Enhancer-trap targeting at the Broad-Complex locus of Drosophila melanogaster.
Here, we describe the exact replacement of a defective unmarked P element by an enhancer-trap transposon marked by the miniwhite gene and carrying lacZ as a reporter gene. The original defective P element was located in an intron of the Broad-Complex (BRC), a key gene involved in metamorphosis. Replacement events resulted from conversions induced by the P-element transposase from a donor enhancer-trap element located on another chromosome. Six independent conversion events were selected. In all converted chromosomes, the enhancer-trap transposon was in the same orientation as the original P element. From the pattern of X-gal staining observed, lacZ expression likely reflects the regulatory influence of BRC enhancers on the convertant transposon. Reversion to wild type was achieved by excision of the enhancer-trap transposon. The six convertants were analyzed in detail at the nucleotide level. The occurrence of a polymorphism at position 33 of the P-element sequences led us to propose a conversion mechanism involving homologous P sequences for repair. This is in contrast to previously analyzed P-element transposase-induced conversion events and proposed models relying on sequence identity between genomic Drosophila sequences. The lack of any homology requirement other than between P element sequences means that our findings can be easily generalized. Targeting a marked P-element derivative at a precise site without loss or addition of genetic information makes it possible to exploit the hundreds of defective P elements scattered throughout the Drosophila genome by replacing them with engineered P elements, already available.
Gonzy-Tréboul
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Laboratoire de Biologie du Développement, Institut Jacques Monod, Centre National de la Recherche Scientifique (CNRS), Paris, France.
Lepesant
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JA
Deutsch
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D000818:Animals; D001483:Base Sequence; D003433:Crosses, Genetic; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004331:Drosophila melanogaster / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005260:Female; D005785:Gene Conversion / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D018390:Gene Targeting / Q000379:methods*; D017344:Genes, Insect / Q000235:genetics; D005804:Genes, Lethal / Q000235:genetics; D007438:Introns / Q000235:genetics; D008297:Male; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D019895:Transposases; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D009713:Nucleotidyltransferases; D019895:Transposases; D001616:beta-Galactosidase