A local, high-density, single-nucleotide polymorphism map used to clone Caenorhabditis elegans cdf-1.
Ras-mediated signaling is required for induction of vulval cell fates during Caenorhabditis elegans development. By screening for suppressors of the multivulva phenotype caused by constitutively active let-60 ras, we identified the mutation n2527. To clone the gene affected by n2527, we developed a method for high-resolution mapping. We took advantage of the genomic DNA sequence of the N2 strain by using DNA sequencing to scan for single-nucleotide polymorphisms (SNPs) at defined genomic positions of the RC301 strain. An average of one polymorphism per 1.4 kb was detected in predicted intergenic regions. Because of this high frequency, DNA sequencing is an efficient method to scan for SNPs. By alternating between identifying SNPs and mapping n2527 using selected recombinants, we generated an SNP map of progressively higher density. An intensive search for SNPs resulted in a local map with an average marker spacing of approximately 4 kb. This was used to map n2527 to a 9.6-kb interval. The small size of this interval made it feasible to use DNA sequencing to identify the molecular lesion. In principle, this approach can be used for high-resolution mapping of any C. elegans mutation. Furthermore, this approach can be applied to other species as the genomic sequence becomes available. The n2527 mutation affects a previously uncharacterized gene that we named cdf-1, as it encodes a predicted protein with significant similarity to members of the cation diffusion facilitator family.
Jakubowski
J
J
Department of Molecular Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.
Kornfeld
K
K
D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans / Q000235:genetics*; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D017931:DNA Primers; D018509:DNA, Helminth / Q000235:genetics; D005260:Female; D016366:Open Reading Frames; D010641:Phenotype; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D014844:Vulva
D017931:DNA Primers; D018509:DNA, Helminth