Programmed DNA rearrangements in the ciliate Oxytricha trifallax

纤毛虫 Oxytricha trifallax 中的程序化 DNA 重排

基本信息

  • 批准号:
    7864144
  • 负责人:
  • 金额:
    $ 5.22万
  • 依托单位:
  • 依托单位国家:
    美国
  • 项目类别:
  • 财政年份:
    2009
  • 资助国家:
    美国
  • 起止时间:
    2009-06-01 至 2011-05-31
  • 项目状态:
    已结题

项目摘要

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.
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发挥着重要作用。Stichotrichous扩张依赖于DNA重排的关键过程的分化。这些纤毛细胞有两种类型的核,即小核(MIC)和大核(MAC)。在交配和分化过程中,生殖系MIC经历大规模的全局缺失和重排,形成体细胞MAC。在Oxytrichatrifallax中,这一过程将1GbMIC基因组(包含基因区和“垃圾”DNA)减少到主要是编码DNA的50Mb MAC基因组。在这个发育过程中去除的DNA片段被称为内部exdsed序列(IBS)。IBS经常中断MIC中的基因区(编码序列),必须将其移除以暴露正确的MAC目的序列(MDS)。除了IBS的存在外,MIC中的MDS片段相对于MAC中的顺序通常是无序的或混乱的。因此,需要基因组重排来正确分选MDS。该提案将通过实验研究Oxytricha中程序化DNA重排的机制,这些实验将测试4种假设,每种假设都解决了当前知识中的主要空白。假设1:常规(非加扰的)IES通过与DDB转座酶所使用的机制类似的机制被去除。初步结果表明,传统的IBScan在去除后被环化。实验将分离反应中间体,以帮助推断常规IBS去除的机理途径。假设2:传统和非传统IBS通过相同的机制被去除。这一假设的检验将是通过显微注射不同的模板将非常规IBS转化为常规IBS的实验,这些模板转换了靶向IBS的身份。假设3:传统的IBS在解扰发生之前被移除。以前在Oxytricha中解决这个问题的尝试是不成功的。qPCR将允许在大核发育期间从不同点监测DNA中的IES:MDS和MDS:MDS连接。假设4:MDSunscrambling或IBS去除不需要指针序列(微同源区域)。虽然指针被认为是必不可少的,在这个过程中,最近的证据表明,母体RNA“模板”指导基因解读。显微注射缺乏指针或轴承合成指针的模板将测试这一假设。健康相关性:DNA重排(缺失、倒位和重复)是导致基因组不稳定的主要因素,与许多人类疾病相关,包括癌症,其中易位和总缺失是癌症和遗传性疾病的重要原因。“热点”之间的重叠可导致肿瘤抑制基因的缺失或促进肿瘤稳定性的基因的复制和随后的过度表达。Oxytricha提供了一个很好的模型系统来研究DNA重排,因为它的重排的幅度和赞助商的能力,在体内重新编程DNA重排。这使得Oxytricha在揭示这一复杂过程和导致人类癌症基因组不稳定性的类似机制方面具有无与伦比的能力。

项目成果

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Brian Patrick Higgins其他文献

Brian Patrick Higgins的其他文献

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{{ truncateString('Brian Patrick Higgins', 18)}}的其他基金

Programmed DNA rearrangements in the ciliate Oxytricha trifallax
纤毛虫 Oxytricha trifallax 中的程序化 DNA 重排
  • 批准号:
    7678654
  • 财政年份:
    2009
  • 资助金额:
    $ 5.22万
  • 项目类别:

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