MECHANISM OF NON-LTR RETROTRANSPOSITION

真核生物基因组充满了由RNA到DNA的反向信息流衍生的序列。这些逆转录物的插入可以诱导自发的遗传疾病和癌症，并通过产生大量重复DNA，在进化过程中完全重塑了真核生物基因组。非长末端重复序列（non-long terminal repeat, non-LTR）逆转录转座元件是负责这些插入的最活跃的机制，但人们对其知之甚少。节肢动物的非ltr元件之一是R2元件。该元件完全插入宿主的28S rRNA基因中，大大简化了其表征。R2编码的单蛋白对其DNA靶点和RNA模板都具有显著的特异性，可以在体外研究其反转录转位机制。这种机制涉及DNA靶点的简单切割和反向转录物直接聚合到被切割的染色体上，因此与已经被很好地描述的逆转录病毒的反转录转位机制有本质的不同。相反，非ltr元件与端粒酶有关，它们的反转录与端粒酶有共同的特征。本提案的具体目的是将R2逆转录转位机制作为所有非ltr元件的模型进行充分表征。提出了蛋白质和核酸成分的详细诱变和蛋白质酶活性的表征。这些体外研究将与黑腹果蝇R2整合反应的体内研究相补充。纯化的R2蛋白/RNA感染到黑腹龙胚后，可以将外源序列整合到28S基因中。该整合系统将使研究核仁rDNA单位内R2元件的转录和翻译成为可能。这些联合研究将解决有关非ltr反转录转位机制的一般问题，以及有关rRNA基因转录调控的问题。最后，越来越多的非ltr元件已被鉴定为在其宿主基因组的独特位点保留不同的插入特异性。由这些元素编码的酶机制看起来与R2相同。我们的长期目标是分离和改变整合反应的目标特异性结合成分，这使我们能够设计系统，我们将在真核生物基因组的其他独特位置插入外源序列。

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.