Mammalian Transposons

导言-哺乳动物的L1元件(长时间散布重复的DNA元件，也称为LINE-1)通过将其RNA转录本复制到DNA中进行复制(反转座)，然后DNA整合到基因组中。~6kb的人类L1元件有4个区域：5个核苷酸非翻译调控区；开放阅读框架(ORF)1编码一个RNA结合蛋白；ORF2编码一个内切酶和逆转录酶；3个核苷酸非翻译序列包含一个保守的富含G的多嘌呤基序。L1元件通过利用被切割的靶DNA的3个启动子OH来启动其转录物的cDNA合成。L1也可以通过这个过程复制其他元件(例如，正弦，如Alu家族)。由于L1活性自哺乳动物出现以来一直存在，并且L1复制的产物大部分被保留，难怪至少30%(860Mb)的人类基因组工作草稿(WDHG)是由L1活动产生的：460Mb是L1DNA，360Mb是Sine DNA。除了它们的绝对质量外，L1分子及其子代可以引起基因重排，使基因失活或改变活性。我们使用了分子生物学、进化论和群体遗传学的工具来研究L1与其宿主之间的相互作用，以及L1的活动如何影响现代人。 最近的发现：对人类正在进行的L1扩增事件的分析-我们早期的研究表明，人类Ta L1家族(L1PA1)，由其他人显示的子宫插入突变的原因，出现在~5Myr之前，随后分化为两个主要的亚家族，TA0和TA1。之前没有描述过的TA1比TA0年轻，现在至少占Ta家族的50%。由于TA1家族是最近才出现的，我们推测人类基因组测序工作将在人类群体中错过大量的TA1插入。这种插入片段可能具有重要的医学意义，可以作为强大的遗传标记，既可以绘制与医学相关的特征图，也可以检查人类群体的历史和结构。同样重要的是，在L1扩增事件被宿主反应(例如选择)重塑或因元素的积累而变得模糊之前，TA1插入片段的全面收集将提供L1扩增事件的初始阶段的实时视图。我们设计了一种公正的方法来收集四个种族的TA1插入片段：非洲侏儒、高加索德鲁人、中国人和美拉尼西亚人。尽管我们的大部分分析仍在进行中，但我们的初步结果是惊人的：首先，我们恢复了先前估计的~300个TA1插入片段中的277个。其次，正如预测的那样，这些插入片段中整整40%没有出现在目前的人类基因组工作草案(WDHG)中。第三，近30%的TA1插入发生在31个离散的基因组区域(热点)。这种聚集在统计上具有重要意义，并且不会通过依赖WDHG的L1组成来识别。对这些热点和含有30个非簇状TA1插入片段的基因座的生物信息学分析尚未揭示这些热点的原因。然而，在61个被检查的基因组座位中，有一半包含在睾丸或早期胚胎发育中高表达的基因。在我们分析的这一点上，很容易推测成功的L1扩增的第一阶段可能涉及到对这些基因组区域的入侵。 L1的适应性进化-我们早些时候发现，L1 ORF 1的螺旋线圈基序在古人类进化的早期经历了几次适应性进化，在非洲类人猿(人类、黑猩猩、大猩猩)的L1进化过程中停止了这一过程。由于卷曲结构域经常介导蛋白质-蛋白质相互作用，我们推测该基序的进化变化可以反映L1与宿主因子的相互作用。我们现在已经证明了这个想法是有价值的。基于细胞培养的逆转录转座分析表明，祖先的螺旋线圈结构域在其他完全现代的元件的背景下是活跃的。因此，自适应修改不是对元素中其他位置的修改的响应，而是可能反映对主体中的修改的响应。我们正在使用细胞质酵母双杂交试验来检测这种可能的L1-宿主相互作用。到目前为止，我们已经证明ORF1蛋白(包括那些包含祖先螺旋线圈基序的蛋白)具有很强的自我作用。非洲类人猿谱系适应性进化的停止表明，在非洲和亚洲(猩猩)类人猿分化后，L1的宿主环境可能发生了巨大变化。确定这样的变化是否也发生在亚洲血统中，应该有助于阐明L1与其宿主之间相互作用的性质。

INTRODUCTION - Mammalian L1 elements (long interspersed repeated DNA elements, also called LINE-1) replicate (retrotranspose) by copying their RNA transcripts into DNA which are then integrated into the genome. The ~6 kb human L1 element has four regions: a 5 prime untranslated (UTR) regulatory region; open reading frame (ORF) 1 encodes an RNA binding protein; ORF2 encodes an endonuclease and reverse transcriptase; the 3 prime UTR contains a conserved G-rich polypurine motif. L1 elements replicate by using the 3 prime OH of the nicked target DNA to prime cDNA synthesis of its transcript. L1 can also replicate other elements (e.g., SINEs such as the Alu family) by this process. As L1 activity has persisted in mammals since their emergence and the products of L1 replication are largely retained, it is not surprising that at least 30% (860 Mb) of the working draft of the human genome (WDHG) was generated by L1 activity: 460 Mb is L1 DNA and 360 Mb is SINE DNA. In addition to their sheer mass, L1 elements, and their SINE offspring, can cause genetic rearrangements and inactivate or alter gene activity. We have used the tools of molecular biology and evolution and population genetics to examine the interaction between L1 and its host and how L1 activity might affect modern humans. RECENT FINDINGS: ANALYSIS OF AN ONGOING L1 AMPLIFICATION EVENT IN HUMANS - Our earlier studies showed that the human Ta L1 family (L1PA1), shown by others to causes in utero insertional mutations, arose ~5 Myr ago and subsequently differentiated