Ribosomal Gene Repeats and Site-Specific Retrotransposons

核糖体基因重复和位点特异性逆转录转座子

• 批准号: 0212452
• 负责人: Thomas Eickbush
• 金额: $ 56.5万
• 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: 2002
• 资助国家: 美国
• 起止时间: 2002-08-01 至 2006-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0212452&HistoricalAwards=false
• 关键词: Ribosomal; Gene; Repeats; Site; Specific

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.

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是插入节肢动物28S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现在是最具特征的转座元件之一。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，可以在相同群体的成员之间以及在黑腹果蝇的19个Harwich突变收集系之间对单个R1和R2插入和缺失进行评分。因此，新的拷贝不断地被插入rDNA基因座，旧的元件从rDNA基因座中被消除。在这个项目中，实验被描述为解决a）重组的力量，带来了这个位点的协同进化，B）R1和R2元件如何受到这些重组的影响，以及c）R1和R2逆转录转座的频率和时间。回答这些问题的努力将侧重于以下具体目标。将来自一个Harwich系的rDNA基因座克隆为一系列重叠的细菌人工染色体（BAC）。虽然在基因组计划中避免了rDNA基因座的克隆，但在本项目中应该是可以实现的，因为已经鉴定了近80个独特标记的R1和R2插入片段作为重叠克隆组装的参考点。根据这张物理图谱，以及该基因座在350代期间如何变化的18个例子，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去除。其次，通过对rRNA基因之间可变基因间间隔区的变化进行评分，可以获得负责rDNA基因座协同进化的重组过程的详细视图。第三，研究幼虫组织中rDNA单位的差异复制。这后一个实验是感兴趣的，因为果蝇幼虫正常生长，即使当他们的rDNA单位的大部分插入在多倍体组织中的复制不足，这些单位有R1和R2插入。最后，为了确定反转录转座的克里思和发育时间，将从一代到下一代监测R1和R2活性。R1反转录转座是足够高的Harwich线，新的事件可以监测每一代。将搜索允许使用R2元件进行类似研究的实验室菌株。这些研究应该提供了一个前所未有的了解生命周期的两个逆转录转座因子。由于R1和R2的靶位点，rDNA基因座和从它形成的核仁，在所有细胞代谢中起着关键作用，这些研究也将提供对这一关键细胞组分的进化，复制和功能的见解。