Identifying and Mapping Parallel Mutations of GAR-3

Abstract

This research identifies a gene that potentially works acetylcholine receptor protein GAR-3. We isolated nematodes (roundworm) Caenorhabditis elegans that are resistant to the drug arecoline, which stimulates behaviors such as spicule protraction (mating behavior). From the isolated worms we created about thirty recombinants, crossing arecoline resistant mutants genetically wildtype worms, and collected their DNA. Testing the chromosomes at different loci can identify a locus that contains mutant DNA; regions of the chromosome that display wildtype DNA are ruled out. An online catalog of single nucleotide polymorphisms allows us to distinguish between the wildtype (HA) and mutant (N2) DNA; there are regions that contain a single base pair difference between the N2 and HA samples. We amplified many DNA regions with polymerase chain reaction and tested each region with a restriction endonuclease that cut either the wildtype or the mutant DNA. We observed each recombinant’s restriction pattern on agarose gel to determine which regions contain 100 percent mutant DNA and could thus statistically be the site of the mutation. We believe the mutation lies in a region on the left end of the X chromosome, based on a larger proportion of N2 DNA compared to regions on the rest of the chromosomes. We believe we can use this data to narrow down the genes in this region and pinpoint which mutation is causing the nonfunctional protein.