Fly-TILL: reverse genetics using a living point mutation resource.
Mutagenesis with ethylmethanesulfonate (EMS) has been the standard for traditional genetic screens, and in recent years has been applied to reverse genetics. However, reverse-genetic strategies require maintaining a viable germline library so that mutations that are discovered can subsequently be recovered. In applying our TILLING (Targeting Induced Local Lesions IN Genomes) method to establish a Drosophila reverse-genetic service (Fly-TILL), we chose to screen the Zuker lines, a large collection of EMS-mutagenized second- and third-chromosome balanced lines that had been established for forward-genetic screening. For the past four years, our Fly-TILL service has screened this collection to provide approximately 150 allelic series of point mutations for the fly community. Our analysis of >2000 point mutations and indels have provided a glimpse into the population dynamics of this valuable genetic resource. We found evidence for selection and differential recovery of mutations, depending on distance from balancer breakpoints. Although this process led to variable mutational densities, we have nevertheless been able to deliver potentially valuable mutations in genes selected by Fly-TILL users. We anticipate that our findings will help guide the future implementation of point-mutation resources for the Drosophila community.
Cooper
Jennifer L
JL
Howard Hughes Medical Institute and Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109-1024, USA.
Till
Bradley J
BJ
Henikoff
Steven
S
D000818:Animals; D003433:Crosses, Genetic; D004252:DNA Mutational Analysis; D004331:Drosophila melanogaster / Q000235:genetics*; D005020:Ethyl Methanesulfonate / Q000737:chemistry; D017344:Genes, Insect / Q000187:drug effects / Q000235:genetics*; D005821:Genetic Techniques; D014644:Genetic Variation; D017354:Point Mutation / Q000235:genetics*
D005020:Ethyl Methanesulfonate