Ribosomal Gene Repeats and Site-Specific Retrotransposons

转座因子在决定真核生物基因组的大小、结构和表达方面起着重要作用。R1和R2是节肢动物28S核糖体RNA基因中插入的两个位点特异性非ltr反转录转座元件。这些元件的特异性使它们能够作为一个方便的模型系统，因此R1和R2现在是表征最好的转座元件之一。利用非ltr逆转录转位经常导致的5'截断序列的多样性，初步实验表明，可以在同一种群成员之间以及在果蝇的19个Harwich突变收集系之间对单个R1和R2的插入和缺失进行标记。因此，新的拷贝不断被插入rDNA位点，旧的元素从rDNA位点中被清除。在这个项目中，实验被描述为解决a)带来该基因座协同进化的重组力，b) R1和R2元素如何受到这些重组的影响，以及c) R1和R2逆转录的频率和时间。回答这些问题的尝试将集中在以下具体目标上。一个Harwich细胞系的rDNA位点将被克隆为一系列重叠的细菌人工染色体（BACs）。虽然在基因组计划中避免了rDNA位点的克隆，但在本项目中应该是可以实现的，因为已经鉴定了近80个唯一标记的R1和R2插入，作为重叠克隆组装的参考点。根据这张物理图谱，以及18个基因座在350代的时间里如何变化的例子，可以确定R1和R2插入基因座的位置，以及它们是否被均匀地移除。其次，通过对rRNA基因间可变基因间隔区域的变化进行评分，可以详细了解rDNA位点协同进化的重组过程。第三，对rDNA单位在幼虫组织中的差异复制进行研究。后一项实验很有趣，因为果蝇幼虫即使通过在多倍体组织中进行R1和R2插入的单位的低复制插入了大部分rDNA单元，也能正常生长。最后，为了确定逆转录转位的速度和发育时机，将从一代到下一代监测R1和R2的活性。在Harwich系中，R1反转位足够高，每一代都可以监测到新的事件。我们将寻找一种实验室菌株，以便用R2元素进行类似的研究。这些研究应该对两个逆转录转座因子的生命周期提供前所未有的理解。由于R1和R2的靶位点，即rDNA位点和由此形成的核仁，在所有细胞代谢中起着关键作用，因此这些研究也将为这一关键细胞成分的进化、复制和功能提供见解。

Transposable elements have played a major role in determining the size, structure and expression of eukaryotic genomes. R1 and R2 are two site-specific non-LTR retrotransposable elements that insert in the 28S ribosomal RNA genes of arthropods. The specificity of these elements has enabled them to serve as a convenient model system, such that R1 and R2 are now among the best characterized transposable elements. Taking advantage of the diverse array of 5' truncations that frequently result from non-LTR retrotransposition, preliminary experiments have shown that individual R1 and R2 insertions and deletions can be scored among members of the same population as well as among 19 Harwich mutation collection lines of Drosophila melanogaster. Therefore new copies are continually being inserted into and old elements eliminated from the rDNA loci. In this project, experiments are described to address a) the recombinational forces that bring about the concerted evolution of this locus, b) how R1 and R2 elements are affected by these recombinations, and c) how often and when R1 and R2 retrotranspose. Attempts to answer these questions will focus on the following specific aims. The rDNA loci from one Harwich line will be cloned as a series of overlapping bacterial artificial chromosomes (BACs). While the cloning of rDNA loci are avoided in genome projects, it should be achievable in this project because nearly 80 uniquely marked R1 and R2 insertions have been identified to serve as reference points for the assembly of overlapping clones. Based on this physical map, as well as the 18 examples of how that locus has changed over a period of 350 generations, it can be determined where R1 and R2 insert into the loci and whether they are uniformly removed. Second, by scoring changes in the variable intergenic spacer region between the rRNA genes, a detailed view can be obtained of the recombinational processes responsible for the concerted evolution of the rDNA locus. Third, a study will be conducted of the differential replication of rDNA units in larval tissues. This latter experiment is of interest because Drosophila larvae grow normally even when large fractions of their rDNA units are inserted by under-replicating in polyploid tissues those units that have R1 and R2 insertions. Finally, to determine the tempo and developmental timing of retrotransposition, R1 and R2 activity will be monitored from one generation to the next. R1 retrotransposition is sufficiently high in the Harwich lines that new events can be monitored per generation. A search will be made for a lab strain that will allow similar studies with the R2 elements. These studies should provide an unprecedented understanding of the life cycle of two retrotransposable elements. Because the target site for R1 and R2, the rDNA locus and the nucleolus that forms from it, plays a key role in all cellular metabolism, these studies will also provide insights into the evolution, replication and function of this critical cellular component.