Human variation in retrotransposon activity

描述(由申请人提供)：这一建议的首要假设是，人类亚群正在获得突变负载，因此可能以不同的速度进化。为了验证这一假设，人们需要研究具有全基因组影响的过程，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就是L1反转录转座子。在这项建议中，我们确定了人类群体中L1逆转座子能力的变异，作为全基因组进化驱动的潜在替代标记。长散布核素-1(LINE-1或L1)是一种丰富的反转录转座子，约占人类DNA的17%。绝大多数L1是化石遗迹，由于失活突变，不能再移动(即逆转座)。我们已经确定，以人类基因组工作草案(HGWD)为代表的单个人类基因组包含大约80-100个活性的L1逆转座子，但只有6个左右占观察到的逆转座子活性的84%。另一方面，我们发现“私有的”或特定于基因组的L1并不少见，这让我们假设，在人类总基因库中，有非常大量的、可能是数百万的高度活跃的L1，而且人类个体之间的L1活性差异很大。这笔赠款代表着两个有经验的小组在人类和哺乳动物反转录转座子领域工作的合乎逻辑的延伸，以努力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有L1的技术，2)高通量、快速分析组织培养中任何L1的相对逆转录转座频率，以及3)确定不同L1是否存在多态的高通量方法。使用这些方法，我们将通过确定各种L1是否存在及其逆转座子能力的等位基因变异来测量个体之间L1逆转座子能力的总变异。我们还将确定在组织培养试验中活跃的在体内转录的L1的比例。利用这些数据，我们将确定L1逆转座能力在不同个体和地理群体之间存在的差异程度。

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.