Comparison of targeted-gene replacement frequencies in Drosophila melanogaster at the forked and white loci.
P element-induced gene conversion has been previously used to modify the white gene of Drosophila melanogaster in a directed fashion. The applicability of this approach of gene targeting in Drosophila melanogaster, however, has not been analyzed quantitatively for other genes. We took advantage of the P element-induced forked allele, f(hd), which was used as a target, and we constructed a vector containing a modified forked fragment for converting f(hd). Conversion frequencies were analyzed for this locus as well as for an alternative white allele, w(eh812). Combination of both P element-induced mutant genes allowed the simultaneous analysis of conversion frequencies under identical genetic, developmental, and environmental conditions. This paper demonstrates that gene conversion through P element-induced gap repair can be applied with similar success rates at the forked locus and in the white gene. The average conversion frequency at forked was 0.29%, and that at white was 0.17%. These frequencies indicate that in vivo gene targeting in Drosophila melanogaster should be applicable for other genes in this species at manageable rates. We also confirmed the homolog dependence of reversions at the forked locus, indicating that P elements transpose via a cut-and-paste mechanism. In a different experiment, we attempted conversion with a modified forked allele containing the su(Hw) binding site. Despite an increased sample size, there were no conversion events with this template. One interpretation (under investigation) is that the binding of the su(Hw) product prevents double-strand break repair.
Lankenau
D H
DH
Department of Developmental Genetics, German Cancer Research Center, Heidelberg, Germany.
Corces
V G
VG
Engels
W R
WR
D018528:ATP-Binding Cassette Transporters; D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D003433:Crosses, Genetic; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D017931:DNA Primers; D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D005091:Exons; D005136:Eye Proteins; D005260:Female; D005785:Gene Conversion; D017344:Genes, Insect; D007301:Insect Hormones / Q000235:genetics*; D007438:Introns; D008297:Male; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D016133:Polymerase Chain Reaction; D013698:Templates, Genetic; D019895:Transposases
D018528:ATP-Binding Cassette Transporters; D017931:DNA Primers; D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D005136:Eye Proteins; D007301:Insect Hormones; D008840:Microfilament Proteins; C084628:f protein, Drosophila; C059254:w protein, Drosophila; D004254:DNA Nucleotidyltransferases; D019895:Transposases