MECHANISM OF NON-LTR RETROTRANSPOSITION

非 LTR 逆转录转座机制

• 批准号: 6604896
• 负责人: Thomas H. Eickbush
• 金额: $ 33.39万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-01 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6604896
• 关键词: Bombycidae; DNA binding protein; DNA footprinting; Drosophilidae; RNA binding protein; RNA directed DNA polymerase; developmental genetics; endonuclease; enzyme activity; eukaryote; gel electrophoresis; gene expression; genetic transcription; genetic translation; northern blottings; nucleic acid repetitive sequence; polymerase chain reaction; ribosomal RNA; site directed mutagenesis; southern blotting; transposon /insertion element

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.

真核生物的基因组中充满了从RNA到DNA的逆向信息流所产生的序列。这些反向转录物的插入可以诱导自发性遗传疾病和癌症，并且随着进化的时间，通过产生大量的重复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）逆转录转座因子编码。该元件专门插入其宿主的28 S rRNA基因，这大大简化了其表征。由R2编码的单一蛋白质对其DNA靶位点和RNA模板都具有显着的特异性，使得能够在体外研究其反转录转座机制。这种机制涉及DNA靶的简单切割和逆转录直接聚合到切割的染色体上，因此与充分描述的逆转录转座的逆转录病毒机制根本不同。相反，非LTR元件与端粒酶相关，并且它们的逆转录转座与端粒酶共享特征，端粒酶是负责端粒的酶。该提案的具体目的是将R2逆转录转座机制充分表征为所有非LTR元件的模型。详细的诱变的蛋白质和核酸组分和表征的蛋白质的酶活性的建议。这些体外研究将与果蝇中R2整合反应的体内研究相补充。纯化的R2蛋白/RNA感染D.黑腹果蝇胚胎可以将外源序列整合到28 S基因中。该整合系统将使研究细胞核仁rDNA单元内R2元件的转录和翻译成为可能。这些综合研究将解决有关非LTR逆转录转座机制的一般问题，以及有关rRNA基因转录调节的问题。最后，越来越多的非LTR元件已被表征，其保留了其宿主基因组中独特位点的不同插入特异性。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特异性结合组分，这使我们能够设计将外源序列插入真核基因组中其他独特位置的系统。