In the past year, we found that using a combination of whole-genome sequencing and SNP (WGS/SNP) mapping is an efficient and relatively rapid technique for the identification of molecular lesions, even in cases where gene mapping is not precise (Jaramillo-Lambert et al., 2015 G3). Using this technique we screened mutants previously characterized as maternal-effect embryonic-lethal but were rescued for viability when mated to wild-type males, suggesting either a sperm or zygotic defect. We identified a new mutation that causes sperm-specific embryonic lethality, it7; mutant males sire dead progeny when mated to wild-type females. Through cell biological analysis we have shown that it7 animals make anucleate sperm. The primary spermatocytes appear to attempt the meiotic divisions but the chromosomes fail to segregate, resulting in anucleate sperm that are capable of fertilizing oocytes; such haploid embryos die during early development. With the help of WGS/SNP mapping we discovered that it7 is an allele of top-2, the C. elegans ortholog of human topoisomerase II, an enzyme that resolves DNA entanglements. We now aim to characterize the male-specific function of TOP-2 during meiosis. Alongside the top-2 work we have continued to study the only strict paternal-effect lethal (PEL) mutant previously identified in C. elegans, SPE-11, through structure function analysis. SPE-11 is a 299 amino acid protein with no obvious protein domains. SPE-11 does have five predicted nuclear localization sequences (NLS) and localizes to germ-line nuclei of the male. We used a GFP::SPE-11 reporter construct that was generated in our lab to analyze mutated NLS and C-terminal deletion constructs for proper SPE-11 localization and rescue of a spe-11 deletion mutant. Previously, we found that NLS1 was neither required for proper localization of SPE-11 nor required for proper function of SPE-11 in the embryo. However, when NLS5 is mutated, SPE-11 is mislocalized and fails to rescue the spe-11 deletion mutant. Interestingly, NLS5 resides in the C-terminus of the SPE-11 protein. Curiously, the majority of existing spe-11 mutant alleles result in C-terminal truncations of the SPE-11 protein, suggesting that the C-terminus is very important for function. To determine which part of the C-terminus is essential for early embryogenesis, we created transgenic lines carrying constructs with truncated versions of SPE-11. We previously showed that two constructs with truncated versions of SPE-11 (GFP::SPE-11 1-289 and GFP::SPE-11 1-294) fail to rescue the embryonic lethality of the spe-11 deletion mutant suggesting that even the most C-terminal end of the protein (i.e. the last five amino acids) are essential for function. Further analysis has identified three critical amino acids in the C-terminus that are required for function. We are now working on identifying SPE-11 interacting proteins through co-immunoprecipitation experiments with the known components of the egg-activation complex and through mass spectrometry. Once interacting proteins are identified we will ask whether the critical C-terminal residues are required for protein-protein interactions.
| Stein, Kathryn K; Golden, Andy (2018) The C. elegans eggshell. WormBook 2018:1-36 |
| Jaramillo-Lambert, Aimee; Fabritius, Amy S; Hansen, Tyler J et al. (2016) The Identification of a Novel Mutant Allele of topoisomerase II in Caenorhabditis elegans Reveals a Unique Role in Chromosome Segregation During Spermatogenesis. Genetics 204:1407-1422 |
| Smith, Harold E; Fabritius, Amy S; Jaramillo-Lambert, Aimee et al. (2016) Mapping Challenging Mutations by Whole-Genome Sequencing. G3 (Bethesda) 6:1297-304 |
| Singaravelu, Gunasekaran; Rahimi, Sina; Krauchunas, Amber et al. (2015) Forward Genetics Identifies a Requirement for the Izumo-like Immunoglobulin Superfamily spe-45 Gene in Caenorhabditis elegans Fertilization. Curr Biol 25:3220-4 |
| Jaramillo-Lambert, Aimee; Fuchsman, Abigail S; Fabritius, Amy S et al. (2015) Rapid and Efficient Identification of Caenorhabditis elegans Legacy Mutations Using Hawaiian SNP-Based Mapping and Whole-Genome Sequencing. G3 (Bethesda) 5:1007-19 |
