title: Inducible protein traps with dominant phenotypes for functional analysis of the Drosophila genome.
abstract: The Drosophila melanogaster genome has been extensively characterized, but there remains a pressing need to associate gene products with phenotypes, subcellular localizations, and interaction partners. A multifunctional, Minos transposon-based protein trapping system called Hostile takeover (Hto) was developed to facilitate in vivo analyses of endogenous genes, including live imaging, purification of protein complexes, and mutagenesis. The Hto transposon features a UAS enhancer with a basal promoter, followed by an artificial exon 1 and a standard 5' splice site. Upon GAL4 induction, exon 1 can splice to the next exon downstream in the flanking genomic DNA, belonging to a random target gene. Exon 1 encodes a dual tag (FLAG epitope and mCherry red fluorescent protein), which becomes fused to the target protein. Hto was mobilized throughout the genome and then activated by eye-specific GAL4; an F1 screen for abnormal eye phenotypes was used to identify inserts that express disruptive fusion proteins. Approximately 1.7% of new inserts cause eye phenotypes. Of the first 23 verified target genes, 21 can be described as regulators of cell biology and development. Most are transcription factor genes, including AP-2, CG17181, cut, klu, mamo, Sox102F, and sv. Other target genes [l(1)G0232, nuf, pum, and Syt4] make cytoplasmic proteins, and these lines produce diverse fluorescence localization patterns. Hto permits the expression of stable carboxy-terminal subfragments of proteins, which are rarely tested in conventional genetic screens. Some of these may disrupt specific cell pathways, as exemplified by truncated forms of Mastermind and Nuf.
authors.affiliation: School of Biological Sciences, Illinois State University, Normal, Illinois 61790.
authors.forename: Swetha
authors.initials: S
authors.lastname: Singari
authors.forename: Naureen
authors.initials: N
authors.lastname: Javeed
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Tardi
authors.forename: Suresh
authors.initials: S
authors.lastname: Marada
authors.forename: Jeff C
authors.initials: JC
authors.lastname: Carlson
authors.forename: Steven
authors.initials: S
authors.lastname: Kirk
authors.forename: Judith M
authors.initials: JM
authors.lastname: Thorn
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Edwards
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D002874:Chromosome Mapping / Q000379:methods*; D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D029721:Drosophila Proteins / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics; D019895:Transposases / Q000235:genetics*
chemical_list: D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; C557617:GAL4 protein, Drosophila; D014157:Transcription Factors; C473499:Minos transposase; D019895:Transposases
keywords: GAL4/UAS; Minos vector; RNA splicing; ectopic expression; transcription factor