Diversity-Generating Retroelements in Phage and Bacterial Genomes

描述(申请人提供)：我们发现了一个新的逆转录元件家族，命名为多样性产生逆转录元件(DGRs)，其功能是使编码蛋白质的DNA序列多样化。DGR的原型是在一个噬菌体基因组中根据它在一个基因中产生可变性的能力来鉴定的，该基因指定了波氏杆菌物种上受体分子的趋向性。波尔德氏菌会引起人类和其他哺乳动物的呼吸道感染。趋向性转换是一个模板依赖的、逆转录酶介导的过程，它在目标基因的特定位置引入核苷酸替换。这种基于盒式磁带的机制理论上能够在单个多肽中产生数万亿个不同的氨基酸序列，提供了大量潜在的配体-受体相互作用。以波氏杆菌噬菌体DGR为标志，我们已经在众多细菌基因组中鉴定出同源元件。特别值得注意的是DGRs，预计将使胃肠道菌群中最丰富的成员之一--类杆菌和齿密螺旋体--表面的蛋白质多样化，而齿密螺旋体是一种与牙周病有关的口腔细菌。原核生物DGRs代表了一种产生蛋白质多样性的全新机制。除了它们作为一个新发现的基因元件家族的基本重要性以及它们在人类健康和疾病中的潜在作用外，DGRs还由于其潜在的应用而引起人们的兴趣。我们的具体目标是：1.对波氏噬菌体中存在的原型多样性产生逆转录元件进行机理分析。2.探索DGR编码的受体蛋白识别配体的受体谱系和结构基础。3.研究DGR在人胃肠道共生类杆菌中的作用。

DESCRIPTION (provided by applicant): We have discovered a new family of retroelements, designated diversity-generating retroelements (DGRs) that function to diversify protein-encoding DNA sequences. The prototype DGR was identified in a bacteriophage genome on the basis of its ability to generate variability in a gene that specifies tropism for receptor molecules on Bordetella species. Bordetella cause respiratory infections in humans and other mammals. Tropism switching is a template-dependent, reverse transcriptase-mediated process that introduces nucleotide substitutions at defined locations within a target gene. This cassette-based mechanism is theoretically capable of generating trillions of different amino acid sequences in a single polypeptide, providing a vast repertoire of potential ligand-receptor interactions. Using the Bordetella phage DGR as a signature, we have identified homologous elements in numerous bacterial genomes. Of particular note are DGRs that are predicted to diversify proteins on the surface of Bacteroides thetaiotaomicron, one of the most abundant members of the gastrointestinal flora, and Treponema denticola, an oral bacterium associated with periodontal disease. Prokaryotic DGRs represent an entirely novel mechanism for generating protein diversity. In addition to their fundamental importance as a newly discovered family of genetic elements and their potential roles in human health and disease, DGRs are of interest as a result of their potential applications. Our specific aims are to: 1. Conduct a mechanistic analysis of the prototype diversity-generating retroelement present in Bordetella bacteriophage. 2. Probe the receptor repertoire and the structural basis of ligand recognition by a DGR-encoded receptor protein. 3. Investigate DGR function in the human gastrointestinal commensal Bacteroides thetaiotaomicron.