Programmed DNA rearrangements in the ciliate Oxytricha trifallax

描述(由申请人提供)： DNA重排在进化过程中产生遗传多样性方面起着重要作用。尾毛的扩张依赖于DNA重排进行关键的分化过程。它们有两种类型的核，即小结节核(MIC)和大结节核(MAC)。在交配和分化过程中，胚系MIC经历了大量的全球缺失和重排，形成了体细胞MAC。在三粒棘豆中，这一过程将1GbMIC基因组(包含基因区域和“垃圾”DNA)减少到50Mb的MAC基因组，该基因组主要编码DNA。在这个发育过程中移除的DNA片段被称为内部外扩序列(IBS)。IB经常中断MIC中的基因组(编码序列)，必须将其移除以暴露正确的MAC目标序列(MDS)。除了IBS的存在外，MIC中的MDS片段通常相对于它们在MAC中的顺序是无序的或被扰乱的。因此，需要基因组重排来正确地对MDS进行排序。这项提议将通过实验来研究Oxytricha中程序化DNA重排的机制，这些实验将测试4个假说，每个假说都解决了当前知识中的主要空白。假设1：通过与DDB转座酶相似的机制去除常规的(非扰乱的)LESS。初步结果表明，常规的IBS在去除后可以循环。实验将分离反应中间产物，以帮助推断传统IBS去除的机理。假设2：常规和非常规IBS是通过相同的机制去除的。对这一假设的检验将是通过显微注射不同的模板来切换靶向IBS的身份，将非常规IBS转换为常规IBS。假设3：常规的IBS在解扰发生之前被移除。之前在Oxytricha上解决这个问题的尝试都是令人费解的。定量聚合酶链式反应将允许在宏观发育过程中从不同的点监测DNA中的IES：MDS和MDS：MDS连接。假设4：指针序列(微同源区域)不是MDSunscurging或IBS去除所必需的。尽管指针在这一过程中被认为是必不可少的，但最近的证据表明，母体RNA“模板”指导着基因的解读。对缺少指针或含有合成指针的模板进行微量注射将检验这一假说。与健康相关：DNA重排(缺失、倒置和复制)是导致基因组不稳定的主要因素，许多人类疾病，包括癌症，移位和大体缺失是导致癌症和遗传性疾病的主要原因。“热点”之间的重组可以导致肿瘤抑制基因的删除或促进肿瘤稳定性的基因的复制和随后的过度表达。Oxytricha为研究DNA重排提供了一个很好的模型系统，因为它的重排幅度和赞助商在体内重新编程DNA重排的能力。这使得Oxytricha在阐明这一复杂的过程和类似的导致人类癌症基因组不稳定性的机制方面是无与伦比的(S)。

DESCRIPTION (provided by applicant): DNA rearrangements play a major role in producing genetic diversity during evolution. Stichotrichous dilates rely on DNA rearrangements for the crucial process of differentiation. These tiliates have two types of nuclei, a micronudeus (MIC)and macronudeus (MAC). Duringmating and differentiation, the germline MICundergoes massive global deletions and rearrangements to form the somatic MAC. In Oxytricha trifallax, this process reduces the 1GbMICgenome (containinggenieregions and "junk" DNA) to a 50Mb MAC genome that is mostly coding DNA. The segments of DNA removed during this developmental process are called internally exdsed sequences (IBS). IBSoften interrupt genieregions (coding sequences) in the MIC and must be removed to expose the correct MAC-destined sequence (MDS). In addition to presence of IBS,the MDS fragmentsin the MIC are often disordered, or scrambled, relative to their order in the MAC. Therefore,genome rearrangements are needed to correctly sort the MDS. This proposal will investigate the mechanism of programmed DNA rearrangements in Oxytricha via experiments that will test 4 hypotheses, each addressing major gaps in current knowledge. Hypothesis 1: Conventional (non-scrambled) lESs are removed via a mechanism similar to that used by DDB transposases. Preliminary results show that a conventional IBScan be circularized upon removal. Experiments will isolate reaction intermediates to help infer the mechanistic pathway of conventional IBS removal. Hypothesis 2: Conventional and non-conventional IBSs are removed via the same mechanism. The test of this hypothesis will be the experimental conversion of a non-conventionalIBSto a conventional IBSthrough microinjection of different templates that switch the identity of a targeted IBS. Hypothesis 3: Conventional IBSs are removed before unscrambling occurs. Previous attempts to address this question in Oxytricha were incondusive. qPCR will permit monitoring of IES:MDS and MDS:MDS junctions in DNA from various points during macronudear development. Hypothesis 4: Pointer sequences (regions of microhomology) are not required for MDSunscrambling or IBS removal. Though pointers have been considered essential in this process, recent evidence suggests that maternal RNA "templates" guide gene unscrambling. Microinjectionof templates lacking pointers or bearing synthetic pointers will test this hypothesis. Health relevance: DNA rearrangements (deletions, inversions, and duplications) are a major factor contributing to genome instability assodated withmany human diseases, induding cancer, with translocations and gross deletionsresponsible for a significant portionof cancer and inherited diseases. Recombinationbetween "hotspots" can result in either deletion of tumor-suppressing genes or duplication, and subsequent over-expression, of genes that promote tumor stability. Oxytricha provides an excellent model system to study DNA rearrangement, because of the magnitude of its rearrangements and the sponsor's ability to reprogram DNA rearrangements in vivo. This makes Oxytricha unparalleled in its ability to shed light on this complex process and similar mechanism(s)responsible for cancerous genomeinstability in humans.