Human transposable element

人类转座因子

基本信息

批准号：
8138944
负责人：
HAIG H. KAZAZIAN
金额：
$ 19.44万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-24 至 2011-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): LINE-1, or L1, elements, are the most prolific and prominent mobile elements in mammalian genomes. In the human and mouse genomes, active L1s and their relics account for 17-20% of the genome mass. By providing activities necessary for mobility of Alu and other elements, they indirectly account for another 10- 12% of these genomes. L1 retrotransposons move through a duplicative "copy and paste" mechanism. In this renewal proposal, we attack a number of unanswered questions concerning the timing and cell type specificity of retrotransposition, the mechanism of L1 insertion into the genome, and the role of siRNA produced by the bidirectional L1 promoter in the control of retrotransposition. In Specific Aim 1, we seek to determine the fraction of total retrotransposition events that occur in male germ cells, female germ cells, and in the early stages of embryonic development. We also wish to determine whether retrotransposition decreases with age, is suppressed after a number of generations, and is increased by defects in methylation or DMA double strand break repair. In Specific Aim 2 we develop further evidence of the role of siRNA derived from the L1 bidirectional promoter. We also characterize the small RNA sequences derived from L1 RNA, and determine whether specific knockdown of L1 transcript levels alters the phenotype of transformed cells. In Specific Aim 3, we will test a novel mechanism of L1 integration. Postulated features of this mechanism are 1) 5' truncation is due to 5' degradation of L1 RNA, 2) template jumping of reverse transcriptase is a key feature of the mechanism, and 3) synthesis of DMA second strand is carried out by a DNA polymerase activity of L1 reverse transcriptase. In Specific Aim 3A we determine which RNA polymerase transcribes L1 and how the RNA is processed (capped and polyadenylated). In Specific Aim 3B we determine the processes involved in L1 RNA degradation, in particular, whether deadenylation, decapping and 5' degradation are involved and whether a significant fraction of L1 RNA is rapidly 5' degraded. These studies will increase greatly our knowledge of the biology of L1 retrotransposition. In addition, they will provide important new information towards the effort to use L1 as a random insertional mutagen in mammals.

描述（由申请人提供）：LINE-1或L1元素是哺乳动物基因组中最多产，最突出的移动元素。在人和小鼠基因组中，活性L1及其遗物占基因组质量的17-20％。通过提供Alu和其他因素的活动所必需的活动，它们间接占这些基因组的10-12％。 L1逆转座子通过重复的“复制和粘贴”机制移动。在此更新建议中，我们攻击了许多有关逆转录定位的时间和细胞类型特异性的未解决问题，L1插入到基因组中的机制以及双向L1启动子在控制逆转序列的控制中产生的siRNA的作用。在特定目标1中，我们试图确定在男性生殖细胞，女生殖细胞以及胚胎发育初期发生的总逆转录事件的比例。我们还希望确定逆转录置位是否随着年龄的增长而降低，几代后会被抑制，并因甲基化或DMA双链破裂修复的缺陷而增加。在特定目标2中，我们进一步证明了siRNA源自L1双向启动子的作用。我们还表征了从L1 RNA得出的小RNA序列，并确定L1转录水平的特定敲低是否会改变转化细胞的表型。在特定目标3中，我们将测试L1整合的新机制。该机制的假设特征是1）5'截断是由于L1 RNA的5'降解，2）逆转录酶的模板跳跃是该机制的关键特征，而3）DMA第二链的合成是由L1逆转录酶的DNA聚合酶活性进行的。在特定的目标3a中，我们确定哪种RNA聚合酶转录L1以及如何处理RNA（限制和聚腺苷酸化）。在特定的目标3B中，我们确定L1 RNA降解所涉及的过程，特别是涉及deadenylation，Decapping和5'降解，以及L1 RNA的大部分是否迅速降解5'。这些研究将大大提高我们对L1逆转录的生物学的了解。此外，他们将提供重要的新信息，以将L1用作哺乳动物中的随机插入诱变。