Mutations in the mariner transposase: the D,D(35)E consensus sequence is nonfunctional.
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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Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, MA 02138, USA.
De Aguiar
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Hartl
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D000818:Animals; D001483:Base Sequence; D016384:Consensus Sequence; D003433:Crosses, Genetic; D004252:DNA Mutational Analysis; D004254:DNA Nucleotidyltransferases / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D004331:Drosophila melanogaster / Q000201:enzymology / Q000235:genetics*; D005816:Genetic Complementation Test; D005854:Germ Cells / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D019895:Transposases
D004251:DNA Transposable Elements; D010446:Peptide Fragments; D004254:DNA Nucleotidyltransferases; D019895:Transposases