Genetics, Vol. 151, 725-737, February 1999, Copyright © 1999

The Gene Search System: A Method for Efficient Detection and Rapid Molecular Identification of Genes in Drosophila melanogaster

Gakuta Tobaa, Takashi Ohsakob, Naomasa Miyataa, Tsuyoshi Ohtsukaa, Ki-Hyeon Seonga, and Toshiro Aigakia,b
a Department of Biological Sciences, Tokyo Metropolitan University, Tokyo 192-0397
b Precursory Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Kyoto 619-0237, Japan

Corresponding author: Toshiro Aigaki, Department of Biological Sciences, Tokyo Metropolitan University, 1-1 Minami-osawa, Hachioji-shi, Tokyo 192-0397, Japan., aigaki-toshiro{at}c.metro-u.ac.jp (E-mail)

Communicating editor: T. C. KAUFMAN

We have constructed a P-element-based gene search vector for efficient detection of genes in Drosophila melanogaster. The vector contains two copies of the upstream activating sequence (UAS) enhancer adjacent to a core promoter, one copy near the terminal inverted repeats at each end of the vector, and oriented to direct transcription outward. Genes were detected on the basis of phenotypic changes caused by GAL4-dependent forced expression of vector-flanking DNA, and the transcripts were identified with reverse transcriptase 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 lethality or defects in adult structure. Molecular analysis of 170 randomly selected insertions with forced expression phenotypes revealed that 21% matched the sequences of cloned genes, and 18% matched reported expressed sequence tags (ESTs). Of the insertions in cloned genes, 83% were upstream of the 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 mapping of the Drosophila genome.




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