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

• Published in: Proceedings of the National Academy of Sciences - PNAS 1997-02, Vol.94 (4), p.1293-1297
• Main Authors: Lohe, A.R. (Harvard University, Cambridge, MA.), De Aguiar, D, Hartl, D.L
• Format: Article
• Language: English
• Subjects: ADN; Amino acids; Animals; Base Sequence; Biological Sciences; Color; Consensus Sequence; Crosses, Genetic; DNA Mutational Analysis; DNA Nucleotidyltransferases - genetics; DNA Transposable Elements - genetics; DROSOPHILA MELANOGASTER; Drosophila melanogaster - enzymology; Drosophila melanogaster - genetics; ENZIMAS; ENZYME; Enzymes; Eyes & eyesight; FENOTIPOS; GENE; GENES; Genetic Complementation Test; Genetic mutation; Genetic transposition; Genetics; Germ cells; Germ Cells - enzymology; Insects; Mariners; Molecular Sequence Data; Mosaicism; MUTACION; MUTACION INDUCIDA; Mutagenesis, Site-Directed; MUTATION; MUTATION PROVOQUEE; Peptide Fragments - genetics; PHENOTYPE; Polymerase Chain Reaction; PROTEINAS AGLUTINANTES; PROTEINE DE LIAISON; Sequence Analysis, DNA; Sequence Deletion; Sequencing; Transgenes; Transposases; Transposons
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.4.1293

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