Minos as a genetic and genomic tool in Drosophila melanogaster

Abstract

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 5'-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient "jump-starting" scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and approximately 55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila.

Figure 1.

Minos donor and helper constructs. Both donors contain the 3xPax6/EGFP dominant marker (Berghammer et al. 1999) and allow plasmid rescue of insertions. The helper construct expresses Minos transposase under heat-shock control and is based on pCaSper. Only the transposon regions are shown.

Figure 2.

Distribution of Minos insertions over the Drosophila genome. The number of insertions on the X chromosome is an underestimate, since 24 integration events were produced by mobilization of an X-linked insertion into the autosomes.

Figure 3.

Sequence composition analysis of Minos insertion sites. (Top) Five nucleotides upstream and downstream of the insertions were aligned for 80 integrants and their informational content was plotted with the SeqLogo program (Schneider and Stephens 1990). (Bottom) Base distribution of the 80 insertion sites.

Figure 4.

Analysis of predicted physical properties of Minos insertion sites. One hundred nucleotides centered around each of the 80 Minos insertions were analyzed and averaged (solid lines). Eighty sequences, also centered around TAs, but randomly taken from the Drosophila genome, were coanalyzed (shaded lines). Arrowheads indicate nucleotide positions where the predicted values between the two groups are different according to a confidence level of >99% (Student's t-test). Bendability is the tendency of DNA to bend toward the major groove. B-DNA twist determines the tightness of the DNA coil. A-philicity is the tendency of the DNA double helix to form A-DNA. Protein-induced deformability describes the propensity of DNA to change conformation upon binding to a protein.

Figure 5.

Hydrogen-bonding analysis of Minos insertion sites compared to Sleeping Beauty and P-element insertion sites. Program HbondView (Liao et al. 2000) was used to illustrate average hydrogen-bonding patterns of base pairs flanking the Minos insertion sites. Six positions for each base pair are color coded as potential hydrogen acceptor (red), donor (blue), or inert (gray). Sleeping Beauty and P graphs are from Liao et al. (2000) and Vigdal et al. (2002), respectively.