MECHANISM OF SEQUENCE SPECIFIC RETROTRANSPOSITION OF R2

逆转录转座因子在所有真核生物基因组中都很丰富。 这些因素无疑发挥了重要作用， 重塑这些基因组，可以解释许多插入突变， 以及染色体重排，被认为是 几种病毒，最重要的是逆转录病毒。 那些 具有长末端重复序列（LTR）的逆转录转座因子是 被充分研究，并被认为是整合到基因组中， 类似于逆转录病毒的机制。 第二类 逆转录转座因子，这里称为非LTR因子，具有 最近被发现，似乎有一个基本的 不同的整合机制。 其具体目的是 建议是研究逆转录转座的机制， 非LTR元件，R2。 此元素专门插入28 S中 各种昆虫的基因。 据了解，R2 一个元件编码一种核酸内切酶，这种核酸内切酶需要RNA才能 特异性切割28 S基因插入位点。 相同蛋白质 还具有逆转录酶活性。具体整合 以及表达整个编码的能力 该元素在E.大肠杆菌使其非常适合测试 目前非LTR逆转录转座模型的基本特征。 将对两者的R2元件进行比较研究。 家蚕和果蝇。 的关键问题 （a）R2蛋白是否能够 特异性结合R2 RNA转录物，如果是这样， （B）所述序列之间的相互作用的性质， 在切割之前和之后将核酸内切酶与DNA靶位点连接， 和（c）是否切割的DNA，其不与所述DNA互补， R2元件，可以作为逆转录的引物。 我们 还将尝试获得R2元件的完全集成 在体内复制到核糖体DNA重复序列中 转录物/R2蛋白复合物导入组织培养细胞。 我们将 使用高灵敏度（PCR）分析来筛选整合。 最后，为我们的长远目标奠定基础， 了解R2的发育规律 反转录转座，我们将试图找到在D。melanogaster R2元件转录的遗传条件， 翻译，以及这是否与新的整合相关 R2收到

Retrotransposable elements are abundant in all eukaryotic genomes. These elements have undoubtly played a significant role in reshaping these genomes, can account for many insertional mutations and chromosomal rearrangements, and are believed to be the origin of several viruses, most significantly retroviruses. Those retrotransposable elements with long-terminal repeats (LTRs) are well-studied and are believed to integrate into the genome by mechanisms similar to retroviruses. A second class of retrotransposable elements, termed here the non-LTR elements, have more recently been discovered and appear to have a fundamentally different mechanism for integration. The specific aim of this proposal is to study the mechanism of retrotransposition of the non-LTR element, R2. This element inserts specifically in the 28S genes of a wide variety of insects. It is known that the R2 element encodes an endonuclease that requires RNA before it can specifically cleave the 28S gene insertion site. The same protein also has reverse transcriptase activity. The specific integration of this element and the ability to express the entire coding capacity of this element in E. coli makes it ideally suited to test basic features of current models of non-LTR retrotransposition. Comparative studies will be conducted with the R2 element of both Bombyx mori and Drosophila melanogaster. The critical questions to be addressed are (a) whether the R2 protein is capable of specifically binding the R2 RNA transcript, and if so what sequences, (b) the nature of the interaction between the endonuclease with the DNA target site before and after cleavage, and (c) whether the cleaved DNA, which is not complimentary to the R2 element, can serve as a primer for reverse transcription. We will also attempt to obtain complete integration of an R2 element into a ribosomal DNA repeat in vivo by injecting the R2-- transcript/R2 protein complex into tissue culture cells. We will use a highly sensitive (PCR) assay to screen for integration. Finally, to lay the foundation for our long term goal of understanding the developmental regulation of R2 retrotransposition, we will attempt to find in D. melanogaster genetic conditions underwhich R2 elements are transcribed and translated, and whether this correlates with the integration of new R2 copies.