Elucida(ng the Mechanism of Purifying Selec(on using C. elegans Adi( Trivedi, Sagen Peterson, Professor Joel Rothman Molecular, Cellular and Developmental Biology, UCSB Mitochondria are the main source of ATP producAon for most cells through a series of redox reacAons. Thus, healthy mitochondria are necessary for growth and survival. Mitochondrial DNA (mtDNA), encoding 12 proteins in these energy producing steps, follows a maternal inheritance paJern, but has a low generaAonal mutaAon rate. The low rate of inherited mtDNA mutaAons suggests the presence of a strong mtDNA selecAon pressure in the germline. Purifying selecAon, as this mechanism is called, ensures healthy mtDNA inheritance, reducing chances of progeny with defecAve mitochondria. We aim to study the rate of purifying selecAon with a focus on germline apoptosis, and to idenAfy the apoptoAc genes involved, beginning with ced3. Germline apoptosis and Purifying Selec(on During oogenesis in C. elegans, >90% potenAal oocytes are killed off, leaving a small subset of cells with mtDNA copies, to develop into mature oocytes 1 . Perhaps this selects against mutant mtDNA, eliminaAng them from the immortal germline. (Fig. 1) Q: How is germline apoptosis regula(ng mtDNA inheritance, and is this a key step of purifying selec(on? Drug Selec(on for N2 Strain In order to measure the rate of purifying selecAon using qPCR in WT and mtDNA mutant strains, it is necessary to develop a selecAon condiAon to differenAate between the progeny that have successfully uptaken WT mitochondria from the ones that have not. We are using a viable mtDNA mutant strain, JR3630, and the laboratory WT strain, N2, and treaAng them with drugs targeAng the mitochondrial respiratory chain (MRC). Gene(cs: MT1522 x JR3630 cross Figure 4: Plasmid map of C. elegans mtDNA showing uaDf5 deleAon 3 Figure 1: Proposed mechanism of purifying selecAon during germline apoptosis in one gonadal arm of C. elegans. Figure 2: Chloramphenicol as selecAon condiAon for N2 strain containing WT mtDNA Table 1: Summary of Drug Selection Screen – Day 3 Measurements Drug No Observable Difference Differential growth observed Concentration Type Chloramphenicol 10ug/mL, 500ug/mL, 1mg/mL JR3630 shows L3 arrest while N2 are gravid adults Doxycycline X Rotenone X Antimycin X Sodium Azide 10uM Reduced Survival of JR3630 Oligomycin X Paraquat 1mM 500mM Reduced health and survival of JR3630 Dinitrophenol (DNP) X CuCl 2 X Ethidium Bromide (EtBr) 25ug/mL JR3630 show slower rate of movement This ced3(n717) ; uaDf5//++ double mutant has nonfuncAonal CED3, a proapoptoAc protein involved in acAvaAon of the germline apoptoAc pathway. We will introduce mitochondria from the laboratory WT strain, N2, and observe levels of both types of mtDNA’s throughout subsequent generaAons using qPCR, to determine purifying selecAon rate changes due to the ced3 mutaAon. Figure 6a: DiagnosAc Digest of F2’s using uaDf5 primers Figure 6b: DiagnosAc Digest of F2’s using n717 primers All F2’s contain the 299 bp band (Fig. 6a) indicaAng presence of the 11 gene mtDNA deleAon, uaDf5. This is consistent with maternal inheritance paJerns. The 459 bp band (Fig.6b) indicates amplificaAon of the ced3 gene through PCR. Sanger sequencing will confirm the exact genotype and presence or absence of the n717 allele of ced3, which contains a point mutaAon. 1 2 3 4 5 6 7 Sodium Azide inhibits cytochrome c oxidase (MRC Complex IV) and Paraquat produces ReacAve Oxygen Species (ROS) 2 . Exposure to both drugs appear to effect the viability of the JR3630 strain more criAcally than the N2 strain. EtBr inhibits mtDNA replicaAon, and treatment of 25ug/mL produces mobility differences. The most consistent difference among concentraAons was seen with the mtDNA translaAon inhibitor, Chloramphenicol (Fig. 2). Molecular Cloning: Mitochondrial GFP to be expressed during early embryo cell division stages Insert + BamH1 Insert + BamH1 Insert + BamH1 + AatII Uncut Insert Figure 7: (Above) In order for mitochondrial GFP to be transcribed during the desired early embryo cell division stages, its sequence must be driven by the pie1 promoter. 5 Figure 8: (Leh) RestricAon digest of insert plasmid. Lane 4 shows the 1604 bp band consistent with mitLS + GFP insert sequence. 1. Gumienny TL, Lambie E, Hartwieg E, Horvitz RH, Hengartner MO. GeneAc control of programmed cell death in the caenorhabdi=s elegans hermaphrodite germline. (1999). Development. 126, 10111022. 2. ZeitounGhandour S, Leszcyszyn Ol, Blindauer CA, Geier FM, Bundy JG and Sturzenbaum SR. C. elegans metallothioneins: response to and defence against ROS toxicity. (2011) Mol Biosyst. 7, 2397406. 3. Tsang WY, and Lemire BD. Stable heteroplasmy but differenAal inheritance of a large mitochondrial deleAon in nematodes. (2002). Biochem. Cell Biol. 80, 645654. 4. Skjeldam HK, et al. Loss of Caenohabdi=s elegans UNG1 uracilDNA glycosylase affects apoptosis in response to DNA damaging agents. (2010). DNA Repair. 9, 861870. 5. Ghosh D, Seydoux G. InhibiAon of TranscripAon by the Caenorhabdi=s elegans germline protein PIE1: GeneAc Evidence for DisAnct Mechanisms TargeAng IniAaAon and ElongaAon Exposure to Chloramphenicol results in the most significant and consistent selecAon for N2 strain so far Will conAnue screen with Chloramphenicol in combinaAon with other MRC targeAng drugs ProducAon of ced3(n717); uaDf5//++ double mutant through molecular geneAcs cross Will be used in qPCR measurements to track rate and involvement of ced3 gene in purifying selecAon Plasmid construct through classical cloning and restricAon digests Will be confirmed through digests, and microinjected into C. elegans. I would like to thank Professor Joel Rothman for providing a unique and sAmulaAng undergraduate research opportunity, and Sagen Peterson for her mentorship and guidance in the lab. I also would like to thank the NIH for support, and the Arnold and Mabel Beckman FoundaAon for the Beckman Scholars Award. Abstract Experimental Studies and Results Introduc(on Acknowledgements Concluding Remarks and Future Direc(ons References Figure 5: CED3 is a proapoptoAc protein in the germline 4 n717 459 bp − uaDf5 299 bp− 1 2 3 4 5 6 7 1 2 3 4 5 6 7 4574 bp− 1604 bp− Figure 3: (Above) The uaDf5 deleAon mutaAon follows maternal Inheritance paJerns, while the ced3 point mutaAon follows a Mendelian Inheritance mode.