into two major subfamilies, Ta0 and Ta1. Ta1, which had not been described earlier, is younger than Ta0 and now accounts for at least 50 % of the Ta family. As the Ta1 family only recently emerged, we reasoned that the human genome sequencing effort would miss a significant number of the Ta1 inserts in the human population. Such inserts could have medical importance as robust genetic markers both for mapping medically relevant traits and for examining the history and structure of human populations. As important, a comprehensive collection of Ta1 inserts will provide a real time view of the initial stages of an L1 amplification event, before it has been remodeled by host responses (e.g., selection) or blurred by the accumulation of elements. We devised an unbiased method to collect Ta1 inserts from four ethnic groups: African pygmy, Caucasian Druze, Chinese, Melanesian. Although most of our analysis is still ongoing, our preliminary results are striking: First, we recovered 277 of the ~300 Ta1 inserts estimated earlier. Second, as predicted, a full 40% of these inserts are not present in the current working draft of the human genome (WDHG). Third, almost 30% of the Ta1 inserts occurred in 31 discrete genomic regions (hotspots). This clustering is statistically significant and would not have been recognized by reliance on the L1 composition of the WDHG. Bioinformatic analysis of these hotspots and the loci harboring 30 non-clustered Ta1 inserts has yet to reveal an explanation for the hot spots. However, half of the 61 examined genomic loci contain genes highly expressed in testis or early embryogenesis. At this point in our analysis it is tempting to speculate that the first stage of a successful L1 amplification may involve invasion of these genomic regions. ADAPTIVE EVOLUTION IN L1 - We had earlier found that the coiled-coil motif of L1 ORF 1 had undergone episodes of adaptive evolution early in hominid evolution and that this ceased during the evolution of L1 in the African apes (human, chimpanzee, gorilla). As coiled-coil domains often mediate protein-protein interaction we reasoned that evolutionary change in this motif could reflect interaction of L1 with host factors. We have now shown that this idea has merit. A cell culture based retrotransposition assay revealed that the ancestral coiled-coil domain is active in the context of an otherwise fully modern element. Thus, the adaptive changes are not a response to changes elsewhere in the element but may reflect a response to changes in the host. We are using the cytoplasmic yeast two hybrid assay to examine such possible L1-host interactions. So far we have shown that ORF1 proteins (including those containing an ancestral coiled-coil motif) strongly self interact. The cessation of adaptive evolution in the African ape lineage suggests that the host environment for L1 may have changed dramatically after African and Asian (orangutan) apes diverged. Determining whether such a change also occurred in the Asian lineage should help illuminate the nature of the interaction between L1 and its host.