Peterson, Sagen et al. (2015) International Worm Meeting "Elucidating the mechanisms for purifying selection of mitochondrial DNA."

Goyal, Sidhartha, Torres Cleuren, Yamila, Peterson, Sagen, Rothman, Joel

[International Worm Meeting, 2015]

The mitochondrial genome (mtDNA) is present in many copies within each cell and follows a maternal pattern of inheritance. mtDNA experiences a high mutation rate which leads to heteroplasmy, in which a mixed population of both wildtype and altered mtDNA exist within the same cell. While each mtDNA can act as a "selfish DNA" element, purifying selection is used to eliminate from the germline mtDNAs that are defective and debilitate mitochondrial function. We are investigating the mechanisms used in purifying selection in the germline. One level at which purifying selection might act is during the early embryonic divisions that establish the germline (P lineage). We have obtained preliminary evidence that the most active mitochondria (those with the highest membrane potential) are enriched in the germline lineage during the early embryonic cell divisions. Following this early phase, we found that germline mitochondria are then fully quiescent (no detectable membrane potential) until after the initiation of larval development. We have taken advantage of a 3 kb mtDNA deletion, uaDf5 [Tsang et al 2002], to investigate further the mechanisms that remove defective mtDNAs. This deletion exhibits stable heteroplasmy, raising the suggestion that it is maintained as a result of complementing mutations on the non-deleted mtDNAs in the strain [Tsang et al 2002]. However, we found that the uaDf5-bearing strain appears to contain fully intact, wild-type mtDNA, suggesting that the deletion may be maintained as a result of replicative advantage. Further, we found that the uaDf5 strain gives rise to arresting embryos with many cell corpses, reminiscent of cells that have undergone programmed cell death. Lines that generate arresting embryos at highest frequency also show extended lifespan in the survivors, consistent with a trade-off between mitochondrial function required for survival and lifespan extension. Finally, we have obtained preliminary results suggesting that physiological state of worms influences the rate of removal of mtDNAs encoding defective proteins. We are investigating the trans-generational dynamics of this apparent purifying selection.

Benjamin Ross (2006) Development & Evolution Meeting "Depletion of vrk-1 by RNAi Produces Chromosome Segregation Defects and Embryonic Lethality in C. elegans"

Benjamin Ross

[Development & Evolution Meeting, 2006]

Residue-specific histone modifications correlate with biological phenomena (Peterson and Laniel 2004). Studies in Drosophila melanogaster have recently identified and characterized nucleasomal histone kinase-1 (NHK-1). NHK-1 has been shown to phosphorylate T119 of histone H2A. T119 is conserved from C. elegans to humans, and phospho-T119 correlates with condensed chromatin in mitosis. nhk-1 mutants have been found as a result of a female sterile screen in Drosophila. Mutant oocytes exhibit a range of defects in meiotic chromosomal organization, including aberrant polar bodies and spindle formation. (Aihara et al. 2004; Cullen et al. 2005; Ivanovska et al. 2005). We are interested in how chromosomes are prepared for entry into meiosis, and what the developmental and maturation consequences are of improper or incomplete preparation. This led us to examine vrk-1, a homolog of nhk-1. We subjected N2 L4s to RNAi by vrk-1 bacterial feeding, and observed 100% embryonic lethality in the F1 progeny. Dissected embryos stained with the centrosomal marker spd-2 and DAPI showed consistently 'messy' chromosome organization and segregation in early embryos. Embryonic phenotypes observed include anaphase bridging, stray chromosomes, misalignment at the metaphase plate, and de-condensation of metaphase chromosomes. In order to generate a more severe phenotype, we used a vrk-1 deletion allele, ok1181. Mutants homozygous for ok1181 are sterile, with vulval and male-tail defects, and appear to have a severely depleted germ-line. ok1181 heterozygotes subjected to vrk-1 RNAi produced 100% dead embryos. Staining of these embryos with DAPI and the SPD-2 antibody revealed an unusual phenotype. Embryos, which appeared to be multi-cellular by DIC imaging and by anti-actin immunostaining, exhibited extremely reduced DAPI staining, relative to un-fed control embryos. spd-2 centrosomal staining was normal in quantity, suggesting normal centrosome replication. We believe that the severe reduction in DNA seen in embryos laid by ok1181 heterozygotes represents a more complete depletion of the vrk-1 gene product by RNAi, compared to N2, with differences in phenotype potentially due to the diminished amount of maternally supplied vrk-1 message in embryos laid by ok1181/+ adults. Other data will be presented at the conference.