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

Abstract

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.

Figure 1

Germline excision of the peach element from w^pch in males bearing a single copy of the mutant hsp70:Mos1 transgene. The bars represent the mean ± 1 binomial standard error; dots indicate no germline excision. The nature and position of the mutations in the hsp70:Mos1 sequence are depicted along the horizontal axis. The triangles at the ends represent the inverted repeats. M at 1 and E at 345 are the amino and carboxyl ends of the putative transposase. NLS is part of a nuclear localization signal, and the aspartates of the D,D(34)D motif are indicated. The bar labeled ns is not significantly different from the control value of 14.7%. The others are all significant even when adjusted for multiple tests.

Figure 2

Germline excision of the peach element in heteroallelic mutant/hsp70:Mos1–182 males laid out as in Fig. 1. The horizontal line indicates a value equal to that observed in hsp70:Mos1/+ controls (14.7%). Unmarked bars are not significantly different from the control value; single asterisks, significantly less than control value; double asterisks, highly significantly less than control value (and also significantly less than one-half the control value). Significance levels corrected for multiple tests.

Figure 3

Structure of the 31-bp insertion in EMS-96. The insertion is indicated by the brackets, and the 26-bp regions of inverted homology are indicated by arrows. The locations of three 7-bp tandem repeats are indicated by a line above the sequence. Numbering refers to the nucleotide sequence of Mos1.