Retention of induced mutations in a Drosophila reverse-genetic resource.
Targeting induced local lesions in genomes (TILLING) is a reverse-genetic method for identifying point mutations in chemically mutagenized populations. For functional genomics, it is ideal to have a stable collection of heavily mutagenized lines that can be screened over an extended period of time. However, long-term storage is impractical for Drosophila, so mutant strains must be maintained by continual propagation of live cultures. Here we evaluate a strategy in which ethylmethane sulfonate (EMS) mutagenized chromosomes were maintained as heterozygotes with balancer chromosomes for >100 generations before screening. The strategy yielded a spectrum of point mutations similar to those found in previous studies of EMS-induced mutations, as well as 2.4% indels (insertions and deletions). Our analysis of 1887 point mutations in 148 targets showed evidence for selection against deleterious lesions and differential retention of lesions among targets on the basis of their position relative to balancer breakpoints, leading to a broad distribution of mutational densities. Despite selection and differential retention, the success of a user-funded service based on screening a large collection several years after mutagenesis indicates sufficient stability for use as a long-term reverse-genetic resource. Our study has implications for the use of balancer chromosomes to maintain mutant lines and provides the first large-scale quantitative assessment of the limitations of using breeding populations for repositories of genetic variability.
Cooper
Jennifer L
JL
Fred Hutchinson Cancer Research Center, Seattle, Washington 98109, USA.
Greene
Elizabeth A
EA
Till
Bradley J
BJ
Codomo
Christine A
CA
Wakimoto
Barbara T
BT
Henikoff
Steven
S
D000483:Alleles; D000818:Animals; D002875:Chromosomes; D003433:Crosses, Genetic; D004252:DNA Mutational Analysis; D004331:Drosophila melanogaster / Q000235:genetics*; D005020:Ethyl Methanesulfonate / Q000737:chemistry; D017344:Genes, Insect / Q000187:drug effects; D005821:Genetic Techniques; D014644:Genetic Variation; D006579:Heterozygote; D008957:Models, Genetic; D016296:Mutagenesis; D009153:Mutagens; D009154:Mutation
D009153:Mutagens; D005020:Ethyl Methanesulfonate
Methods Mol Biol. 2003;236:205-20
14501067
Nature. 2007 Jul 12;448(7150):151-6
17625558
Nature. 2007 Jan 4;445(7123):82-5
17203060
Genetics. 2004 May;167(1):207-16
15166148
Genome Res. 2007 May;17(5):649-58
17416746
Nat Rev Genet. 2006 Jul;7(7):524-36
16755288
Nucleic Acids Res. 2005 May 13;33(9):2806-12
15894797
Genetics. 2000 Nov;156(3):1169-73
11063692
Nat Biotechnol. 2000 Apr;18(4):455-7
10748531
Genetics. 2007 Aug;176(4):2151-63
17565942
Methods Cell Biol. 2004;77:91-112
15602907
Genetics. 1999 Dec;153(4):1825-38
10581288
Biol Reprod. 2008 Jan;78(1):2-12
17901073
PLoS Genet. 2005 Nov;1(5):e67
16299588
Genetics. 2004 May;167(1):203-6
15166147
Genome Res. 2005 May;15(5):718-23
15867432
Plant J. 2006 Feb;45(4):684-94
16441355
BMC Plant Biol. 2008 Jan 24;8:9
18218134
Nat Biotechnol. 2005 Jan;23(1):75-81
15580263
Methods Mol Biol. 2006;351:51-8
16988425
Genome Biol. 2007;8 Suppl 1:S9
18047701
Curr Opin Genet Dev. 2006 Dec;16(6):545-52
17055251
Genetics. 2003 Jun;164(2):731-40
12807792
Nature. 1990 May 10;345(6271):170-2
2110627
BMC Plant Biol. 2007 Apr 11;7:19
17428339
Genomics. 2004 Feb;83(2):332-4
14706462
Plant Physiol. 2000 Jun;123(2):439-42
10859174
Genome Res. 2003 Mar;13(3):524-30
12618384