Genome Reorganization in Hypotrichous Ciliated Protozoa

发育性或程序性的DNA重排发生在各种生物体中。在某些情况下，所涉及的分子机制已被很好地理解，但关于发生在许多真核生物中的大规模重排过程的信息却很少。次毛纤毛原生动物在有性生殖过程中经历了广泛的基因组重组。生发微核的复制消除了大约100,000个DNA间隙段或内部消除序列（IES），并伴随着侧面序列的重新连接，形成转录活性大核。被淘汰的ess由两个大的，相关的转座子家族，以及短的独特的DNA片段组成。染色体断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的末端，称为端粒。已经开发了模型来解释这种广泛重排所涉及的机制，这些模型将被测试。进一步表征这两个主要重排过程的分子机制将被完成。高度敏感的结扎介导的PC程序将用于寻找体内重排中间体，这些中间体经历了染色体断裂，但不包括端粒添加。将进行一系列类似的PCR分析，以确定IES切除过程中的中间产物。将寻求从适当分期发展的大核中提取的体外染色体断裂活性。最后，使用部分纯化的端粒酶制剂进行额外的体外研究，以测试当前的从头端粒合成模型。克拉苏是为数不多的案例之一，其中有强有力的证据表明转座因子参与发育DNA重排。对这一过程的进一步了解将使这一机制得到验证，并为转座子及其宿主基因组的共同进化提供相关信息。最终，这些研究还可能导致在体内和体外产生人工染色体的实际使用工具，以及提出减轻染色体断裂有害影响的策略。

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.