Animals homozygous for mutations in the unc-23 gene exhibit progressive dystrophy of the anterior body wall musculature and have a diagnostic bent-head phenotype. This phenotype is not observed when animals are grown in liquid culture (Bullerjahn and Riddle, pers. Comm.). Neither muscle cell positioning nor myofilament assembly is affected in liquid grown unc-23 animals. Muscle cell attachment, however, is affected since a small amount of stress applied results in detachment of the muscle cells from the hypodermis. The results of immunological staining of unc-23 animals with antibodies to basement membrane and hypodermal components suggests that the primary defect in unc-23 animals is located within the hypodermis. We have used PCR/deficiency mapping to molecularly map unc-23 to a region of nine cosmids. Our transformation rescue attempts, using the cosmids within this region, has to date been unsuccessful. We are now adding other methods such as YAC injections and RNAi to our list of approaches in order to rescue unc-23 . To further characterize the role of unc-23 in muscle cell attachment and identify other interacting proteins, we screened for suppressors of unc-23(e25) animals. We have identified 7 dominant and 5 recessive suppressors. We have determined that four of these are intergenic suppressors. We will report on the mapping and further characterization of these suppressors.

Au, V. et al. (2019) International Worm Meeting "Identifying and characterizing novel targets for anthelmintic development in C. elegans."

Moerman, D.G., Au, V.

[

International Worm Meeting,

2019]

Parasitic nematodes affect agricultural crops, livestock, and over a billion people worldwide. Mass drug adminstration programs are currently being employed in developing countries to treat and prevent human helminthiasis. Although temporarily effective, this approach is predicted to lead to the development of anthelmintic resistance. Extensive use of the few available classes of anthelmintics in agriculture has already created widespread resistance to these drugs. Having a larger repertoire of drugs to treat helminthiasis may curb the anticipated rise in drug resistance. One possible approach to find novel anthelmintics is to identify and characterize novel targets for drug development. Using WormBase Parasite, we identified 384 nematode-specific orthologues between C. elegansand eight parasitic nematodes. Of these orthologues, 45 are putative druggable targets. A handful of these targets were knocked-out or over-expressed to mimic the effects of receptor antagonists or agonists, respectively. These mutants were phenotyped for lethality, morphological differences, impaired locomotion, and reduced fecundity. ChEMBL, a large database of bioactive compounds and their targets, allow us to predict which compounds may interact with these putative anthelmintic drug targets based on sequence similarity and known interactions with related targets. These molecules may then be validated in C. elegans, and ultimately in parasitic nematodes, leading to the discovery of new drugs for the treatment of helminth infections.

Mok, C.K. et al. (2019) International Worm Meeting "PhenoMIP: a deep phenotyping approach to assay C. elegans gene function."

Moerman, D.G., Belmarez, G, Mok, C.K., Edgley, M, Waterston, R.H.

[

International Worm Meeting,

2019]

The C. elegans genome is compact, measuring just over 100MB. With approximately 20,000 predicted genes, this eukaryotic genome can generate a complex organism with a highly-predictable and well-defined cell lineage. Despite the depth of knowledge regarding this organism, the function of nearly half of its genes remains unclear. Although there are many forward and reverse genetic techniques available, all of them require culturing each strain individually. To improve the ability to phenotypically characterize genetic variants, we sought to develop a method to assay many variants in parallel. [AR1] The Million Mutation Project (MMP) yielded a library of 2007 mutagenized strains and 40 wild isolates with fully sequenced genomes encompassing in toto more than 1.4M single nucleotide variants. We hypothesized that a number of these mutations could impact organismal fitness across a wide range of environmental conditions. Based on genomic data, we designed a series of molecular inversion probes (MIPs) that targeted strain-specific variants. We naively pooled sets of 40-60 MMP strains or wild isolates and cultured these for four to ten generations under conditions that varied by food source or temperature. Using the phenotype of population fitness, we analysed overall population composition at multiple timepoints via sequencing (phenoMIPs). Our analysis of phenoMIP libraries, generated population growth phenotypes allowing us to assign strains into one of multiple fitness categories. From this data, we mapped several slow-growth mutants to specific loci via MIP-MAP. We further observed strain-specific alterations to population fitness that were dependent upon culture conditions. Amongst the wild isolate strains, we also tested for RNAi-specific intra-strain growth differences to identify potential genetic interactions. In particular we identified a suppression of lethality on emb-27 RNAi by the wild isolate ED3052 and mapped this to the left arm of LGIII. By phenoMIPs, we examined 202 MMP strains grown across 8 pooling experiments. Our data suggests that amongst the MMP mutant strains, there may be as many as 77% of strains with reduced fitness [AR2] phenotypes on one of three common bacterial food sources. We believe that phenoMIPs is a robust, cost-efficient methodology for identifying differential fitness phenotypes that could further elucidate the function of the many unexplored genes and regulatory regions within the C. elegans genome.

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