Genome Reorganization in Hypotrichous Ciliated Protozoa

发育中的或程序化的DNA重排发生在各种生物体中。在某些情况下，所涉及的分子机制是众所周知的，但关于发生在许多真核生物中的大规模重排过程的信息很少。假毛状纤毛虫原生动物在有性繁殖过程中经历了广泛的基因组重组。生发微核的拷贝消除了大约100,000个DNA间质片段或内部消除序列(IES)，同时伴随着侧翼序列的重新连接，形成了转录活性的大核。被消除的ISE包括两个相关的转座子大家族，以及短的、独特的DNA片段。染色体碎裂产生基因大小的大核DNA分子。简单的重复序列被添加到基因大小片段的末端作为端粒。已经开发了模型来解释这种广泛的重排所涉及的机制，并将对这些模型进行测试。将对两个主要重排过程的分子机制进行进一步的表征。高灵敏的连接介导的PC程序将被用来寻找已经经历了染色体碎裂而不是端粒添加的体内重排中间体。将进行类似的一系列聚合酶链式反应分析，以确定IES切除过程中的中间产物。在体外，从适当分期的发育的大核中提取的提取物中的染色体断裂活性将被寻找。最后，还将对部分纯化的端粒酶制剂进行额外的体外研究，以测试当前的从头合成端粒模型。E.Crassus是少数几个有强有力证据表明转座元件参与发育DNA重排的案例之一。对这一过程的进一步了解将使这一机制得到验证，并提供与转座子及其宿主基因组共同进化相关的信息。最终，这些研究还可能带来在体内和体外产生人造染色体的实用工具，以及提出减轻染色体断裂的有害影响的策略。

Developmental, or programmed, DNA rearrangement occurs in a variety of organisms. The molecular mechanisms involved are, in some cases, well understood but little information exists for the large-scale rearrangement processes that occur in numerous eukaryotes. The hypotrichous ciliated protozoan, Euplotes crassus, undergoes extensive genome reorganization during sexual reproduction. A copy of the germinal micronucleus eliminates about 100,000 interstitial segments of DNA or internal eliminated sequences (IES) with concomitant rejoining of flanking sequences to form the transcriptionally active macronucleus. The eliminated IESs consists of two large, related families of transposons, as well as short unique segments of DNA. Chromosome fragmentation generates the gene size macronuclear DNA molecules. Simple repeats are added to the ends of the gene size pieces as telomeres. Models have been developed to explain the mechanisms involved in this extensive rearrangement and the models will be tested. Further characterization of the molecular mechanisms of the two major rearrangement processes will be done. Highly sensitive ligation-mediated PC procedures will be used to search for in vivo rearrangement intermediates that have undergone chromosome fragmentation, but not telomere addition. A similar series of PCR analysis will be performed to identify intermediates in the IES excision process. In vitro chromosome fragmentation activity in extracts derived from appropriately staged developing macronuclei will be sought. Finally, additional in vitro studies with partially purified telomerase preparations will be performed to test the current model of de novo telomere synthesis.E. crassus is one of the few cases where there is strong evidence for the involvement of transposable elements in developmental DNA rearrangement. Further insight into this process will allow this mechanism to be validated as well as providing information relevant to the co-evolution of transposons and their host genomes. Ultimately these studies may also lead to tools that are of practical use in generating artificial chromosomes both in vivo and in vitro, as well as suggesting strategies for mitigating the harmful effects of chromosome breaks.