The Drosophila Gene Disruption Project: Progress Using Transposons With Distinctive Site Specificities

Abstract

The Drosophila Gene Disruption Project (GDP) has created a public collection of mutant strains containing single transposon insertions associated with different genes. These strains often disrupt gene function directly, allow production of new alleles, and have many other applications for analyzing gene function. Here we describe the addition of 7600 new strains, which were selected from .140,000 additional P or piggyBac element integrations and 12,500 newly generated insertions of the Minos transposon. These additions nearly double the size of the collection and increase the number of tagged genes to at least 9440, approximately two-thirds of all annotated protein-coding genes. We also compare the site specificity of the three major transposons used in the project. All three elements insert only rarely within many Polycomb-regulated regions, a property that may contribute to the origin of "transposon-free regions" (TFRs) in metazoan genomes. Within other genomic regions, Minos transposes essentially at random, whereas P or piggyBac elements display distinctive hotspots and coldspots. P elements, as previously shown, have a strong preference for promoters. In contrast, piggyBac site selectivity suggests that it has evolved to reduce deleterious and increase adaptive changes in host gene expression. The propensity of Minos to integrate broadly makes possible a hybrid finishing strategy for the project that will bring .95% of Drosophila genes under experimental control within their native genomic contexts.

Drosophila P elements preferentially transpose to replication origins

“…However, the other two genes, which appear no different in structure, had ORC sites at their promoters, and both contained multiple P insertions. P element insertions are absent in many "repressed" genomic regions regulated by Polycomb Group (PcG) proteins (14). Consequently, we determined if such regions also were deficient in ORC-binding sites and found that they were not.…”

“…We analyzed 18,213 independent transposition events of the EY P element, which were shown previously to be representative of P transposition generally (13,14). Comparable datasets for two other DNA transposons, the mariner-like Minos element and the piggyBac element, were used for comparison (14,16).…”

“…The Gene Disruption Project (GDP) has accumulated extensive data on the site specificity of several transposons, including P elements, as a byproduct of generating gene disruption strains for public use in about two-thirds of all Drosophila genes (13,14). By analyzing more than 100,000 individual transposition events from more than 10 independent screens based on a variety of engineered P elements, the highly nonrandom selectivity of P element insertion has been thoroughly documented.…”

“…Although P element integration sites are GC rich, their primary sequences do not explain this insertional bias (15). Other wellstudied DNA transposons such as piggyBac share a general preference for genes and promoters, but piggyBac hotspots overlap P element hotspots to only a limited extent (14,16). P elements may target genes that contain open chromatin and/or are actively transcribed in early germ cells where transposition occurs (4,17).…”

Methods to study Drosophila immunity

“…Transposon mutagenesis, albeit suffering from insertion bias [72], allows for easy retrieval of positional information, and forms the basis for a downstream toolkit of genetic applications including imprecise excision knock-out, Gal4-UAS overexpression of flanking genes, or element replacement by targeting vectors, to name but a few [73][74][75][76][77]. Extensive libraries of P-element-based transposon insertions are available through stock centers, along with deletion and duplication lines [78][79][80][81]. Finally, targeted gene knock-out using optimized targeting plasmids in combination with CRISPR will greatly accelerate full KO coverage of the fly genome [82].…”