransposition capability in the human population as a potential surrogate marker for genome-wide, evolutionary drive. Long Interspersed Nuclear Element-1 (LINE-1 or L1) is an abundant retrotransposon that comprises ~17% of human DNA. The vast majority of L1s are fossil relics and can no longer move (i.e., retrotranspose) because of inactivating mutations. We have determined that an individual human genome as represented by the human genome working draft (HGWD) contains roughly 80-100 active L1 retrotransposons, but that only about 6 account for 84% of the observed retrotransposition activity. On the other hand, we have found that "private" or genome-specific L1s are not uncommon, leading us to hypothesize that there are a very large number, perhaps millions, of highly active L1s in the total human gene pool and that the variation in L1 activity among individual human beings is substantial. This grant represents a logical extension of the work of two experienced groups in the field of human and mammalian retrotransposons in an effort to answer an important question in human biology. The groups bring important areas of technical expertise, namely, 1) a technique to display the human-specific L1s of any individual in a denaturing polyacrylamide gel, 2) a high throughput, rapid assay of the relative retrotransposition frequency of any L1 in tissue culture, and 3) high throughput methods to determine presence/absence polymorphisms of different L1s. Using these methods, we will measure the total variation in L1 retrotransposition capability among individuals by determining both presence/absence of various L1s and allelic variation in their retrotransposition capability. We will also determine the fraction of L1s active in the tissue culture assay that are transcribed in vivo. Using this data, we will determine the extent of variation in L1 retrotransposition capability that exists among different individuals and geographic groups.