title: Target-selected mutant screen by TILLING in Drosophila.
abstract: The availability of the full Drosophila genomic DNA sequence prompts the development of a method to efficiently obtain mutations in genes of interest identified by their sequence homologies or biochemically. To date, molecularly characterized mutations have been generated in around 6000 of the approximately 15,000 annotated fly genes, of which around one-third are essential for viability. To obtain mutations in essential and nonessential genes of interest, we took a reverse genetics approach, based on the large-scale detection of point mutations by Cel-I-mediated heteroduplex cleavage. A library of genomic DNA from 2086 EMS-mutagenized lines was established. The library was screened for mutations in three genes. A total of 6.1 Mb were screened, and 44 hits were found in two different mutagenesis conditions. Optimal conditions yielded an average of one mutation every 156 kb. For an essential gene tested, five of 25 mutations turned out to cause lethality, confirming that EMS mutagenesis leads to high frequency of gene inactivation. We thereby established that Cel-I-mediated TILLING can be used to efficiently obtain mutations in genes of interest in Drosophila.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
authors.forename: Sylke
authors.initials: S
authors.lastname: Winkler
authors.forename: Anja
authors.initials: A
authors.lastname: Schwabedissen
authors.forename: Dana
authors.initials: D
authors.lastname: Backasch
authors.forename: Christian
authors.initials: C
authors.lastname: Bökel
authors.forename: Claudia
authors.initials: C
authors.lastname: Seidel
authors.forename: Stefanie
authors.initials: S
authors.lastname: Bönisch
authors.forename: Maximilian
authors.initials: M
authors.lastname: Fürthauer
authors.forename: Antje
authors.initials: A
authors.lastname: Kuhrs
authors.forename: Laura
authors.initials: L
authors.lastname: Cobreros
authors.forename: Michael
authors.initials: M
authors.lastname: Brand
authors.forename: Marcos
authors.initials: M
authors.lastname: González-Gaitán
mesh_terms: D000818:Animals; D001483:Base Sequence; D003433:Crosses, Genetic; D017931:DNA Primers; D004331:Drosophila melanogaster / Q000235:genetics*; D004720:Endonucleases / Q000378:metabolism; D015723:Gene Library; D017344:Genes, Insect / Q000235:genetics*; D016678:Genome; D020180:Heteroduplex Analysis / Q000379:methods*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004720:Endonucleases; C406922:CEL I nuclease