Abstract
Caenorhabditis elegans, especially the N2 isolate, is an invaluable biological model system. Numerous additional natural C. elegans isolates have been shown to have unexpected genotypic and phenotypic variations which has encouraged researchers to use next generation sequencing methodology to develop a more complete picture of genotypic variations among the isolates. To understand the phenotypic effects of a genomic variation (GV) on a single gene, in a variation-rich genetic background, one should analyze that particular GV in a well understood genetic background. In C. elegans, the analysis is usually done in N2, which requires extensive crossing to bring in the GV. This can be a very time consuming procedure thus it is important to establish a fast and efficient approach to test the effect of GVs from different isolates in N2. Here we use a Mos1-mediated single-copy insertion (MosSCI) method for phenotypic assessments of GVs from the variation-rich Hawaiian strain CB4856 in N2. Specifically, we investigate effects of variations identified in the CB4856 strain on tac-1 which is an essential gene that is necessary for mitotic spindle elongation and pronuclear migration. We show the usefulness of the MosSCI method by using EU1004 tac-1(or402) as a control. or402 is a temperature sensitive lethal allele within a well-conserved TACC domain (transforming acidic coiled-coil) that results in a leucine to phenylalanine change at amino acid 229. CB4856 contains a variation that affects the second exon of tac-1 causing a cysteine to tryptophan change at amino acid 94 also within the TACC domain. Using the MosSCI method, we analyze tac-1 from CB4856 in the N2 background and demonstrate that the C94W change, albeit significant, does not cause any obvious decrease in viability. This MosSCI method has proven to be a rapid and efficient way to analyze GVs.
Highlights
C. elegans is central to biomedical, molecular, cell and developmental biology research, and is among the best genetically and molecularly characterized and understood model organisms
We were interested in variations that are expected to cause a significant disruption of essential protein-coding genes. One such variation was identified within tac1 and we wanted to test the phenotype of this variation in the N2 background. tac-1 is an essential gene and the only member of the transforming acidic coiled-coil (TACC) protein family in C. elegans whose function is crucial for pronuclear migration and mitotic spindle elongation [30,31,32]
In CB4856 we identified a number of single nucleotide changes that affect tac-1
Summary
C. elegans is central to biomedical, molecular, cell and developmental biology research, and is among the best genetically and molecularly characterized and understood model organisms. Mos1-mediated single-copy insertion (MosSCI) is a recently developed method in C. elegans that allows integration of transgenes as single copies at a defined genomic site [27]. We used whole-genome sequencing to identify GVs that disrupt protein-coding genes in CB4856 which is a wildisolate strain of C. elegans from Hawaii (Vergara, Tarailo-Graovac, et al in preparation).
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