Repair of P element ends following hybrid element excision leads to recombination in Drosophila melanogaster

Liang, X; Sved, J. A.

doi:10.1038/hdy.2008.87

Cited by 2 publications

(3 citation statements)

References 17 publications

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“…In one case the hybrid element frequently resolves by integration (Gray et al, 1996), although also by repair of P element ends (Liang and Sved, 2009). In the other, the present case, the resolution is almost entirely through repair.…”

Section: Discussionmentioning

confidence: 99%

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Properties of re-arranged P elements in Drosophila melanogaster

Liang

Sved

2009

Heredity

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P elements, both complete and incomplete, contain a left and right end, normally depicted as pointing away from each other. Here, we examine the properties of P elements that may be described as 're-arranged elements' or 'inside-out elements', containing inverted ends. Two such structures exist, having either ends pointing towards each other, 'headto-head or H-H', or ends pointing in the same direction 'head-to-tail or H-T'. We show that both structures are unstable in the presence of P element transposase. For the H-H element there is a high frequency of deletion of the intervening material and almost exact rejoining of element ends with the 4 bp CATG palindromic end sequence shared by the two element ends. This result is predicted by the Beall and Rio model of P element excision. For the H-T element there is a high frequency of exact excision of the entire inverted right-end, a result again predicted by the Beall and Rio model. Both structures lead to recombination in the way expected from a normal element. The rates of recombination are, however, much lower than might be expected from the organization of ends, a result that can be explained in terms of the low likelihood of insertion into a chromosomal region lacking another P element end. We also investigate the properties of combinations of re-arranged and normal elements, and show that there is a directionality property when left and right ends are combined in trans that can be explained in terms of strand repair.

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Section: Discussionmentioning

confidence: 99%

“…The determination by Beall and Rio (1997) of the action of P element transposase provides a simple explanation for the preponderance of this type of deletion (Liang and Sved, 2009).…”

Section: Introductionmentioning

confidence: 99%

Properties of re-arranged P elements in Drosophila melanogaster

Liang

Sved

2009

Heredity

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“…This strategy is possible because pWalkman sequences contain two P-element ends. Although these P-element ends are in an "inside-out" orientation when compared to their natural orientation, they can nevertheless be acted on by the P transposase to generate a DSB (Liang and Sved 2009). To test the efficacy of this approach, we introduced a source of P transposase into the duplicated lines and screened for potential reduction events as before.…”

Section: Reductions Induced By P-element Transposasementioning

confidence: 99%

Long-Range Targeted Manipulation of the Drosophila Genome by Site-Specific Integration and Recombinational Resolution

Wesolowska

Rong

2013

Genetics

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Significant advances in genomics underscore the importance of targeted mutagenesis for gene function analysis. Here we have developed a scheme for long-range targeted manipulation of genes in the Drosophila genome. Utilizing an attP attachment site for the phiC31 integrase previously targeted to the nbs gene, we integrated an 80-kb genomic fragment at its endogenous locus to generate a tandem duplication of the region. We achieved reduction to a single copy by inducing recombination via a site-specific DNA break. We report that, despite the large size of the DNA fragment, both plasmid integration and duplication reduction can be accomplished efficiently. Importantly, the integrating genomic fragment can serve as a venue for introducing targeted modifications to the entire region. We successfully introduced a new attachment site 70 kb from the existing attP using this two-step scheme, making a new region susceptible to targeted mutagenesis. By experimenting with different placements of the future DNA break site in the integrating vector, we established a vector configuration that facilitates the recovery of desired modifications. We also show that reduction events can occur efficiently through unequal meiotic crossing over between the large duplications. Based on our results, we suggest that a collection of 1200 lines with attachment sites inserted every 140 kb throughout the genome would render all Drosophila genes amenable to targeted mutagenesis. Excitingly, all of the components involved are likely functional in other eukaryotes, making our scheme for long-range targeted manipulation readily applicable to other systems. DROSOPHILA melanogaster is an excellent organism for the genetic analysis of cellular and developmental processes. For the past two decades, methods of gene targeting by homologous recombination have facilitated the engineering of crafted mutations (Gloor et al. 1991;Rong and Golic 2000;Rong et al. 2002;Gong and Golic 2003). The wealth of information provided by bioinformatic analyses has further aided geneticists in the design of mutations. Gene targeting in flies, however, is quite labor intensive, discouraging its use in systematic mutagenesis, which involves more than one targeted allele, although success stories have revealed the benefits of such approaches (Gronke et al. 2010;Spitzweck et al. 2010).Recently, several laboratories have demonstrated that, by combining homologous recombination with the efficient phiC31-mediated site-specific recombination method, the need for repetitive gene targeting of individual alleles to the endogenous locus can be eliminated (Gao et al. 2008;Huang et al. 2009;Weng et al. 2009;Iampietro et al. 2010). We developed the site-specific integrase-mediated repeated targeting (SIRT) method, in which an attP attachment site for phiC31 integrase is first targeted to the genome in the proximity of the locus of interest using gene targeting by homologous recombination. Further modifications are carried out by integration of a plasmid that carries the modif...

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Repair of P element ends following hybrid element excision leads to recombination in Drosophila melanogaster

Cited by 2 publications

References 17 publications

Properties of re-arranged P elements in Drosophila melanogaster

Properties of re-arranged P elements in Drosophila melanogaster

Long-Range Targeted Manipulation of the Drosophila Genome by Site-Specific Integration and Recombinational Resolution

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