Sequence Specific non-LTR Retrotransposable Elements

R1和R2是插入节肢动物28S核糖体RNA基因的两个位点特异性的非LTR逆转座子元件。最近的实验表明，尽管R1和R2具有显著的进化稳定性，但它们在种群中的更替速度很快(即在rDNA基因座上插入新的元件和消除旧元件的速度很快)。因此，当前提案的一个主要焦点是设计实验，以确定控制果蝇rDNA基因座内R1和R2水平的参数。利用非LTR逆转录转座经常导致的5‘端截断的多样性阵列，初步实验表明，在黑腹盘藻的Harwich突变收集系中，可以对单个R2插入和缺失进行评分。哈里奇系作为31个独立的小种群已经保持了250多代。通过对这31个品系进行简单的PCR分析，可以监测到数百个插入/缺失事件，这些事件应该能够分别测量X和Y染色体rDNA基因座上的R1和R2事件的频率，并确定它们是以爆发式还是以低构成率发生的。还将进行初步尝试，以确定为什么某些果蝇物种组保留了两个不同的R1元素谱系，而其他物种组只保留了一个R1谱系。将分析具有多个R1的两个物种的R1和R2周转和rDNA单位一致性，以检验较低水平的协同进化能够保护多个家族的假设。果蝇幼虫组织中R1和R2的插入水平(相对于成虫水平)降低，这是因为在幼虫多倍体(通常是多线)组织的形成过程中，含有这些插入的rDNA单位复制不足。第二系列实验旨在直接研究导致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单位或将新序列插入这些单位。由于R1和R2的逆转录转座和进化与rRNA基因的逆转录转座和进化密切相关，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。最古老的元件是特定于位点的，编码的蛋白质类似于R2。这项建议中的第三组实验旨在识别这些原始位点特定的非LTR元件的额外例子，特别是在像贾第虫这样的原始真核血统中。从这些研究中获得的信息将有助于深入了解第一批真核基因组的起源，并有助于评估非LTR逆转座子与其最接近的近亲：端粒酶和第二组内含子的关系。转座子是真核生物基因组中丰富的成分。现在有许多例子表明，这些元件在决定这些基因组的大小、结构和调节方面发挥了重要作用。越来越多的转座元件，特别是逆转录转座元件，已经被证明可以通过对基因组中特定位置的专一性来减少或控制它们对宿主的影响。R1和R2是插入节肢动物28S核糖体RNA基因的两个位点特异性的非LTR逆转座子元件。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、进化和逆转座子机制的研究已经取得了长足的进展，使R1和R2成为目前最具特征的逆转座元件之一。对这些元素进化的广泛研究表明，在节肢动物门的整个历史中，R1和R2一直是rDNA基因座稳定、活跃的组成部分，估计周期超过6亿年。在这项提案中，描述了一系列不同的实验，目的是更好地了解这些元素的非凡稳定性和起源。

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.