Genome Reorganization in Hypotrichous Ciliated Protozoa

在许多情况下，真核生物DNA重排过程中的分子机制尚不清楚。本实验室一直在研究发生在次毛纤毛原生动物长尾蚤（Euplotes crasssus）中的广泛基因组重组。在其生命周期中，这种生物体将其染色体微核的一个拷贝转化为只包含短线性基因大小的DNA分子的大核。在这一过程中，大约有10万个DNA间隙片段被删除，包括短片段（内部消除序列；IESs）和高度重复的转座子样片段。差异筛选方法将用于分离在大核发育过程中特异性表达的基因和蛋白质。发育cDNA文库也将使用重排DNA片段作为探针进行表达和筛选，试图直接识别编码与重排位点相互作用的蛋白质的克隆。将对分离的基因进行发育表达时间分析，并进行研究以确定其基因产物是否定位于发育中的巨核。这样的基因，如果被发现，将被完全排序，以进一步了解它们的功能。DNA重排被用作构建功能性蛋白质编码区的手段，因此可以被视为发育调节基因表达的手段。为了了解真核生物的这一过程，该实验室已经并将继续研究发生在次毛纤毛原生动物（Euplotes crasssus）中的广泛基因组重组。该领域的研究将包括确定在经历大核发育的细胞中的多个细胞核中，哪一个负责产生发育调节的转录本，以及检查发育特异性基因中可能参与其协调调节的共享序列元件。＊＊＊

9414416 Klobutcher The molecular mechanisms involved in DNA rearrangement processes of eukaryotic organisms are, in many instances, poorly understood. This laboratory has been studying the extensive genome reorganization that occurs in hypotrichous ciliated protozoan Euplotes crassus. During its life cycle, this organism transforms a copy of its chromosomal micronucleus into a macronucleus that contains only short linear, gene-sized DNA molecules. On the order of 100,000 interstitial segments of DNA are deleted during this process, including short elements(internal eliminated sequences; IESs) and highly repetitive, transposon-like elements. Differential screening approaches will be used to isolate genes and proteins that are expressed specifically during macronuclear development. The developmental cDNA library will also be expressed and screened using segments of DNA that undergo rearrangement as probes in an attempt to directly identify clones that encode proteins that interact with rearrangement sites. The isolated genes will be analyzed in regard to time of developmental expression and studies will be done to determine if their gene products localize to the developing macronucleus. Such genes, if found, will be completely sequenced to gain further insight into their function. %%% DNA rearrangement is used as a means of constructing functional protein coding regions and thus can be viewed as a means of developmentally regulating gene expression. To learn about such processes in a eukaryotic organism, this laboratory has been, and will continue, to study the extensive genome reorganization that occurs in the hypotrichous ciliated protozoan, Euplotes crassus. Studies in this area will include determining which of the multiple nuclei in a cell undergoing macronulear development is responsible for producing developmentally regulated transcripts and examining developmental-specific genes for shared sequence elements that are potentially involved in their coordinate regulation. ***