Role of Meiotic Genes in the Basal Eukaryote Giadia lambia

描述(申请人提供)：这项拟议研究的目标是以肠道寄生虫蓝氏贾第鞭毛虫为模式生物研究减数分裂的进化。在进化方面，贾第鞭毛虫是可用于研究的最基本的真核生物，它最后一次与人类或酵母共享共同祖先是在15亿年前；因此，它是这个项目的理想模式生物。贾第鞭毛虫被认为是一种无性生物，但最近在其基因组中发现的减数分裂基因同源基因增加了贾第虫经历减数分裂或与减数分裂相关的进化过程的可能性。减数分裂双链断裂修复(DSBR)和同源染色体间同源重组(HR)所涉及的基因和过程是从体细胞修复DNA损伤的过程进化而来的。因此，这些候选减数分裂基因(CMG)在贾第虫中的存在使人们能够在进化的背景下研究DNA损伤修复的机制。了解这些机制很重要，因为许多基因的突变通常涉及DNA损伤修复和减数分裂导致基因组不稳定和癌症。本研究的具体目的是：(1)寻找贾第鞭毛虫类减数分裂过程的分子证据。(2)寻找贾第鞭毛虫减数分裂样过程的细胞学证据。(3)寻找贾第鞭毛虫在体内外交配的证据。GFP表达数据显示，6个CMG显示出与减数分裂类似功能一致的时间和空间表达模式。将通过创造显性/负性突变体来研究参与DNA损伤/修复和核融合的选定CMG的生物学功能。初步的荧光原位杂交(FISH)数据支持囊内发生核融合事件。通过对激子进行FISH，以及使用免疫荧光显微镜和透射电子显微镜监测包囊过程中的核行为，将完成核融合和核之间核材料交换的更多证据。将利用小鼠模型系统寻找贾第鞭毛虫减数分裂事件的活体证据。由于DNA损伤是在减数分裂过程中诱导的，因此这些过程的修改可能是致癌的。如果DNA修复机制失效，DSB的诱导可能对细胞有害，并可能导致基因组改变。该项目将极大地提高DNA损伤修复领域的知识，因为它与基因组稳定性有关。总体而言，该项目将有助于更深入地了解DNA损伤修复途径中涉及的减数分裂过程的进化。

DESCRIPTION (provided by applicant): The goal of this proposed research is to study the evolution of meiosis using the intestinal parasite Giardia lamblia as a model organism. Evolutionary, Giardia is the most basal eukaryote available for study having last shared a common ancestor with man or the yeasts over 1.5 billion years ago; thus it is an ideal model organism for this project. Giardia is believed to be an asexual organism, but the recent discovery of meiotic gene homologs in its genome raises the possibility that Giardia either undergoes meiosis or a process that is evolutionary related to meiosis. It has been proposed that the genes and processes involved in meiotic double stranded break repair (DSBR) and homologous recombination (HR) between homologous chromosomes evolved from processes used by somatic cells to repair DNA damage. Therefore, the presence of these candidate meiotic genes (CMGs) in Giardia allows the study of the mechanisms involved in repairing DNA damage in an evolutionary context. Understanding these mechanisms is important because mutations in many genes normally involved in DNA damage repair and meiosis lead to genome instability and cancer. The specific aims of this research are: (1) To discover molecular evidence for meiotic-like processes in Giardia. (2) To discover cytological evidence for meiotic- like processes in Giardia. (3) To search for evidence of Giardia mating both in vitro and in vivo. GFP expression data reveal that six CMGs show temporal and spatial expression patterns consistent with a meiotic-like function. The biological functions of selected CMGs involved in DNA damage/repair and nuclear fusion will be investigated by creating dominant/negative mutants. Preliminary fluorescent in situ hybridization (FISH) data support a nuclear fusion event in cysts. Additional evidence of nuclear fusion and exchange of nuclear material between nuclei will be accomplished by performing FISH on excyzoites, as well as monitoring nuclear behavior during the encystation process using both immunofluorescent microscopy and TEM. In vivo evidence of a meiotic event in Giardia will be sought with the use of a mouse model system. Because DNA damage is induced in cells undergoing meiosis, modifications of these processes can be oncogenic. If DNA repair mechanisms fail, the induction of DSBs can be deleterious to the cell and could result in genome alterations. This project will greatly enhance knowledge in the field of DNA damage repair as it relates to genome stability. Overall, this project will lead to a deeper understanding of the evolution of meiotic processes involved in DNA damage repair pathways.