Ribosomal gene loci dynamics and specific retrotransposons

所有高等真核生物基因组都是由大量的转座因子组成的，这些转座因子不断地试图进一步扩大它们的数量。这些元素可以被认为是细胞内的寄生虫。虽然一般认为转座因子或多或少是随机插入的，但越来越多的因子已被证明通过进化位点特异性来最小化它们对宿主的影响。本研究中使用的模型系统是R1和R2非ltr反转录转座元件，它们特异性插入到许多动物的28S rRNA基因中，但在昆虫中研究得最多。真核生物的数百个串联排列的rRNA基因（rDNA位点）经历了动态的重组过程，以消除位点内的变异。然而，纵观它们的历史，R1和R2在rRNA基因中稳定地维持着它们的存在，这表明它们很好地适应了利用细胞设计的重组和调节机制来合成rRNA。本研究的目的是在三个水平上研究几种果蝇的rDNA基因及其r1和R2插入。第一个目标是确定特定rDNA位点在特定时间段内发生的变化。以5'标记的R1和R2元素为参考点，在一系列重叠的重组DNA克隆上，可以恢复分离超过400代的两个重复系的rDNA位点的大片段。对扩展区域的详细比较将使我们能够对重组的具体模式和引起已知在这些线之间发生的许多变化的反转位事件的分布有一个初步的看法。其次，将筛选D. simulans的自然种群，以确定R2元素活跃的频率，并表征rDNA位点的大小和结构。这些研究将在拟马鳖中进行，因为拟马鳖的姐妹种在Y染色体上没有rDNA单元，并且很容易获得具有活性R2元素的种群。第三，通过对12种果蝇和其他昆虫的全基因组“霰弹枪”测序工作获得的公共数据将用于对各种动物rDNA位点内的核苷酸变异进行评分。目标将是建立可用于比较rDNA位点协同进化的机制和效率的方法。这些研究将使我们能够更好地了解大小的波动、表达的调节以及随着时间的推移，在rDNA位点中产生序列均匀性和片段变化的重组过程。它们还将增加我们对高等生物和这些细胞内寄生虫之间达到的微妙平衡的理解。所有高等生物的基因组都处于与内部寄生虫的持续战斗中，这些寄生虫被称为可移动或转座因子。来自不同生物体的总基因组DNA中有10%到90%是由这些元素组成的。本研究的重点是一个模型系统，该系统具有移动元件特异性插入基因组的一个位置的优势：连续重复核糖体RNA基因（rDNA位点）。模式生物是几种果蝇（果蝇）。研究包括短期实验室实验、群体实验和利用基因组序列数据跟踪rDNA位点插入和删除的移动元件，以及这些插入如何导致rDNA位点的变化。这些知识将有助于研究人员为了人类的利益而控制和利用这些元素。该项目还将进一步促进高中科学教师、本科生和研究生的教育。

All higher eukaryotic genomes are composed to a large measure of transposable elements that continually attempt to expand their numbers even further. These elements can be thought of as intracellular parasites. While it is generally assumed that transposable elements insert more or less at random, a growing number of elements have been shown to minimize their effects on the host by evolving site-specificity. The model systems used in this research are the R1 and R2 non-LTR retrotransposable elements, which insert specifically into the 28S rRNA genes of many animals but have been studied most extensively in insects. The hundreds of tandemly arranged rRNA genes (the rDNA loci) of eukaryotes undergo dynamic processes of recombination to eliminate variation within the locus. Yet throughout their history, R1 and R2 have stably maintained their presence in the rRNA genes indicating they are well-adapted to exploit the recombinational and regulatory mechanisms devised by the cell to synthesize rRNA. The objectives of this research are to study the rDNA genes and their R1and R2 insertions in several Drosophila species at three levels. The first objective is to define the changes that have occurred in specific rDNA loci over defined periods of time. Large segments of the rDNA loci from two replicate lines of D. melanogaster that have been separated by over 400 generations will be recovered on a series of overlapping recombinant DNA clones assembled using the 5' marked R1 and R2 elements as reference points. Detailed comparisons of the extended regions will enable a first view of the specific patterns of recombination and of the distribution of the retrotransposition events that have given rise to the many changes known to have occurred between these lines. Second, natural populations of D. simulans will be screened to determine the frequency with which R2 elements are active as well as to characterize the size and structure of the rDNA loci. These studies will be conducted in D. simulans because this sister species of D. melanogaster has no rDNA units on the Y chromosome and populations with active R2 elements are readily obtained. Third, public data made available through whole genome shot-gun sequencing efforts of 12 Drosophila species as well as those of other insects will be used to score the nucleotide variation within the rDNA loci of various animals. The goal will be to establish the approaches that can be used to compare the mechanism and efficiency of concerted evolution of the rDNA loci. These studies will enable a greater understanding of the fluctuation in size, the regulation of expression, and the recombinational processes that give rise to both the sequence uniformity and the segmental changes that occur in the rDNA locus over time. They will also increase our understanding of the delicate balance that is reached between higher organisms and these intracellular parasites. The genomes of all higher organisms are in a constant battle with internal parasites called mobile or transposable elements. Between 10% and 90% of total genomic DNA from different organisms is composed of these elements. This research focuses on a model system that has the advantage that the mobile elements specifically insert into one location of the genome: the tandemly repeated ribosomal RNA genes (rDNA locus). The model organisms are several fruit fly species (Drosophila). The studies include short term laboratory experiments, population experiments, and the utilization of genomic sequence data to follow the mobile elements being inserted into and deleted from the rDNA locus, as well as how the rDNA locus changes as a result of these insertions. This knowledge will help researchers both control and exploit these elements for the advantage of mankind. The project will also serve to further the education of high school science teachers, undergraduates, and graduate students.