Knockout Targeting of the Drosophila Nap1 Gene and Examination of DNA Repair Tracts in the Recombination Products
a Department of Zoology, University of Heidelberg, D-69120 Heidelberg, Germanye@qg, 百拇医药
b Research Group Epigenetics, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germanye@qg, 百拇医药
c Department of Developmental Genetics, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germanye@qg, 百拇医药
ABSTRACTe@qg, 百拇医药
We used ends-in gene targeting to generate knockout mutations of the nucleosome assembly protein 1 (Nap1) gene in Drosophila melanogaster. Three independent targeted null-knockout mutations were produced. No wild-type NAP1 protein could be detected in protein extracts. Homozygous Nap1KO knockout flies were either embryonic lethal or poorly viable adult escapers. Three additional targeted recombination products were viable. To gain insight into the underlying molecular processes we examined conversion tracts in the recombination products. In nearly all cases the I-SceI endonuclease site of the donor vector was replaced by the wild-type Nap1 sequence. This indicated exonuclease processing at the site of the double-strand break (DSB), followed by replicative repair at donor-target junctions. The targeting products are best interpreted either by the classical DSB repair model or by the break-induced recombination (BIR) model. Synthesis-dependent strand annealing (SDSA), which is another important recombinational repair pathway in the germline, does not explain ends-in targeting products. We conclude that this example of gene targeting at the Nap1 locus provides added support for the efficiency of this method and its usefulness in targeting any arbitrary locus in the Drosophila genome.
THE completion of the genome sequence provides unlimited access to all genes of Drosophila melanogaster (ADAMS et al. 2000 ). Nevertheless, despite nearly a century of Drosophila genetics, there are many Drosophila genes for which corresponding mutants are still unavailable. Means to overcome the drawback had been site-selected transposon mutagenesis (BALLINGER and BENZER 1989 ; KAISER and GOODWIN 1990 ) and RNA-mediated interference (RNAi; KENNERDELL and CARTHEW 1998 ). While transposon mutagenesis involves elaborate PCR screening, RNAi generates only gene-specific phenocopies of loss-of-function mutations and does not always cause a true null phenotype. Therefore, methods of gene knockout targeting have been developed. Drosophila gene targeting is accomplished by two alternative techniques (GLOOR et al. 1991 ; RONG and GOLIC 2000 ). Both take advantage of the fly's endogenous homologous recombination machinery in the germline. One method utilizes a P-element-induced double-strand break in a target gene, which then is repaired from an ectopic donor construct by means of synthesis-dependent strand annealing (SDSA; NASSIF et al. 1994). P-induced gap repair was developed by Engels and colleagues (GLOOR et al. 1991(Susanne Lankenau Thorsten Barnickel Joachim Marhold Frank Lyko Bernard M. Mechler and Dirk-Henner La)
b Research Group Epigenetics, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germanye@qg, 百拇医药
c Department of Developmental Genetics, Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germanye@qg, 百拇医药
ABSTRACTe@qg, 百拇医药
We used ends-in gene targeting to generate knockout mutations of the nucleosome assembly protein 1 (Nap1) gene in Drosophila melanogaster. Three independent targeted null-knockout mutations were produced. No wild-type NAP1 protein could be detected in protein extracts. Homozygous Nap1KO knockout flies were either embryonic lethal or poorly viable adult escapers. Three additional targeted recombination products were viable. To gain insight into the underlying molecular processes we examined conversion tracts in the recombination products. In nearly all cases the I-SceI endonuclease site of the donor vector was replaced by the wild-type Nap1 sequence. This indicated exonuclease processing at the site of the double-strand break (DSB), followed by replicative repair at donor-target junctions. The targeting products are best interpreted either by the classical DSB repair model or by the break-induced recombination (BIR) model. Synthesis-dependent strand annealing (SDSA), which is another important recombinational repair pathway in the germline, does not explain ends-in targeting products. We conclude that this example of gene targeting at the Nap1 locus provides added support for the efficiency of this method and its usefulness in targeting any arbitrary locus in the Drosophila genome.
THE completion of the genome sequence provides unlimited access to all genes of Drosophila melanogaster (ADAMS et al. 2000 ). Nevertheless, despite nearly a century of Drosophila genetics, there are many Drosophila genes for which corresponding mutants are still unavailable. Means to overcome the drawback had been site-selected transposon mutagenesis (BALLINGER and BENZER 1989 ; KAISER and GOODWIN 1990 ) and RNA-mediated interference (RNAi; KENNERDELL and CARTHEW 1998 ). While transposon mutagenesis involves elaborate PCR screening, RNAi generates only gene-specific phenocopies of loss-of-function mutations and does not always cause a true null phenotype. Therefore, methods of gene knockout targeting have been developed. Drosophila gene targeting is accomplished by two alternative techniques (GLOOR et al. 1991 ; RONG and GOLIC 2000 ). Both take advantage of the fly's endogenous homologous recombination machinery in the germline. One method utilizes a P-element-induced double-strand break in a target gene, which then is repaired from an ectopic donor construct by means of synthesis-dependent strand annealing (SDSA; NASSIF et al. 1994). P-induced gap repair was developed by Engels and colleagues (GLOOR et al. 1991(Susanne Lankenau Thorsten Barnickel Joachim Marhold Frank Lyko Bernard M. Mechler and Dirk-Henner La)