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

Transposable elements are frequently used in Drosophila melanogaster for imprecise excision screens to delete genes of interest. However, these screens are highly variable in the number and size of deletions that are recovered. Here, we show that conducting excision screens in mus309 mutant flies that lack DmBlm, the Drosophila ortholog of the Bloom syndrome protein, increases the percentage and overall size of flanking deletions recovered after excision of either P or Minos elements.TRANSPOSABLE elements have a rich history as mutagenesis tools in Drosophila melanogaster (reviewed in Ryder and Russell 2003). Initially, researchers focused their efforts on the use of nonautonomous P-element transposons for gene disruption (Cooley et al. 1988). However, P elements have insertion biases, preferring to transpose into euchromatic regions, the 5′ regions of genes (Tsubota et al. 1985; Kelley et al. 1987), and to target sequence motifs similar to the octamer GGCCAGAC (O''Hare and Rubin 1983). These biases make it unlikely that full genome saturation will be reached using P-element mutagenesis. 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 (Hacker et al. 2003; 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).In spite of the growing number of transposon insertions in the Drosophila genome, many are inserted in regions that do not completely abolish gene function, such as 5′-UTRs and introns. This can make it difficult to discern the true null phenotypes of genes. Furthermore, there still exist a sizable number of genes for which no transposon insertions are available. To address these issues, many transposons have been constructed with additional characteristics, such as FRT sites, that make generation of molecularly defined deletions by site-specific recombination relatively straightforward (Parks et al. 2004; Thibault et al. 2004; Ryder et al. 2007). However, until saturation of the genome with these designer transposons is achieved, their utility in creating single-gene deletions remains limited.A more general approach for generating single-gene deletions that has proven successful is the use of P elements in imprecise excision screens. Excision of a P element creates a DNA double-strand break with 17 nucleotide noncomplementary ends (Beall and Rio 1997). If the ends of the break are degraded prior to repair, a deletion of DNA flanking the original insertion site is created (reviewed in Hummel and Klambt 2008). On average, the frequency of flanking deletions recovered from imprecise excision screens is ∼1%. However, this frequency varies tremendously by locus and depends on a multitude of factors that are not well understood, including chromatin structure and local sequence context. Therefore, generation of suitable deletion mutants frequently involves screening many hundreds of independent lines.An alternative method that uses P elements to generate deletions involves screening for events associated with male recombination. These events, which probably arise through a hybrid element insertion mechanism, generate one-sided deletions of sizes ranging from several base pairs to several kilobases (Preston and Engels 1996). This method, although powerful, involves screening a large number of flies and requires two sequential screens to generate bidirectional deletions.P-element-induced double-strand breaks are preferentially repaired through homologous recombination using a sister chromatid or a homologous chromosome as a template (Engels et al. 1990). Previously, we and others have demonstrated that the Drosophila Bloom protein ortholog (DmBlm), a RecQ DNA helicase encoded by mus309, is involved in homology-directed repair of these breaks (Beall and Rio 1996; McVey et al. 2004a). In the absence of DmBlm, repair of a P-element-induced break on a plasmid or at a chromosomal locus frequently results in a large, flanking deletion. Several groups have applied this observation to imprecise excision screens using P elements and have successfully recovered multiple deletions (Astrom et al. 2003; Johansson et al. 2007; Y. Rong, unpublished data). However, a direct comparison between imprecise excision screens carried out in wild-type vs. mus309 mutant backgrounds has not been published, and little is known regarding the use of this technique with other types of transposable elements. In this study, we used three different transposons to test the hypothesis that the use of a mus309 mutant background in imprecise excision screens would result in a greater yield of deletions and that these deletions would be larger than those recovered from a wild-type background.

The mus309 mutant background increases the frequency and size of flanking deletions following P-element excision:

Previously, we have shown that repair of a double-strand break created by excision of the P{w^a} transposon, located at 13F1–13F4 on the X chromosome, is deletion prone in the absence of DmBlm (McVey et al. 2004a). This is likely due to a requirement for DmBlm in D-loop unwinding during homologous recombination (Bachrati et al. 2006; Weinert and Rio 2007). We have speculated that an unknown endonuclease may cleave D-loops in the absence of DmBlm, resulting in deletions flanking the P-element insertion site. To determine whether these observations can be generalized to imprecise excision screens, we tested two additional P-element insertions. One of these, P{EPgy2}Trf4-1^EY14679, is inserted within a 1-kb intron of the Trf4-1 gene on the X chromosome (Figure 1A). The other, P{EPgy2}mus205^EY20083, is inserted in a small intron in the mus205 gene on chromosome 2 (Figure 1B). Both of these EY elements contain wild-type copies of the yellow and white genes (Bellen et al. 2004). Thus, flies possessing EPgy2 elements have a wild-type body color and pigmented eyes. For our excision screens, we generated males containing the P element and a constitutively expressed transposase source, Δ2-3 (Robertson et al. 1988). To test the effect of DmBlm absence, we also conducted the screens in heteroallelic mus309^D2/mus309^N1 mutants (Kusano et al. 2001; McVey et al. 2007).Open in a separate windowFigure 1.—Frequency and size of deletions accompanying imprecise P-element excision is increased in the mus309 mutant background. Crosses to generate males possessing both the P transposase and the desired P element were carried out in bottles containing standard cornmeal-based food at 25°. Excision events occurring in the male premeiotic germlines of flies carrying (A) P{EPgy2}Trf4-1^EY14679 and the P{ry⁺, Δ2-3}99B transposase or (B) P{EPgy2}mus205^EY20083 and the CyO, H{w⁺, Δ2-3} transposase were recovered in male progeny (for Trf4-1^EY14679) or over a deficiency spanning the region (for mus205^EY20083). Only one excision per male germline was analyzed to ensure that all events were independent. Genomic DNA was isolated and subjected to PCR analysis using primers specific to the P inverted repeats or to sequences flanking each P element. A and B show a genomic region with genes represented as boxes, intergenic regions as lines, and P elements as inverted triangles. Deletions were recovered from both wild-type (top panels) and mus309^D2/mus309^N1 mutant males (bottom panels) for Trf4-1^EY14679, while deletions were recovered only from mus309 mutant males for mus205^EY20083. Solid lines represent confirmed deletions, broken lines represent potential deletions, and arrows represent deletions that extend farther than was tested by PCR. Numbers in parentheses indicate the number of excisions recovered.First, we determined whether loss of DmBlm affected the fertility of males in which P-element excision was occurring. We found no significant difference in the percentage of wild-type vs. mus309 males that were sterile, as defined by an inability of an individual male to produce more than five adult offspring (