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GENOME REORGANIZATION IN HYPOTRICHOUS CILIATED PROTOZOA

地下纤毛原生动物的基因组重组

基本信息

批准号：
3282756
负责人：
LAWRENCE Anthony KLOBUTCHER
金额：
$ 8.52万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
1984
资助国家：
美国
起止时间：
1984-04-01 至 1988-02-29
项目状态：
已结题

项目摘要

Although a number of gene and genome rearrangement processes have been defined in eukaryotic organisms, the mechanisms that mediate these events are poorly understood. We plan to study the extensive genome reorganization process that occurs in hypotrichous ciliated protozoa, such as Oxytricha nova and Stylonychia mytilus, in order to extend our knowledge of eukaryotic rearrangement processes as well as to define the types of DNA sequences and enzymes that mediate such events. In these organisms a copy of the chromosomal micronuclear genome is transformed into a macronucleus containing small gene-size DNA molecules. This genome rearrangement process occurs at a defined time in the organism's life cycle and involves steps of DNA sequence elimination, sequence addition, and sequence rearrangement. We plan to study this genome rearrangement process by constructing recombinant DNA clones of macronuclear DNA molecules and the regions of the micronuclear chromosome from which they are derived. These materials will be characterized in order to define DNA sequences that are retained and eliminated during development. We will also clone and characterize intermediate DNA forms generated during the reorganization process in order to determine the sequence of events or steps involved in producing gene-size macronuclear DNA molecules from micronuclear chromosomes. Finally, we will develop two systems that will allow us to study the particular DNA sequences and enzymes mediating genome reorganization. In the first, we will directly microinject cloned micronuclear DNA sequences into developing macronuclei and follow their fate in order to precisely define sequences involved in rearrangement. Our second approach will be to develop in vitro assays for enzymes involved in rearrangement to ultimately purify and characterize their activities.

尽管一些基因和基因组重排过程已经在真核生物中定义的，调节这些事件的机制人们对此了解甚少。我们计划研究广泛的基因组重组发生在下毛纤毛原虫中的重组过程，如作为Oxytricha nova和Stylonchia Mytilus，为了扩展我们的知识真核重排过程以及定义DNA的类型调节这类事件的序列和酶。在这些生物体中有一个拷贝的染色体微核基因组转化为大核含有基因大小的小DNA分子。这种基因组重排过程发生在生物体生命周期的特定时间，包括 DNA序列消除、序列添加和序列测定的步骤重新编排。我们计划研究这种基因组重排过程，通过构建大核DNA分子的重组DNA克隆及其区域它们衍生出的微核染色体。这些材料将以确定保留的DNA序列和在开发过程中被淘汰。我们还将克隆和刻画重组过程中产生的中间DNA形式按顺序确定生产过程中涉及的事件或步骤的顺序来自微核染色体的基因大小的大核DNA分子。最后，我们将开发两个系统，使我们能够研究介导基因组重组的特定DNA序列和酶。在……里面首先，我们将直接显微注射克隆的微核DNA序列发展成大核并跟随它们的命运，以便准确地定义重排过程中涉及的序列。我们的第二个方法将是建立参与重排的酶的体外检测方法，最终提纯并描述他们的活动。