Functional Roles for Tetrahymena RAD51 During Conjugation and the Cell Cycle

这种有纤毛的原生动物拥有一种不同寻常的基因组组织，它有效地将体细胞和生殖系遗传功能的劳动分配给两个不同的细胞核。微核是生殖核，在营养生长过程中有丝分裂，是二倍体(四膜虫为2N=10)，转录上是沉默的。相比之下，大核是体细胞核，无性分裂，包含所有基因的多个副本，在转录上是活跃的。在四膜虫中，有性繁殖或接合是一个严格控制的过程，其进程在大约12个小时内遵循可预测的模式。在两个交配型相反的四膜虫细胞形成接合对后不久，微核经历了一系列的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，然后融合产生合子的二倍体核。在接合后期，亲本的大核被破坏，一个新的大核从合子微核的副本中发育出来。嗜热四膜虫的巨核发育是以预先编程和控制的方式进行的，它涉及一系列定点特异的染色体断裂和DNA缺失事件。四膜虫RAD51的表达在不同的环境条件和发育阶段不同。暴露于DNA损伤剂后，RAD51的表达增加。此外，RAD51的水平在营养细胞周期和接合过程中都有所不同。RAD51基因座的破坏会导致接合细胞不能完成减数分裂，并导致营养细胞周期缺陷，从而导致亚二倍体细胞的积累。生殖系纯合子RAD51缺失菌株的接合导致接合后的第一次营养细胞分裂受阻，导致停滞在脱离接合的发育阶段。为了更准确地破译Rad51p在接合过程中的作用，一个有条件的、温度敏感的(T.S.)四膜虫RAD51的等位基因将被鉴定。将对一株酵母RAD51缺失菌株进行质粒编码的四膜虫Rad51p的互补检测。成功的互补将使直接筛查四膜虫RAD51T.S成为可能。酵母中的等位基因。如果四膜虫RAD51同源物不能补充酵母RAD51缺失菌株，则将突变质粒编码的酵母RAD51，将其导入RAD51缺失菌株，并筛选T.S。等位基因。保守氨基酸的错义突变(S)，可授予T.S.酵母Rad51p的表型将被引入到四膜虫同系物中，随后将在四膜虫RAD51无效外接子中表达。RAD51条件突变体可以在有限的温度下在接合过程中失活，这将有助于深入了解四膜虫发育控制的基因组重排是如何介导的，包括那些导致rDNA回文形成的基因组重排。对这些发育控制的重组的更完整的理解将有助于深入了解其他生物如何实现类似的过程，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.