Mutations in the mariner transposase: The D,D(35)E consensus sequence is nonfunctional

AUTOR(ES)

Lohe, Allan R.

FONTE

The National Academy of Sciences of the USA

RESUMO

Genetic analysis of eukaryote transposases and comparison with their prokaryote counterparts have been greatly hindered by difficulty in isolating mutations. We describe a simple eye-color screen that facilitates isolation and analysis of mutations in the mariner transposase in Drosophila melanogaster. Use of ethyl methanesulfonate and site-directed mutagenesis has identified 18 residues that are critical for in vivo excision of a target mariner element. When the mutations were examined in heterozygous mutant/nonmutant genotypes, more than half of the mutant transposase proteins were found to reduce the activity of the wild-type transposase, as assayed by the frequency of germline excision of a target element. Remarkably, transposase function is obliterated when the D,D(34)D acidic, ion-binding domain is replaced with the consensus sequence D,D(34)E found in the nematode Tc1 transposase and in many other transposases in the superfamily. A number of mutations strongly complement wild-type transposase in a dominant-negative manner, suggestive of subunit interactions in the excision reaction; these mutations are located in a small region that includes part of the D,D(34)D motif. Transposase function also is eliminated by a mutation in the inferred initiation codon and by a mutation in a putative nuclear localization signal.

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