Somatic Chromosome Origins and Organization in the Genome of Tetrahymena thermophila

在许多不同的生物中，包括脊椎动物免疫球蛋白和酵母交配型的基因，已经在基因水平上发现了发育中的基因组重排。纤毛原生动物提供了一个极端的例子，在这种情况下，整个基因组都会发生巨大的发育变化。所有有性能力的纤毛虫都含有两个独立的核，一个是沉默的生发微核，另一个是活跃转录的体细胞大核。嗜热四膜虫具有二倍体微核，具有5条中部着丝粒染色体，这些染色体经历减数分裂和有丝分裂，转录上是沉默的。它的大核是在广泛的基因组改变后接合过程中的微核衍生而来的，含有约45个拷贝的200条无丝分裂染色体，这些染色体已经丢失了基因组的10%-15%，由拷贝数校正系统维持，活跃地转录，并分离出碎片化基因组的单倍型。在这个由两部分组成的项目中，重排的位点将被分离出来，并用于在微核遗传图谱上定位大核基因组，从而创建两个相关的遗传图谱。在第一部分中，将创建不同大小的生发缺失，并将其映射到特定的微核染色体。由于微核不表达，杂核体中的缺失将保持不变，与纯合突变型微核和野生型大核一起，并相互杂交以识别重叠。来自缺失菌株的DNA将作为模板，用已经绘制的微核特异随机扩增序列制成的引物，将缺失的DNA放在现有的微核减数分裂图谱上。在第二部分中，染色体断裂位点(CBS)两侧的序列将被克隆和测序。这些位置发生在发育过程中从微核染色体中切下大核染色体的地方，因此标志着微核基因组中相邻的大核染色体的边界。微核特异DNA的范围将在每个克隆中确定，克隆将通过探测脉冲场电泳胶来识别相邻的大核染色体。大核染色体的长度和连接处的微核特定序列将结合在一起，以创建投影在微核基因组上的大核染色体的物理图谱。这个项目的最终结果将是收集一系列非常有用的遗传试剂(缺失集)，并将物理距离和特定的大核位置添加到四膜虫的遗传图谱中。这些信息将极大地促进位置克隆和互补克隆。此外，所有断裂点的序列收集将增加我们对这种发育导向的染色体重排的了解。

Developmental genome rearrangements have been found at the gene level in a number of diverse organisms, including genes for vertebrate immunoglobin and yeast mating type. The ciliated protozoa provide an extreme case, in which an entire genome is subject to massive developmental alteration. All sexually competent ciliates contain two separate nuclei, a silent germinal micronucleus and an actively transcribed somatic macronucleus. Tetrahymena thermophila has a diploid micronucleus with five metacentric chromosomes that undergo meiosis and mitosis and are transcriptionally silent. It's macronucleus, derived from the micronucleus during conjugation following extensive genome alteration, contains about 45 copies of 200 amitotic chromosomes that have lost 10-15% of the genome, are maintained by a copy number correction system, are actively transcribed, and segregate haplotypes of the fragmented genome. In this two-part project, the sites for the rearrangement will be isolated and used to locate the macronuclear genome on the micronuclear genetic map, creating two relational genetic maps. In the first part, germinal deletions of various sizes will be created and mapped to specific micronuclear chromosomes. Because the micronucleus is not expressed, deletions will be maintained in heterokaryons, with homozygous mutant micronuclei and wild type macronuclei, and crossed to each other to identify overlaps. DNA from deletion strains will be used as template for PCR, with primers made from already mapped micronuclear specific RAPD sequences, to place the deletions on the existing micronuclear meiotic map. In the second part, the sequences that flank chromosome breakage sites (cbs) will be cloned and sequenced. These sites occur where macronuclear chromosomes are cut out of micronuclear chromosomes during development, and therefore mark the boundaries of adjacent macronuclear chromosomes in the micronuclear genome. The extent of micronuclear specific DNA will be determined in each clone and the clones will be used to identify adjoining macronuclear chromosomes, by probing pulse field electrophoresis gels. Length of macronuclear chromosomes and micronuclear specific sequences at the junctions will be combined to create a physical map of the macronuclear chromosomes projected on the micronuclear genome. The meiotic map location of the breakage sites will be identified by the mapped deletions, created in the first part, to relate the physical and genetic maps.The end result of this project will be a collection of extremely useful genetic reagents (the deletion set) and the addition of physical distances and specific macronuclear locations to the genetic map of Tetrahymena. This information will greatly facilitate positional cloning and cloning by complementation. In addition, the collection of sequences at all the break points will add to our knowledge this developmentally directed chromosome rearrangement.