Nuclear inheritance and genetic exchange in Giardia intestinalis, a divergent eukaryote with two nuclei
The divergent eukaryotic parasite Giardia intestinalis is a major cause of diarrheal disease worldwide. The Giardia life cycle consists of two major stages: the binucleate trophozoite, which is found attached to the wall of the small intestine, and the infectious cyst, which is able to persist for weeks in the environment. The two nuclei in the trophozoite are equivalent and remain independent during mitosis. Although Giardia is presumed to be asexual based on the lack of an observed sexual cycle or gametes, its genome contains low levels of heterozygosity. This identity between nuclei is surprising because an asexual organism with two nuclei would be expected to accumulate differences due to genetic drift.
Interestingly, the Giardia genome also contains seven homologs of meiosis-specific genes (HMGs): Spo11, Hop1, Dmc1a, Dmc1b, Hop2, Mnd1, and Mer3. In sexual organisms, these genes are specifically required for meiotic homologous recombination. Is Giardia using these genes for a cryptic sexual or parasexual process? Or, as an early-diverging eukaryote, is it using them for a process such as DNA damage repair, which is thought to have been the predecessor of meiotic homologous recombination? Furthermore, how is Giardia able to maintain two independent nuclei with low levels of heterozygosity? In this dissertation, I describe my efforts during my graduate studies to answer these questions. To do so, I set out to examine the functions of the HMGs in Giardia and to determine whether genetic exchange occurs between nuclei at some point in the Giardia life cycle.
Before I could test the functions of the HMGs, I needed molecular tools for gene knockdown in Giardia. Attempts to knock down genes in Giardia using RNAi have not been successful, and knockouts are infeasible due the parasite's tetraploid genome content. Thus, in Chapter 2, I describe my development of morpholinos as a new tool for gene knockdown in Giardia. When electroporated into trophozoites, these translation-blocking oligonucleotides are able to reduce the protein levels of their targets for up to 72 hours.
Next, I examined the behavior of the nuclei and cytoskeleton throughout the Giardia life cycle, with the goal of determining whether the quadrinucleate cyst is formed by a mitotic division or cell fusion and whether the nuclear pairs remain associated throughout encystation and excystation. In Chapter 3, I describe my use of cell mixing experiments and immunofluorescence staining of cytoskeletal components to demonstrate that cysts are formed by an incomplete mitotic division, and the parental nuclear pairs remain associated throughout encystation and excystation. Thus, any genetic exchange in the cyst occurs between nuclei derived from the same parent cell, and nuclear sorting does not appear to be a method by which Giardia reduces heterozygosity between nuclei.
Finally, in Chapter 4, I demonstrate that nuclear fusion and genetic exchange occur between nuclei in the cyst, a process we have termed "diplomixis." Although three of the HMGs (Dmc1b, Hop2, Mnd1) are expressed in the trophozoite, suggesting that they may play roles in somatic DNA damage repair/recombination, three HMGs (Spo11, Hop1, and Dmc1a) are only expressed in the cyst. Additionally, using integrated chromosomal markers, I have demonstrated that the nuclei fuse and exchange genetic material during encystation. Homologous recombination between nuclei, presumably catalyzed by the HMGs, could be the mechanism by which Giardia maintains low levels of allelic heterozygosity. These findings are significant not only for our knowledge of the basic biology and life cycle of this important parasite, but also for our understanding of the evolution of eukaryotic DNA damage repair and meiosis.