Large-Scale Functional Annotation and Expanded Implementations of the P{wHy} Hybrid Transposon in the Drosophila melanogaster Genome

Abstract

Whole genome sequencing of the model organisms has created increased demand for efficient tools to facilitate the genome annotation efforts. Accordingly, we report the further implementations and analyses stemming from our publicly available P{wHy} library for Drosophila melanogaster. A two-step regime-large scale transposon mutagenesis followed by hobo-induced nested deletions-allows mutation saturation and provides significant enhancements to existing genomic coverage. We previously showed that, for a given starting insert, deletion saturation is readily obtained over a 60-kb interval; here, we perform a breakdown analysis of efficiency to identify rate-limiting steps in the process. Transrecombination, the hobo-induced recombination between two P{wHy} half molecules, was shown to further expand the P{wHy} mutational range, pointing to a potent, iterative process of transrecombination-reconstitution-transrecombination for alternating between very large and very fine-grained deletions in a self-contained manner. A number of strains also showed partial or complete repression of P{wHy} markers, depending on chromosome location, whereby asymmetric marker silencing allowed continuous phenotypic detection, indicating that P{wHy}-based saturational mutagenesis should be useful for the study of heterochromatin/positional effects.

Genetic Manipulation of Genes and Cells in the Nervous System of the Fruit Fly

“…Deletions can be generated through X-ray mutagenesis, imprecise excision of a P element or Minos (see above), excision of sequence between any two P element transposons located at different positions of the same chromosome (Cooley et al, 1990;Parks et al, 2004;Pare et al, 2009), the deletion-generator strategy (Huet et al, 2002;Mohr and Gelbart, 2002;Myrick et al, 2009), or through Flp-mediated recombination between two FRT sites each located in a transposon located at different positions of the same chromosome (Ryder et al, 2004;Parks et al, 2004;Ryder et al, 2007;Cook et al, 2010a). FRT deletions now cover 98% of the chromosomes (Cook et al, 2010b).…”

Modeling Amyloid Diseases in Fruit Fly Drosophila Melanogaster

“…The largest extension in Drosophila researches belongs to vectors, framed on the basis of mobile P-element [Adams & Sekelsky, 2002;Bellen et al, 2004]; however property of the P-element to be built in only certain genome sites, limits the mutagenesis in the whole genome. Therefore now approaches with use of other mobile elements preferring insertion sites distinct from the P-element, in particular, hobo [Huet et al, 20002;Myrick et al, 2009;Smith et al, 1993] and Minos [Metaxakis et al, 2005], are developed. It is necessary to notice that use of Drosophila models allows avoiding such restrictions arising in action with human material, as incomplete family pedigrees, genetic heterogeneity of population, duration of the sampling.…”

Saturation of the Human Phenome

“…Attempts at saturation mutagenesis using P-elements have been widely performed in D. melanogaster [ 93 ], however it is well documented that this transposon shows significant site specificity thus is not a good mutagen for true saturation. Efforts are currently under way to generate genome-wide saturation with transposable elements demonstrating less site specificity than the original P-element [ 94 - 96 ]. Although the larger number of genes in the fly makes whole genome pairwise genetic mapping a daunting prospect, nonetheless de novo mutagenesis in genetically compromised fly strains is a routinely used tool to identify genetic interactions and members of shared physiological or regulatory pathways [ 97 , 98 ].…”