AN EVOLUTIONARY LINK BETWEEN TELOMERES AND TRANSPOSONS

描述(申请人提供)：端粒分子生物学比最初认为的要复杂得多；对它的理解有赖于对进化变异的研究，而果蝇端粒是显著的变异：果蝇缺乏端粒酶。相反，果蝇端粒是两个非LTR反转录转座子HET-A和START的长串联阵列，而不是几乎所有其他生物中端粒酶产生的简单重复序列的阵列。HET-A和START是最早发现的在细胞结构中具有真正作用的转座元件；因此，它们为更好地理解端粒和非LTR反转录转座子提供了机会，反转录元件是逆转录元件的一大类。 在发现D.virilis具有转座子端粒后，我们的主要目标之一是利用该物种与D.Blackogaster之间约60My的差异，通过序列分析和跨物种细胞生物相互作用分析，寻找其宿主细胞内转座子结构和相互作用的保守特征。另一个主要目标来自我们的D.Blackogaster研究，该研究提供了第一个关于非LTR反转录转座子的Gag蛋白在细胞内定位的信息，并确定了可能参与HET-A和TARTGgs在端粒和运输中定位的蛋白质。我们将使用分子研究来确定这些蛋白质与GAG和/或端粒DNA的参与。我们还将使用分子技术来研究那些通过遗传分析检测到端粒关联的蛋白质。 因此，我们现在建议(1)比较来自远亲物种的端粒转座子的序列和细胞生物学，以加深对转座子端粒进化的了解；(2)通过使用RNAi特异性地耗尽转录本来了解端粒转座子的发育调控转录的意义，从而确定哪些细胞类型显示出表型效应，然后研究这些影响；(3)分析以端粒为靶点的反转录转座子的细胞生物学，利用遗传学和生化技术来确定参与决定反转录转座子从细胞浆到其染色体末端靶的路径的蛋白质的分子相互作用，以及(4)将端粒转座子阵列形成的染色质与其他端粒中发现的染色质进行比较。

DESCRIPTION (provided by applicant): Telomere molecular biology is far more complex than originally thought; understanding it is aided by study of evolutionary variants, and Drosophila telomeres are remarkable variants: Drosophila lack telomerase. Instead, Drosophila telomeres are long tandem arrays of two non-LTR retrotransposons, HeT-A and TART, not the arrays of simple repeats generated by telomerase in almost all other organisms. HeT-A and TART are the first transposable elements found with a bona fide role in cell structure; thus, they offer opportunity for better understanding of both telomeres and non-LTR retrotransposons, a major class of retroelements. Having discovered that D.virilis has transposon telomeres, one of our major goals is to use the approximately 60 MY divergence between this species and D. melanogaster to find conserved features in transposon structure and interactions within their host cells by sequence analysis and by cross-species cell biological interaction assays. Another major goal derives from our D. melanogaster studies that provided the first information on the intracellular Iocalizations of Gag proteins from non-LTR retrotransposons and have identified proteins that may be involved in the localization of HeT-A and TARTGags, both at the telomere and in transit. We will use molecular studies to determine the involvement of these proteins with Gag and/or telomere DNA. We wilt also use molecular techniques to study proteins whose telomeric association was detected by genetic analysis. Thus, we now propose (1) to compare the sequences and cell biology of telomere transposons from distantly related species to deepen insight into the evolution of transposon telomeres, (2) to understand the significance of the developmentally regulated transcription of telomere transposons by using RNAi to specifically deplete transcripts and thereby determine which cell types show phenotypic effects and then to study those effects, (3) to analyze the cell biology of retrotransposon targeting to telomeres, using genetic and biochemical techniques to determine the molecular interactions of proteins involved in determining the path of the retrotransposon from the cytoplasm to its target at the end of the chromosome, and (4) to compare the chromatin formed by telomeric transposon arrays with that found in other telomeres.