MECHANISM OF SEQUENCE SPECIFIC RETROTRANSPOSITION OF R2

R2序列特异性逆转录转座机制

• 批准号: 2459400
• 负责人: Thomas H. Eickbush
• 金额: $ 24.05万
• 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 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459400
• 关键词: Bombycidae; DNA binding protein; Drosophilidae; RNA binding protein; RNA directed DNA polymerase; developmental genetics; endonuclease; enzyme activity; gel electrophoresis; gene expression; genetic transcription; genetic translation; northern blottings; nucleic acid sequence; polymerase chain reaction; ribosomal RNA; southern blotting; transposon /insertion element

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.

真核基因组中充满了来自反向流动的序列 从RNA到DNA的信息。负责逆转座子的元件 因为这些反向转录本无疑在 基因组的一般形状，可以解释许多当前插入 突变和染色体重排，被认为是 大量的病毒，例如。逆转录病毒。最丰富的，但很差的 理解，这些逆转座子的一类是非长的 末端重复(非LTR)逆转录转座元件。最好的 特征性的非LTR元件是昆虫的R2元件。首字母 已经定义了R2逆转位机制中的步骤，因为R2 特异性地插入到沙门氏菌28S核糖体基因的独特序列中 它的主人。R2 RNA模板的逆转录由缺口启动 在DNA靶点，因此逆转录转座是通过一种机制 与人们熟知的逆转录病毒机制完全不同。 这种靶标启动的逆转录机制被认为是由 大多数其他非LTR元件，第二类内含子，An可能是起源 处理过的假基因，甚至可能是内含子。具体的 这项建议的目的是充分描述R2逆转录转座 机制作为所有非LTR要素的模型。直接酶学研究 这一机制将使用纯化的R2蛋白和核酸进行 组件。这些体外研究将与体内研究相辅相成 果蝇整合反应的研究。纯净的 R2蛋白质和R2 RNA模板已被证明整合了R2序列 在体细胞和生殖系细胞中插入28S基因 胚胎。这一体内整合系统的进一步发展将 使研究R2元件的转录和翻译成为可能 在rDNA单位的背景下。这些研究将涉及一般情况 关于非LTR逆转位机制的问题以及 关于基因转录和重组机制的几个问题 RRNA基因。最后，这些研究将作为 开发可提供以下独特优势的转换系统 将外源序列插入基因组的特定序列中， RDNA单位。因为将使用来自飞蛾的R2蛋白质和RNA模板 为了整合果蝇中的序列，该系统可以作为通用的 昆虫和可能的其他生物的转化系统。