Functional Roles for Tetrahymena RAD51 During Conjugation and the Cell Cycle

纤毛原生动物拥有一个不寻常的基因组组织，它有效地将体细胞和生殖系遗传功能的劳动划分在两个不同的细胞核之间。微核是种系细胞核，在营养生长期间进行有丝分裂，是二倍体（四膜虫的2N = 10），并且是转录沉默的。相反，巨核是体细胞核，无丝分裂，包含所有基因的多个拷贝，并且转录活跃。在四膜虫中，有性繁殖或结合是一个严格控制的过程，其进程遵循一个可预测的模式，大约需要12个小时。当两个相对交配类型的四膜虫细胞形成偶联对后，微核经历一系列减数分裂和有丝分裂。在这些核分裂之后，接合体交换单倍体核，然后融合产生合子二倍体核。在结合后期，亲本大核被破坏，一个新的大核从合子微核的拷贝中发育出来。巨核发育以一种预先编程和控制的方式进行，涉及一系列位点特异性染色体断裂和DNA缺失事件。对嗜热四膜虫高度保守的DNA链转移蛋白Rad51（细菌recA重组酶的真核同源物）的功能进行了研究。四膜虫RAD51的表达在不同的环境条件和发育阶段有所不同。暴露于DNA损伤剂后，RAD51表达增加。此外，RAD51水平在营养细胞周期和接合过程中都有变化。体细胞RAD51位点的破坏导致偶联细胞无法完成减数分裂，营养细胞周期缺陷导致次二倍体细胞的积累。纯合子rad51零株的种系接合导致接合后的第一次营养细胞分裂受阻，导致接合发育阶段停滞。为了更精确地破译Rad51p在偶联过程中的作用，我们将鉴定四膜虫RAD51的一个条件温度敏感等位基因。质粒编码的四膜虫Rad51p将用于酵母rad51零菌株的互补测试。成功的互补将使直接筛选酵母四膜虫rad51ts等位基因成为可能。如果四膜虫RAD51同源物不能与酵母RAD51无效菌株互补，质粒编码的酵母RAD51将被诱变，引入到RAD51无效菌株中，并筛选t.s.等位基因。使酵母Rad51p具有ts表型的保守氨基酸的错义突变将被引入到四膜虫同源物中，随后在四膜虫rad510交换子中表达。一个可以在限制性温度下偶联过程中失活的RAD51条件突变体将为深入了解四膜虫中发育控制的基因组重排（包括导致rDNA回文形成的重排）是如何介导的提供线索。对这些发育控制的重组的更全面的了解将有助于了解在其他生物体中如何实现类似的过程，例如脊椎动物免疫球蛋白基因的重排。利用单细胞模式生物，如四膜虫，可以更容易地探索和理解DNA序列重排的机制。

The ciliated protozoa possess an unusual genome organization that effectively divides the labor of somatic and germline genetic functions between two distinct nuclei. The micronucleus is the germline nucleus, divides mitotically during vegetative growth, is diploid (2N = 10 for Tetrahymena), and is transcriptionally silent. In contrast, the macronucleus is the somatic nucleus, divides amitotically, contains multiple copies of all genes, and is transcriptionally active. Sexual reproduction, or conjugation, is a tightly controlled process in Tetrahymena whose progression follows a predictable pattern over approximately twelve hours. Soon after two Tetrahymena cells of opposite mating type form a conjugating pair, the micronuclei undergo a series of meiotic and mitotic divisions. Following these nuclear divisions, conjugants exchange haploid nuclei, which then fuse to produce a zygotic, diploid nucleus. Late in conjugation the parental macronuclei are destroyed, and a new macronucleus develops from a copy of the zygotic micronucleus. Macronuclear development proceeds in a pre-programmed and controlled manner that involves a series of site-specific chromosome breakage and DNA deletion events.A functional study of the highly conserved DNA strand transfer protein Rad51 (the eukaryotic homolog to the bacterial recA recombinase) from Tetrahymena thermophila has been initiated. Tetrahymena RAD51 expression varies under a number of different environmental conditions and developmental stages. RAD51 expression increases following exposure to DNA damaging agents. In addition, RAD51 levels vary during both the vegetative cell cycle and conjugation. Disruption of the somatic RAD51 locus results in failure of conjugating cells to complete meiosis, and in vegetative cell cycle defects that lead to an accumulation of hypodiploid cells. Conjugation of germline, homozygous rad51 null strains lead to a block of the first vegetative cell division following conjugation, resulting in an arrest at the exconjugant developmental stage. In order to more precisely decipher the role of Rad51p during conjugation, a conditional, temperature sensitive (t.s.) allele of Tetrahymena RAD51 will be identified. Plasmid-encoded Tetrahymena Rad51p will be tested for complementation of a yeast rad51 null strain. Successful complementation will make it possible to directly screen for Tetrahymena RAD51 t.s. alleles in yeast. If the Tetrahymena RAD51 homolog fails to complement a yeast rad51 null strain, plasmid-encoded yeast RAD51 will be mutagenized, introduced into a rad51 null strain, and screened for a t.s. allele. Missense mutations of conserved amino acid(s) that confer a t.s. phenotype to the yeast Rad51p will be introduced to the Tetrahymena homolog, which will subsequently be expressed in Tetrahymena rad51 null exconjugants. A RAD51 conditional mutant that can be inactivated during conjugation at the restrictive temperature will provide insight into how developmentally controlled genome rearrangements in Tetrahymena, including those leading to rDNA palindrome formation, are mediated.A more complete understanding of these developmentally controlled recombination will provide insight into how similar processes are achieved in other organisms, such as the rearrangement of vertebrate immunoglobulin genes. Mechanisms by which DNA sequences are rearranged can be more easily explored and understood using a single-celled model organism such as Tetrahymena.