MECHANISM OF NON-LTR RETROTRANSPOSITION

真核生物基因组充满了从RNA到DNA的信息反向流动所产生的序列。插入这些逆转录本可导致自发性遗传疾病和癌症，并随着进化时间的推移，通过产生大量重复的DNA而完全重塑真核基因组。负责这些插入的最活跃但知之甚少的机制是由非长末端重复序列(Non-Ltr)逆转录转座元件编码的。节肢动物中最具特征的非LTR元件之一是R2元件。该元件专门插入到宿主的28S rRNA基因中，大大简化了其特性。R2编码的单一蛋白对其DNA靶点和RNA模板都具有显着的特异性，使体外研究其逆转录转座机制成为可能。这种机制包括DNA靶标的简单切割和反向转录产物直接聚合到被切割的染色体上，因此与众所周知的逆转录病毒逆转录病毒转座机制有根本的不同。相反，非LTR元件与端粒酶相关，并且它们的逆转录转座具有端粒酶的共同特征，端粒酶是负责端粒的酶。这项提议的具体目的是充分说明R2逆转座机制是所有非LTR元件的模式。对蛋白质和核酸成分进行了详细的诱变，并对蛋白质的酶活性进行了表征。这些体外研究将与果蝇R2整合反应的体内研究相辅相成。纯化的R2蛋白/RNA感染黑腹果蝇胚胎后，可以将外源序列整合到28S基因中。这一整合系统将使细胞核仁rDNA单元内R2元件的转录和翻译研究成为可能。这些联合研究将解决关于非LTR逆转录转座机制的一般问题，以及关于rRNA基因转录调控的问题。最后，越来越多的非LTR元件被鉴定为对其宿主基因组中的独特位置保留了不同的插入特异性。由这些元件编码的酶机制似乎与R2相同。我们的长期目标是分离和改变整合反应的靶标特异结合成分，这使我们能够设计出将外源序列插入到真核基因组中其他独特位置的系统。

Eukaryotic genomes are filled with sequences derived by the reverse flow of information from RNA to DNA. Insertion of these reverse transcripts can induce spontaneous genetic disorders and cancer and have over evolutionary time completely reshaped eukaryotic genomes by generating enormous amounts of repetitive DNA. The most active, yet poorly understood, machinery responsible for these insertions is encoded by the non-long terminal repeat (non-LTR) retrotransposable elements One of the best characterized non-LTR elements is the R2 element of arthropods. This element exclusively inserts into the 28S rRNA genes of its host which greatly simplifies its characterization. The single protein encoded by R2 has remarkable specificity for both its DNA target site and RNA template enabling in vitro studies of its retrotransposition mechanism. This mechanism involves simple cleavage of the DNA target and the polymerization of the reverse transcript directly onto the cleaved chromosome, and thus is fundamentally different from the well-described retroviral mechanism of retrotransposition. Instead non-LTR elements are related to and their retrotransposition shares features with telomerase, the enzyme responsible for telomeres. The specific aim of this proposal is to fully characterize the R2 retrotranspostion mechanism as a model for all non-LTR elements. Detailed mutagenesis of the protein and nucleic acid components and characterization of the enzymatic activities of the protein are proposed. These in vitro studies will be complemented with in vivo studies of the R2 integration reaction in Drosophila melanogaster. Purified R2 protein/RNA when infected into D. melanogaster embryos can integrate foreign sequences into the 28S genes. This integration system will enable studies of the transcription and translation of R2 elements within the rDNA units of cell's nucleolus. These combined studies will address general questions about the mechanism of non-LTR retrotransposition, as well as questions concerning transcription regulation of the rRNA genes. Finally, a growing number of non-LTR elements have been characteriz4ed that retain different insertion specificities for unique sites in their host's genome. The enzymatic machinery encoded by these elements appears identical to that of R2. Our long term goal is to isolate and alter the target specific binding component of the integration reaction , which enable us to design systems that we will insert foreign sequences at other unique locations in eukaryotic genomes.