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Genetics, Vol 145, 267-279, Copyright © 1997
INVESTIGATIONS |
Mobilization of a Minos Transposon in Drosophila melanogaster Chromosomes and Chromatid Repair by Heteroduplex Formation
B. Arca, S. Zabalou, T. G. Loukeris and C. Savakis
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas Present address: Istituto di Parassitologia, Fondazione ``Istituto Pasteur-Cenci Bolognetti'', Universita di Roma ``La Sapienza'', P.le Aldo Moro 5, 00185 Rome, Italy.
Transposase-mediated mobilization of the element Minos has been studied in the Drosophila melanogaster genome. Excision and transposition of a nonautonomous Minos transposon in the presence of a Minos transposase gene was detected with a dominant eye color marker carried by the transposon. Frequencies of excision in somatic tissues and in the germ line were higher in flies heterozygous for the transposon than in homozygotes or hemizygotes. Transposition of a X chromosome-linked insertion of Minos into new autosomal sites occurred in 1-12% of males expressing transposase, suggesting that this system is usable for gene tagging and enhancer trapping in Drosophila. Sequence analysis of PCR-amplified donor sites after excision showed precise restoration of the original target sequence in ~75% of events in heterozygotes and the presence of footprints or partially deleted elements in the remaining events. Most footprints consisted of the four terminal bases of the transposon, flanked by the TA target duplication. Sequencing of a chromosomal donor site that was directly cloned after excision showed a characteristic two-base mismatch heteroduplex in the center of the 6-bp footprint. Circular extrachromosomal forms of the transposon, presumably representing excised Minos elements, could be detected only in the presence of transposase. A model for chromatid repair after Minos excision is discussed in which staggered cuts are first produced at the ends of the inverted repeats, the broken chromatid ends are joined, and the resulting heteroduplex is subsequently repaired. The model also suggests a simple mechanism for the production of the target site duplication and for regeneration of the transposon ends during reintegration.
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