MUTATIONS IN THE MARINER TRANSPOSASE - THE D,D(35)E CONSENSUS SEQUENCE IS NONFUNCTIONAL

Genetic analysis of eukaryote transposases and comparison with their p rokaryote counterparts have been greatly hindered by difficulty in iso lating mutations. We describe a simple eye-color screen that facilitat es isolation and analysis of mutations in the mariner transposase in D rosophila melanogaster. Use of ethyl methanesulfonate and site-directe d mutagenesis has identified 18 residues that are critical for in vivo excision of a target mariner element. When the mutations were examine d in heterozygous mutant/nonmutant genotypes, more than half of the mu tant transposase proteins were found to reduce the activity of the wil d-type transposase, as assayed by the frequency of germline excision o f a target element. Remarkably, transposase function is obliterated wh en the D,D(34)D acidic, ion-binding domain is replaced with the consen sus sequence D,D(34)E found in the nematode Tc1 transposase and in man y other transposases in the superfamily. A number of mutations strongl y complement wild-type transposase in a dominant-negative manner, sugg estive of subunit interactions in the excision reaction; these mutatio ns are located in a small region that includes part of the D,D(34)D mo tif. Transposase function also is eliminated by a mutation in the infe rred initiation codon and by a mutation in a putative nuclear localiza tion signal.