[an error occurred while processing this directive] BDGP: Experimental Strategy of the P Element Gene Disruption Project

Gene Disruption Project

Experimental Strategy of the P Element Gene Disruption Project

The scale of the gene disruption experiments we successfully carried out--inactivating more than 25% of all vital genes--is unprecedented in multicellular organisms, and is likely to provide unique insights into the organization of eukaryotic genomes. Papers describing the design and some initial results (Spradling et al., 1995) and final results (Spradling et al., 1999) have been published.

Insertional mutagenesis can efficiently associate complementation groups with specific open reading frames on a genome-wide scale. Consequently, collections of mutant strains bearing gene disruptions provide a resource for functional genomics that is of paramount importance. Our gene disruption project generated a large collection of strains that each contained a single P element insertion altering the expression of a different genomic open reading frame. The strain library contains 1045 strains disrupting at least 1,000 different genes, corresponding to more than 25% of the estimated 3600 genes on the Drosophila autosomes essential for adult viability. The insertions defining 67% of the genes in the collection were verified by genetic tests to cause the associated recessive mutant phenotype. Moreover, sequences flanking the insertions in more than 90% of the lines were determined to exactly position them in the genome, and to identify 376 potentially affected transcripts within the BDGP collection of EST sequences. These insertion-flanking sequences have been deposited in the dbSTS division of GenBank. Strains listed as Primary in the Spradling et al. 1999 paper are available from the Bloomington Stock Center and have already assisted the research community in characterizing more than 200 Drosophila genes. Second alleles of many genes are maintained at the Carnegie Institution (chromosome III) or at Berkeley (chromosome II), and have also been available on request since 1993. Our results suggest that it may soon be possible to bring virtually every Drosophila open reading frame under experimental control. In particular, this project will guarantee that as large scale sequencing of Drosophila and human genomes progresses, a significant fraction of human genes with Drosophila homologues will have been associated with P-element induced mutations that can provide insight into their function during Drosophila and human development.

Our gene disruption library was designed to target distinct loci that cause a recognizable lethal, semi-lethal, sterile or visible phenotype. 3,879 candidate lines were collected from seven separate single P element mutagenesis screens (see Spradling et al., 1995). Each such line contained one or more unlocalized P insertions on an autosome bearing a newly-induced scorable recessive phenotype. The insertion library was derived from these lines by: 1) localizing the insertions by in situ hybridization to polytene chromosomes at high resolution; 2) identifying strains with allelic insertions by inter se complementation crosses; 3) verifying that insertions were responsible for the mutant phenotype by crossing to chromosomes bearing deficiencies; 4) sequencing DNA flanking the insertions and comparing the sequences to each other, to EST libraries, and to genomic and protein sequence databases. Single insert-bearing strains that appear to disrupt distinct genes based on these criteria constitute the "primary collection".