Investigating the expression of PEZO-1 protein and its involvement with the Emo oocyte phenotype in C. elegans Open Access

Lee, Esther Hae-Min (2016)

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In the nematode Caenohabiditis elegans, ovulation is usually tightly coupled to fertilization in the spermatheca after which, like many other animals including humans, meiosis is completed and the zygote begins embryonic development. When defective sperm are present, fertilization in the spermatheca does not occur shortly after ovulation. Such ovulated eggs exit meiosis and begin repeated rounds of mitotic-like replication cycles of their DNA in the absence of cell division, a process named endomitosis (Emo). I investigated the Emo oocyte phenotype and its relationship to the mechanosensory protein PEZO-1 encoded by the C10C5.1 gene in C. elegans. Prior work suggested that loss of C10C5.1 function resulted in a nonEmo phenotype, where oocytes would remain paused in meiosis I, even after ovulation and exiting the spermatheca. The C10C5.1 gene is the first gene of a four-gene CEOP4328 operon. I have used million mutation strains, crosses, RT-PCR, and RNAi methods to ensure that only the C10C5.1 gene is involved with the Emo phenotype in C. elegans. I have also begun to develop tools that will allow analyses of tissue-specific PEZO-1 expression in the germline and somatic tissues of C. elegans.

Table of Contents

Chapter 1: Introduction (1)

A. Caenorhabditis elegans Reproductive System (1)

B.C. elegans as model organism and in vitro fertilization (3)

C. Discovery of the nonEmo phenotype and genetic analysis identifying C10C5.1 as the affected gene (6)

D. C10C5.1 and PEZO-1 mechanosensory protein (9)

Chapter 2: Confirming that C10C5.1 is the only gene in CEOP4328 Operon Involved in the nonEmo Phenotype (12)

A. Introduction (12)

B. Methods (13)

C. Results and discussion (14)

Chapter 3: Creation of OD95 fer-1(b232ts) I; ltIs37 IV; ltIs38 strain for visualization of RNA interference (RNAi) caused oocyte phenotypes (15)

A. Introduction (15)

B. Methods (16)

C. Results (16)

D. Discussion (17)

Chapter 4: RNAi methods (18)

A) Introduction (18)

B) Use of eri-1 and rrf-3 to differentially enhance RNAi (19)

B.1) Methods (19)

B.2) Results and discussion (21)

C) Creation of a RNAi hairpin construct to selectively target PEZO-1 production in the germline (23)

C.1) Methods (24)

C.2) Results (25)

Chapter 5: Creation of a pie-1::sid-1::pie-1 expression clone to enhance RNAi in the Germline (26)

A) Introduction (26)

B) Methods (27)

C) Results (27)

D) Discussion (27)

Chapter 6: Conclusions and future directions (28)

Figures and References (32)

References (58)

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