Sequence Specific non-LTR Retrotransposable Elements

序列特异性非 LTR 逆转录转座元件

• 批准号: 9974606
• 负责人: Thomas Eickbush
• 金额: $ 45.6万
• 依托单位: University of Rochester
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9974606&HistoricalAwards=false
• 关键词: Sequence; Specific; non; LTR; Retrotransposable

R1 and R2 are two site-specific non-LTR retrotransposable elements that insert in the 28S ribosomal RNA genes of arthropods. Recent experiments have indicated that despite their remarkable evolutionary stability, R1 and R2 rapidly turnover in a population (i.e new elements are inserted and old elements eliminated from the rDNA loci at a high rate). Thus a major focus of the current proposal is experiments designed to determine the parameters that control the level of R1 and R2 within the rDNA loci of Drosophila. Taking advantage of the diverse array of 5' truncations that frequently result from non-LTR retrotransposition, preliminary experiments have shown that individual R2 insertions and deletions can be scored in the Harwich mutation collection lines of D. melanogaster. The Harwich lines have been maintained as 31 small, separate populations for over 250 generations. The hundreds of insertion/deletion events that can be monitored by simply PCR assays of these 31 lines should enable separate measurements of the frequency of R1 and R2 events on the rDNA loci of the X and Y chromosomes, and determine whether they occur in bursts or at low constitutive rates. Initial attempts will also be made to determine why certain species groups of Drosophila have retained two distinct lineages of R1 elements, while other species groups have retained only one R1 lineage. R1 and R2 turnover and rDNA unit uniformity in two species with multiple R1s will be analyzed to test the hypothesis that lower levels of concerted evolution enable the preservation of multiple families. Larval tissues of Drosophila have reduced levels of R1 and R2 insertions (relative to the adult levels) because rDNA units with these insertions are under-replicated during the formation of the polyploid (usually polytene) tissues of larvae. A second series of experiments is designed to directly study the mechanism responsible for this under-replication. The molecular/genetic tools are now available to mark individual rDNA units or to insert new sequences into these units. Because the retrotransposition and evolution of R1 and R2 are so intimately tied to that of the rRNA genes, these studies should also provide insights, as well as supply new tools, for the study of the evolution, replication and expression of the rRNA genes themselves. Finally, all non-LTR retrotransposons can be divided into a limited number of distinct, ancient lineages. The oldest elements are site-specific and encode proteins similar to R2. A third set of experiments in this proposal is designed to identify additional examples of these original site-specific non-LTR elements, particularly in primitive eukaryotic lineages like Giardia. Information obtained from these studies will provide insights into the origins of the first eukaryotic genomes, and help to evaluate the relationship of non-LTR retrotransposable elements to their closest relatives: telomerase and group II introns. Transposable elements are abundant components of eukaryotic genomes. Numerous examples now exist to indicate that these elements have played a significant role in determining the size, structure and regulation of these genomes. A growing number of transposable elements, particularly the retrotransposable elements, have been shown to minimize, or control, their effects on the host by becoming specific for defined locations within the genome. R1 and R2 are two site-specific non-LTR retrotransposable elements that insert in the 28S ribosomal RNA genes of arthropods. The specificity and sequence uniformity of these elements has enabled them to serve as a convenient model system. Considerable progress has been made in studies of their distribution, evolution and retrotransposition mechanism, such that R1 and R2 are now among the best characterized retrotransposable elements. Extensive studies on the evolution of these elements suggest that R1 and R2 have been stable, active components of the rDNA loci for the entire history of the Arthropod phylum, a period estimated to be over 600 million years. In this proposal, a varied set of experiments are described with the objective of better understanding the remarkable stability and the origins of these elements.

R1和R2是插入节肢动物28 S核糖体RNA基因的两个位点特异性非LTR反转录转座元件。 最近的实验表明，尽管R1和R2具有显着的进化稳定性，但它们在种群中的周转速度很快（即新元件插入，旧元件以高速率从rDNA基因座中消除）。 因此，目前的建议的一个主要重点是实验设计，以确定控制果蝇rDNA基因座内的R1和R2的水平的参数。 利用经常由非LTR反转录转座产生的多种多样的5'截短，初步实验已经表明，可以在D.黑腹菌 Harwich品系作为31个独立的小种群已经维持了250多代。 通过对这31个细胞系进行简单的PCR分析可以监测数百个插入/缺失事件，这应该能够单独测量X和Y染色体的rDNA基因座上R1和R2事件的频率，并确定它们是以爆发形式发生还是以低组成率发生。 最初的尝试也将确定为什么果蝇的某些物种组保留了两个不同的谱系的R1元件，而其他物种组只保留了一个R1谱系。 R1和R2的营业额和rDNA单位的均匀性在两个物种与多个R1的分析，以测试的假设，即较低水平的协同进化，使多个家庭的保存。 果蝇的幼虫组织中R1和R2插入的水平降低（相对于成虫水平），因为在幼虫的多倍体（通常为多线）组织形成过程中，具有这些插入的rDNA单位复制不足。 第二个系列的实验旨在直接研究这种复制不足的机制。 分子/遗传工具现在可用于标记单个rDNA单元或将新序列插入这些单元。 由于R1和R2的逆转录转座和进化与rRNA基因的进化密切相关，这些研究也应该为研究rRNA基因本身的进化、复制和表达提供新的工具。 最后，所有非LTR反转录转座子可以分为有限数量的不同的，古老的谱系。 最古老的元件是位点特异性的，编码类似于R2的蛋白质。 第三组实验旨在确定这些原始位点特异性非LTR元件的其他例子，特别是在原始真核细胞谱系中，如贾第虫。 从这些研究中获得的信息将提供深入了解第一个真核生物基因组的起源，并有助于评估非LTR逆转录转座因子与其最近的亲属：端粒酶和II组内含子的关系。 转座因子是真核生物基因组的重要组成部分。 现在有许多例子表明，这些元件在决定这些基因组的大小、结构和调控方面发挥了重要作用。 越来越多的转座因子，特别是反转录转座因子，已被证明可以通过对基因组内特定位置的特异性来最小化或控制它们对宿主的影响。 R1和R2是插入节肢动物28 S核糖体RNA基因的两个位点特异性非LTR反转录转座元件。 这些元素的特异性和序列一致性使它们能够作为一个方便的模型系统。 对它们的分布、进化和反转录转座机制的研究取得了很大进展，R1和R2是目前最具代表性的反转录转座因子之一。 对这些元素进化的广泛研究表明，R1和R2在节肢动物门的整个历史中一直是rDNA基因座的稳定、活性成分，估计这一时期超过6亿年。 在这一建议中，一组不同的实验进行了描述，目的是更好地了解显着的稳定性和这些元素的起源。