In the past year, we performed a non-complementation screen in hopes of recovering a strong temperature-sensitive allele of spe-11 that could be used for a genetic suppressor screen. Thus far, we have recovered three new alleles of spe-11. All were found to be non-conditional alleles that produce truncated protein products, like most of the existing spe-11 alleles. These new alleles behave like the reference allele and thus we believe they too are null alleles. We are continuing this screen in hopes of identifying a temperature-sensitive allele of spe-11 that can be used in a suppressor screen. Alternatively, we hope to at least identify a missense allele, which would also be a good candidate for a suppressor screen. In another approach to isolate missense alleles of spe-11, we have been using the TALEN technology. This approach holds great promise for C. elegans as it should allow us to target specific regions of any gene for deletions and missense mutations. We have generated spe-11 constructs and are currently injecting them to generate lines. In the absence of the ideal conditional allele for a suppressor screen, we have initiated a biochemical approach to identify proteins that physically interact with the SPE-11 protein. We will purify SPE-11 from lysates using a monoclonal antibody we recently generated. Alternatively, we will also purify epitope-tagged SPE-11 from transgenic animals expressing epitope-tagged SPE-11. The purified SPE-11 will be subjected to mass spectrometry to identify factors that interact. Once such factors are identified, we will validate those genes using RNAi to determine whether they have spe-11-like phenotypes. We will also generate GFP transgenes to then determine the expression of these proteins in live animals. The above genetic and biochemical approach should help identify factors that function with SPE-11. Given that SPE-11 is a novel protein, the identification of its genetic and physical partners will shed light on the mechanisms by which SPE-11 controls eggshell development and early embryogenesis. Our non-complementation screen also has led us to consider another approach to identify SPE-11 interactors. The screen has suggested the existence of non-allelic non-complementing alleles (enhancer mutants) and thus we are also planning to isolate these enhancer alleles in the future as they may be quite informative as to the genetic pathways in which spe-11 functions. We have recently initiated a structure-function study to determine which regions or domains of SPE-11 are essential for development. Since we have spe-11 transgenes that completely rescue its various phenotypes, we can now test whether SPE-11 variants with specific domains deleted are still capable of rescue. These studies are underway. We are also pursuing a study to determine whether a few maternal effect lethal (mel) mutants identified over 30 years ago might really be paternal effect lethal (pel) mutants. A few mel mutants were shown to be rescued by male mating, suggesting that wild-type sperm can rescue the embryonic lethal phenotype. This could be interpreted to mean that the original mutant is actually a pel mutant and not a mel mutant. The ultimate test is to show that mutant males sire dead progeny when mated to wild type hermaphrodites (or females). We are currently pursuing three mel mutants that we suspect might really be pel mutants. Once confirmed, we will further characterize the pel phenotypes, determine expression patterns, and start suppression screens on those that are temperature-sensitive. We hope to also determine whether these genes function with spe-11 or control distinct processes.
| 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 |
