The gene search system: A method for efficient detection and rapid molecular identification of genes in Drosophila melanogaster

We have constructed a P-element-based gene search vector for efficient dete ction of genes in Drosophila melanogaster. The vector contains two copies o f the upstream activating sequence (UAS) enhancer adjacent to a core promot er, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the ba sis of phenotypic changes caused by GAL4-dependent forced expression of vec tor-flanking DNA, and the transcripts were identified with reverse transcri ptase PCR (RT-PCR) using the vector-specific primer and followed by direct sequencing. The system had a greater sensitivity than those already in use for gain-of-function screening: 64% of the vector insertion lines (394/613) showed phenotypes with forced expression of vector-flanking DNA, such as l ethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% mat ched the sequences of cloned genes, and 18% matched reported expressed sequ ence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of t he protein-coding region. We discovered two new genes that showed sequence similarity to human genes, Ras-related protein 2 and microsomal glutathione S-transferase. The system can be useful as a tool for the functional mappi ng of the Drosophila genome.