Extensive, Nonrandom Diversity of Excision Footprints Generated by Ds-Like Transposon Ascot-1Suggests New Parallels with V(D)J Recombination

ABSTRACT

Upon insertion, transposable elements can disrupt or alter gene function in various ways. Transposons moving through a cut-and-paste mechanism are in addition often mutagenic when excising because repair of the empty site seldom restores the original sequence. The characterization of numerous excision events in many eukaryotes indicates that transposon excision from a given site can generate a high degree of DNA sequence and phenotypic variation. Whether such variation is generated randomly remains largely to be determined. To this end, we have exploited a well-characterized system of genetic instability in the fungus Ascobolus immersus to perform an extensive study of excision events. We show that this system, which produces many phenotypically and genetically distinct derivatives, results from the excision of a novel Ds-like transposon,Ascot-1, from the spore color gene b2. A unique set of 48 molecularly distinct excision products were readily identified from a representative sample of excision derivatives. Products varied in their frequency of occurrence over 4 orders of magnitude, yet most showed small palindromic nucleotide additions. Based on these and other observations, compelling evidence was obtained for intermediate hairpin formation during the excision reaction and for strong biases in the subsequent processing steps at the empty site. Factors likely to be involved in these biases suggest new parallels between the excision reaction performed by transposons of the hAT family and V(D)J recombination. An evaluation of the contribution of small palindromic nucleotide additions produced by transposon excision to the spectrum of spontaneous mutations is also presented.

ACKNOWLEDGMENTS

We thank Anne Dumay, Fatima Graia, and Marc-AndréSélosse for their contributions to the work, Denise Zickler for help with photography, and Christian Cibert, Shelly Esposito, Vincenzo Rocco, and François Taddei for valuable suggestions during preparation of the manuscript. Special thanks are due to Michael Ronemus for critical reading of the manuscript.

This work was supported by grants from the Association pour la Recherche sur le Cancer (contract 6200) and the Groupement de Recherches et d’Etudes sur les Génomes (contract 44/95).

ADDENDUM

Since this paper was submitted for review, evidence for the formation of 3′ overhangs as a result of hairpin resolution has been obtained for V(D)J recombination (25a).