Introduction of the transposable element Minos into the germ line of Drosophila melanogaster.

Abstract

A transposon based on the transposable element Minos from Drosophila hydei was introduced into the genome of Drosophila melanogaster using transformation mediated by the Minos transposase. The transposon carries a wild-type version of the white gene (w) of Orosophila inserted into the second exon of Minos. Transformation was obtained by injecting the transposon into preblastoderm embryos that were expressing transposase either from a Hsp7O-Minos fusion inserted into the genome via P-element-mediated transformation or from a coinjected plasmid carrying the Hsp7O-Minos fusion. Between 1% and 6% of the fertile injected individuals gave transformed progeny. Four of the insertions were cloned and the DNA sequences flanking the transposon ends were determined. The "empty" sites corresponding to three ofthe insertions were amplified from the recipient strain by PCR, cloned, and sequenced. In all cases, the transposon has inserted into a TA dinucleotide and has created the characteristic TA target site duplication. In the absence of transposase, the insertions were stable in the soma and the germ line. However, in the presence of the Hsp7O-Minos gene the Minos-w transposon excises, resulting in mosaic eyes and germ-line reversion to the white phenotype. Minos could be utilized as an alternative to existing systems for transposon tagging and enhancer trapping in Drosophila; it might also be of use as a germ-line transformation vector for non-Drosophila insects.Transposable elements have been used as vectors for stable germ-line transformation in Drosophila melanogaster. The mobile element P, present in some D. melanogaster populations, was the first element shown to insert into germ-line chromosomes in embryos (1). Typically, P-element-mediated transformation is achieved by coinjecting into preblastoderm embryos a mixture of two plasmids: one expressing transposase but unable to transpose and one carrying a gene (or genes) of interest flanked by the inverted terminal repeats of the element. Transformants are detected among the progeny of the injected individuals through the expression of dominant phenotypes. Germ-line transformation has revolutionized Drosophila research through the introduction of powerful methodologies such as analysis of in vitro mutagenized genes, gene cloning by transposon tagging, and enhancer trapping.Two other elements unrelated to P, hobo and mariner, have been shown to be able to transpose in the genome of D.melanogaster. hobo is found in some but not all D. melanogaster populations and causes hybrid dysgenesis (2, 3). Defective hobo elements containing foreign DNA can transpose into germline chromosomes from plasmids if coinjected into preblastoderm embryos along with a full-length element that can provide transposase (4). mariner, an element found in Drosophila mauritiana but not in D. melanogaster, has been introduced into D. melanogaster and shown to induce in trans mobilization of a nonautonomous mariner element (5, 6). Efforts to transfer the Drosophila germ-line transformation me...

Evolution of the Drosophila Feminizing Switch Gene Sex-lethal

“…Laying by parents aged at least 1 week after eclosion was at 25°, while DNA injection was at 18°. The Minos transposase source was pHSS6hsMi2 (Loukeris et al 1995a). Vector and transposase DNA were mixed together in injection buffer (5 mm KCL and 0.1 mm Na Phosphate, pH 6.8) at a final concentration of 800 and 200 ng/ml, respectively.…”

“…The Minos transformation vector was pMiw1 (Loukeris et al 1995a) modified by cleanly replacing the w-promoter-driven mini-white marker that had been inserted at an EcoRI site by a 4.5-kb EcoRI fragment from pW8 (Klemenz et al 1987) that contained the mini-white gene driven by the hsp70 promoter instead. The Mi{mel/sisAsisB,w 1mC } transgene carried a 5.1-kb EcoRI-XbaI sc fragment from pJEP200 (Erickson and Cline 1991) and a 4.2-kb ScaI sisA fragment from pJE301 (Erickson and Cline 1993) inserted at a NotI site just upstream of the w 1 marker.…”

Minos as a Genetic and Genomic Tool in Drosophila melanogaster

“…Analysis of Minos insertion sites: BLAST (Altschul et al 1990) analysis was used to place the 92 insertions recovered plus 4 previously published insertions (Loukeris et al 1995a) on the Drosophila genome, according to release 3 of the D. melanogaster database (Celniker et al 2002). Seven insertions were in repetitive regions.…”

“…Minos is a member of the Tc1/mariner family of transposable elements with 255-bp long terminal inverted repeats, which flank a single gene encoding transposase. The Minos transposase has been shown to catalyze, in most cases, precise excision and integration of the element without involvement of flanking DNA (Loukeris et al 1995a;Arca et al 1997). Most excision events either are precise (i.e., the original, preinsertion sequence is restored) or leave behind a characteristic 6-bp footprint, consisting of four terminal nucleotides of the Minos element followed by the duplicated TA, which is generated by the element upon insertion; complex events involving partial loss of the element are rare (Arca et al 1997).…”

Removal of the Bloom Syndrome DNA Helicase Extends the Utility of Imprecise Transposon Excision for Making Null Mutations in Drosophila

“…Therefore, mutational systems that utilize transposable elements with different insertion biases have been developed. These include Hobo (Smith et al 1993); the lepidopteran-derived piggyBac element, which inserts at TTAA sites Horn et al 2003); and Minos, a Tc-1/mariner-like element originally isolated from Drosophila hydei that inserts at TA dinucleotides (Franz and Savakis 1991;Loukeris et al 1995). Using a combination of these transposons, the Drosophila Gene Disruption Project has generated inserts in 60% of the 14,850 annotated genes (Spradling et al 1999;Bellen et al 2004).…”