MECHANISM OF SEQUENCE SPECIFIC RETROTRANSPOSITION OF R2

真核生物的基因组中充满了来自反向流动的序列 从RNA到DNA的信息。 负责的逆转录转座因子 因为这些反向转录物无疑在 基因组的一般形状，可以解释许多目前的插入 突变和染色体重排，并被认为是起源 许多病毒，例如。逆转录病毒 最丰富的，但很少 据了解，这些逆转录转座因子的类别是非长链的。 末端重复（非LTR）逆转录转座因子。 最好的 特征性的非LTR元件是昆虫的R2元件。 初始 R2反转录转座机制中的步骤已经被定义，因为R2 特异性地插入到28S核糖体基因的独特序列中， 它的宿主 R2 RNA模板的逆转录通过切口引发 在DNA靶位点，因此反转录转座是通过一种机制 与已被充分描述的逆转录病毒机制完全不同。 这种靶向启动的逆转录机制被认为是用于 大多数其他非LTR元件，II组内含子，可能是起源 西内斯，加工假基因，甚至可能是内含子。 具体 该提案的目的是充分表征BCR2逆转录转座 机制作为所有非LTR元素的模型。 直接酶促研究 该机制将使用纯化的R2蛋白和核酸进行 件. 这些体外研究将与体内研究相补充。 果蝇整合反应的研究。 纯化 R2蛋白和R2 RNA模板已显示整合R2序列 插入到D. melanogaster 胚胎 这种体内整合系统的进一步发展将 使R2元件的转录和翻译的研究成为可能 在rDNA单位的上下文中。 这些研究将解决一般 非LTR逆转录转座机制的问题，以及 关于转录和重组机制的问题， rRNA基因。 最后，这些研究将作为 开发一种转换系统，提供以下独特优势： 将外源序列插入到基因组的特定序列中， rDNA单位。 因为将使用来自蛾的R2蛋白和RNA模板， 为了在果蝇中整合序列，该系统可以用作通用的 昆虫和可能的其他生物的转化系统。

Eukarytic genomes are filled with sequences derived from the reverse flow of information from RNA to DNA. The retrotransposable elements responsible for these reverse transcripts have undoubtedly played a significant role in the general shaping of genomes, can account for many current insertional mutations and chromosomal rearrangements, and are believed to be the origin of numerous viruses, eg. retroviruses. The most abundant, yet poorly understood, class of these retrotransposable elements are the non-long terminal repeat (non-LTR) retrotransposable elements. The best characterized non-LTR elements are the R2 elements of insects. The initial steps in the R2 retrotransposition mechanism have been defined because R2 specifically inserts into a unique sequence in the 28S ribosomal genes of its host. Reverse transcription of the R2 RNA template is primed by a nick at the DNA target site, thus retrotransposition is by a mechanism completely different from that of the well-described retroviral mechanism. This target primed reverse transcription mechanism is believed to used by most other non-LTR elements, the group II introns, an is likely the origin of SINEs, processed pseudogenes and maybe even of introns. The specific aim of this proposal is to fully characterize athe R2 retrotransposition mechanism as a model for all non-LTR elements. Direct enzymatic studies of the mechanism will be conducted using purified R2 protein and nucleic acid components. These in vitro studies will be complemented with in vivo studies of the integration reaction in Drosophila melanogaster. Purified R2 protein and R2 RNA templates have been shown to integrate R2 sequences into the 28S genes of somatic and germline cells of D. melanogaster embryos. Further development of this in vivo integration system will enable studies of the transcription and translation of the R2 element within the context of the rDNA units. These studies will address general questions of the mechanism of non-LTR retrotransposition, as well as questions concerning the transcription and recombinational mechanism of the rRNA genes. Finally, these studies will serve as the basis for the development of a transformation system that offers the unique advantage of inserting foreign sequences into a specific sequence of the genome, the rDNA units. Because R2 protein and RNA templates from a moth will be used to integrate sequences in a fruit fly this system may serve as a general transformation system for insects and possibly other organisms.