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

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调控决定簇。 复制子（复制起始位点及其顺式作用调控决定簇）的彻底遗传解剖已在有限数量的实验生物中实现。 先前对嗜热四膜虫的研究集中在21 kb rDNA微型染色体上，该微型染色体在发育中的大核中的单个S期期间扩增5000倍，但在随后的每次营养细胞分裂期间仅复制一次。 这项新研究项目的目标是鉴定和解剖四膜虫中额外的染色体复制子。 通过比较未扩增的复制子与rDNA的遗传结构，可以深入了解细胞周期控制。 这些调查也将作为一个切入点，探讨如何保持基因平衡的纤毛虫大核，缺乏传统的有丝分裂分离机制。 该项目的初始阶段包括通过2D凝胶电泳在小的（~100 kb）非rDNA大核染色体中定位复制起点，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，以鉴定保守元件并确定复制起点与相邻基因的物理和可能的功能关系。 这些复制子的遗传组织将被使用两种互补的策略来剖析。 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。 在发育中的大核中的这些Cbs位点处的程序化DNA片段化将用于绘制所检查的复制子的物理限制。 作为这种方法的一种变体，将使用标记拯救来恢复缺陷复制子的功能。 在互补方法中，将来自大小选择的（IOOkb）染色体的限制性片段插入工程化的可选择载体中以产生质粒文库。 这些文库将被转化到四膜虫中，以鉴定能够支持自主复制的人工微型染色体。DNA复制是一个高度调节的过程，确保染色体复制一次，并且每次细胞分裂仅复制一次。 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）。 高等真核生物中染色体复制子的高度复杂性，以及这些系统中天然或人工微型染色体的缺乏阻碍了这一领域的研究。 四膜虫逆转遗传分析的顺从性将在这里被利用，以确定复杂的真核复制子是如何组织的，并探讨如何全球细胞周期调控机制可以差异复制起点。 这个项目的一个好处是实验方法的多样性，这将为学生提供新的（基因组学）和更传统的（分子遗传学）技术的接口独特的学习机会。

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.