Chromosome Homeostasis: Identification and Functional Analysis of Chromosomal Replicons in the Amitotic Tetrahymena Macronucleus

在真核生物中，对复制起始的调控知之甚少，主要是由于难以识别顺式作用的调控决定因素。在有限数量的实验生物体中，对复制子（复制起始位点及其顺式调控决定因子）进行了彻底的遗传解剖。先前对嗜热四膜虫的研究主要集中在21 kb rDNA的小染色体上，该染色体在发育中的大核的单个S期扩增5000倍，但在随后的每次营养细胞分裂中只复制一次。这个新的研究项目的目标是鉴定和解剖额外的染色体复制子在四膜虫。通过比较未扩增到rDNA的复制子的遗传组织，可以获得对细胞周期控制的见解。这些研究也将作为一个切入点，探索在缺乏传统有丝分裂分离机制的纤毛虫大核中如何维持基因平衡。该项目的初始阶段包括通过2D凝胶电泳绘制小（~100 kb）非rdna大核染色体的复制起点，并克隆跨越这些位点的DNA。这些区域将进行比较序列和功能基因组分析，以确定保守的元素，并确定复制起源与邻近基因的物理和可能的功能关系。然后，这些复制子的遗传组织将使用两种互补的策略进行剖析。第一种方法将采用一种新的方法，其中染色体断裂序列（Cbs）元件将靶向生殖系（微核）基因组中的特定位置。在发育中的宏核中这些Cbs位点上的程序化DNA片段将用于绘制所检查的复制子的物理极限。作为这种方法的一种变体，标记拯救将用于恢复有缺陷的复制子的功能。在一种互补的方法中，从大小选择（100 kb）染色体的限制性内切片段将插入到工程选择载体中以生成质粒文库。这些文库将转化为四膜虫，以鉴定能够支持自主复制的人工小染色体。DNA复制是一个高度调控的过程，确保每次细胞分裂染色体复制一次且仅复制一次。偏离严格的细胞周期控制可导致小DNA片段的选择性扩增（过度复制）。高等真核生物染色体复制子的高度复杂性以及这些系统中缺乏天然或人工小染色体阻碍了这一领域的研究。四膜虫对反向遗传分析的适应性将在这里被利用来确定复杂的真核生物复制子是如何组织的，并探索全球细胞周期调节机制是如何不同地施加于复制起源的。这个项目的一个好处是实验方法的多样性，这将为学生提供独特的学习机会，在新的（基因组学）和更传统的（分子遗传学）技术的接口。

The regulation of replication initiation is poorly understood in eukaryotes, largely due to the difficulty in identifying cis-acting regulatory determinants. A thorough genetic dissection of replicons (replication initiation sites and their cis-acting regulatory determinants) has been achieved in a limited number of experimental organisms. Prior research on Tetrahymena thermophila focused on the 21 kb rDNA minichromosome, which is amplified 5000-fold during a single S phase in the developing macronucleus, yet is replicated just once during each subsequent vegetative cell division. The goal of this new research project is to identify and dissect additional chromosomal replicons in Tetrahymena. By comparing the genetic organization of replicons that are not amplified to the rDNA, insights into cell cycle control should be obtained. These investigations will also serve as an entry point to explore how genic balance is maintained in the ciliate macronucleus, which lacks traditional mitotic segregation machinery. The initial stage of the project involves the mapping of replication origins in small (~100 kb), non-rDNA macronuclear chromosomes by 2D gel electrophoresis, and cloning of DNA that spans these sites. These regions will be subjected to comparative sequence and functional genomic analysis, to identify conserved elements and determine the physical and possibly functional relationship of replication origins to neighboring genes. The genetic organization of these replicons will then be dissected using two complementary strategies. The first approach will employ a novel method in which chromosome breakage sequence (Cbs) elements will be targeted to specific positions in the germline (micronuclear) genome. Programmed DNA fragmentation at these Cbs sites in the developing macronucleus will be used to map the physical limits of the replicons under examination. As a variation to this approach, marker rescue will be used to restore function to defective replicons. In a complementary approach, restriction fragments from size-selected (100 kb) chromosomes will be inserted into an engineered selectable vector to generate plasmid libraries. These libraries will be transformed into Tetrahymena to identify artificial minichromosomes capable of supporting autonomous replication.DNA replication is a highly regulated process, assuring that chromosomes are duplicated once and only once per cell division. Deviations from strict cell cycle control can result in selective amplification (over-replication) of small DNA segments. The high degree of complexity of chromosomal replicons in higher eukaryotes, and the absence of natural or artificial minichromosomes in these systems has hampered research in this area. The amenability of Tetrahymena to reverse genetic analysis will be exploited here to determine how complex eukaryotic replicons are organized and to explore how global cell cycle regulatory mechanisms can be differentially imposed upon replication origins. A bonus of this project is the diversity of experimental approaches, which will provide students with unique learning opportunities at the interface of new (genomics) and more traditional (molecular genetic) technologies.