Lori Friedman

Systematic generation of high-resolution deletion coverage of the Drosophila melanogaster genome

In fruit fly research, chromosomal deletions are indispensable tools for mapping mutations, characterizing alleles and identifying interacting loci. Most widely used deletions were generated by irradiation or chemical mutagenesis. These methods are labor-intensive, generate random breakpoints and result in unwanted secondary mutations that can confound phenotypic analyses. Most of the existing deletions are large, have molecularly undefined endpoints and are maintained in genetically complex stocks. Furthermore, the existence of haplolethal or haplosterile loci makes the recovery of deletions of certain regions exceedingly difficult by traditional methods, resulting in gaps in coverage. Here we describe two methods that address these problems by providing for the systematic isolation of targeted deletions in the D. melanogaster genome. The first strategy used a P element–based technique to generate deletions that closely flank haploinsufficient genes and minimize undeleted regions. This deletion set has increased overall genomic coverage by 5–7%. The second strategy used FLP recombinase and the large array of FRT-bearing insertions described in the accompanying paper to generate 519 isogenic deletions with molecularly defined endpoints. This second deletion collection provides 56% genome coverage so far. The latter methodology enables the generation of small custom deletions with predictable endpoints throughout the genome and should make their isolation a simple and routine task.

Target discovery in the postgenomic era

Genetic screens can reveal new pathway modifier genes that would be difficult to uncover using other experimental approaches. The conservation of biochemical pathways and ability to quickly screen large numbers of candidate target genes strongly supports the use of model system genetics. We have carried out large-scale genetic screens in Drosophila melanogaster, Caenorhabditis elegans, and cells to identify modifier genes of cancer-related pathways and phenotypes. Genetic screens can identify the function of novel genes, and establish functional links between genes that may have been previously identified, but whose role in a process was not understood. Invertebrate genetic screens are carried out in animals or cells with mutations in cancer genes that produce a measureable phenotype. In Drosophila we typically perform tissue-specific screens, in the eye or the wing, so that the cancer mutations do not affect viability or fertility of the organism. These sensitized genetic backgrounds are then screened to identify genes that modify the visible phenotype. Modifier genes can be identified through reverse genetic approaches, including transposon insertions and RNA interference. The availability of fully sequenced genomes and the use of reverse genetic tools such as RNA interference enables genetic screens to be focused on classes of proteins which are amenable to drug discovery, thus enhancing the efficiency of target identification. Genetic screens will help build a better understanding of signal transduction pathways and gene function on a large scale.