MECHANISM OF SEQUENCE SPECIFIC RETROTRANSPOSITION OF R2

R2序列特异性逆转录转座机制

• 批准号: 2181663
• 负责人: Thomas H. Eickbush
• 金额: $ 18.22万
• 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 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2181663
• 关键词: Bombycidae; DNA binding protein; DNA footprinting; Drosophilidae; RNA binding protein; RNA directed DNA polymerase; developmental genetics; endonuclease; enzyme activity; gene expression; genetic translation; high performance liquid chromatography; nucleic acid sequence; polymerase chain reaction; southern blotting; transposon /insertion element

Retrotransposable elements are abundant in all eukaryotic genomes. These elements have undoubtly played a significant role in reshaping these genomes, can account for many insertional mutations and chromosomal rearrangements, and are believed to be the origin of several viruses, most significantly retroviruses. Those retrotransposable elements with long-terminal repeats (LTRs) are well-studied and are believed to integrate into the genome by mechanisms similar to retroviruses. A second class of retrotransposable elements, termed here the non-LTR elements, have more recently been discovered and appear to have a fundamentally different mechanism for integration. The specific aim of this proposal is to study the mechanism of retrotransposition of the non-LTR element, R2. This element inserts specifically in the 28S genes of a wide variety of insects. It is known that the R2 element encodes an endonuclease that requires RNA before it can specifically cleave the 28S gene insertion site. The same protein also has reverse transcriptase activity. The specific integration of this element and the ability to express the entire coding capacity of this element in E. coli makes it ideally suited to test basic features of current models of non-LTR retrotransposition. Comparative studies will be conducted with the R2 element of both Bombyx mori and Drosophila melanogaster. The critical questions to be addressed are (a) whether the R2 protein is capable of specifically binding the R2 RNA transcript, and if so what sequences, (b) the nature of the interaction between the endonuclease with the DNA target site before and after cleavage, and (c) whether the cleaved DNA, which is not complimentary to the R2 element, can serve as a primer for reverse transcription. We will also attempt to obtain complete integration of an R2 element into a ribosomal DNA repeat in vivo by injecting the R2-- transcript/R2 protein complex into tissue culture cells. We will use a highly sensitive (PCR) assay to screen for integration. Finally, to lay the foundation for our long term goal of understanding the developmental regulation of R2 retrotransposition, we will attempt to find in D. melanogaster genetic conditions underwhich R2 elements are transcribed and translated, and whether this correlates with the integration of new R2 copies.

逆转座子在所有真核生物基因组中都有丰富的存在。 毫无疑问，这些因素在 改变这些基因组，可以解释许多插入突变 和染色体重排，并被认为是 在几种病毒中，最重要的是逆转录病毒。那些 具有长末端重复序列(LTRs)的逆转座子是 经过充分的研究，并被认为通过 类似于逆转录病毒的机制。第二类 逆转座子，在这里被称为非LTR元件，具有 最近被发现，似乎从根本上说 不同的整合机制。这样做的具体目的是 建议研究逆转位的机制。 非LTR元件，R2。这一元素专门插入到28s 各种各样的昆虫的基因。众所周知，R2 元素编码一种需要RNA才能使用的内切酶 特异地切割28S基因插入位点。同样的蛋白质 还具有逆转录酶活性。具体的整合 以及表达整个编码的能力 这种元素在大肠杆菌中的容量使其非常适合于测试 目前非LTR型逆行转位模型的基本特点。 将与两者的R2元素进行比较研究 家蚕和黑腹果蝇。关键问题 要解决的问题是：(A)R2蛋白是否能够 特别结合R2 RNA转录本，如果是这样的话 序列，(B)两者之间相互作用的性质 切割前后DNA靶位的核酸内切酶， 以及(C)被切割的DNA是否与 R2元件，可以作为反转录的引物。我们 还将尝试获得R2元素的完全集成 通过注射R2-进入体内的核糖体DNA重复序列 转录本/R2蛋白复合体导入组织培养细胞。我们会 使用高度敏感的(聚合酶链式反应)分析来筛选整合。 最后，为我们的长期目标奠定基础 理解R2的发育规律 逆转座，我们将尝试在D.Blackogaster中找到 R2元件转录和转录的遗传条件 翻译，以及这是否与新的 R2复制。