Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1999 Feb;151(2):725–737. doi: 10.1093/genetics/151.2.725

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

G Toba 1, T Ohsako 1, N Miyata 1, T Ohtsuka 1, K H Seong 1, T Aigaki 1
PMCID: PMC1460511  PMID: 9927464

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (352.7 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Banfi S., Borsani G., Rossi E., Bernard L., Guffanti A., Rubboli F., Marchitiello A., Giglio S., Coluccia E., Zollo M. Identification and mapping of human cDNAs homologous to Drosophila mutant genes through EST database searching. Nat Genet. 1996 Jun;13(2):167–174. doi: 10.1038/ng0696-167. [DOI] [PubMed] [Google Scholar]
  3. Bellen H. J., O'Kane C. J., Wilson C., Grossniklaus U., Pearson R. K., Gehring W. J. P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev. 1989 Sep;3(9):1288–1300. doi: 10.1101/gad.3.9.1288. [DOI] [PubMed] [Google Scholar]
  4. Bier E., Vaessin H., Shepherd S., Lee K., McCall K., Barbel S., Ackerman L., Carretto R., Uemura T., Grell E. Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector. Genes Dev. 1989 Sep;3(9):1273–1287. doi: 10.1101/gad.3.9.1273. [DOI] [PubMed] [Google Scholar]
  5. Brand A. H., Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development. 1993 Jun;118(2):401–415. doi: 10.1242/dev.118.2.401. [DOI] [PubMed] [Google Scholar]
  6. Brunner D., Dücker K., Oellers N., Hafen E., Scholz H., Klämbt C. The ETS domain protein pointed-P2 is a target of MAP kinase in the sevenless signal transduction pathway. Nature. 1994 Aug 4;370(6488):386–389. doi: 10.1038/370386a0. [DOI] [PubMed] [Google Scholar]
  7. Chen F., Barkett M., Ram K. T., Quintanilla A., Hariharan I. K. Biological characterization of Drosophila Rapgap1, a GTPase activating protein for Rap1. Proc Natl Acad Sci U S A. 1997 Nov 11;94(23):12485–12490. doi: 10.1073/pnas.94.23.12485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chiba A., Snow P., Keshishian H., Hotta Y. Fasciclin III as a synaptic target recognition molecule in Drosophila. Nature. 1995 Mar 9;374(6518):166–168. doi: 10.1038/374166a0. [DOI] [PubMed] [Google Scholar]
  9. Cooley L., Kelley R., Spradling A. Insertional mutagenesis of the Drosophila genome with single P elements. Science. 1988 Mar 4;239(4844):1121–1128. doi: 10.1126/science.2830671. [DOI] [PubMed] [Google Scholar]
  10. DeJong J. L., Morgenstern R., Jörnvall H., DePierre J. W., Tu C. P. Gene expression of rat and human microsomal glutathione S-transferases. J Biol Chem. 1988 Jun 15;263(17):8430–8436. [PubMed] [Google Scholar]
  11. Deák P., Omar M. M., Saunders R. D., Pál M., Komonyi O., Szidonya J., Maróy P., Zhang Y., Ashburner M., Benos P. P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: correlation of physical and cytogenetic maps in chromosomal region 86E-87F. Genetics. 1997 Dec;147(4):1697–1722. doi: 10.1093/genetics/147.4.1697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hariharan I. K., Carthew R. W., Rubin G. M. The Drosophila roughened mutation: activation of a rap homolog disrupts eye development and interferes with cell determination. Cell. 1991 Nov 15;67(4):717–722. doi: 10.1016/0092-8674(91)90066-8. [DOI] [PubMed] [Google Scholar]
  13. Harrison D. A., Binari R., Nahreini T. S., Gilman M., Perrimon N. Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and developmental defects. EMBO J. 1995 Jun 15;14(12):2857–2865. doi: 10.1002/j.1460-2075.1995.tb07285.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jimenez B., Pizon V., Lerosey I., Béranger F., Tavitian A., de Gunzburg J. Effects of the ras-related rap2 protein on cellular proliferation. Int J Cancer. 1991 Sep 30;49(3):471–479. doi: 10.1002/ijc.2910490327. [DOI] [PubMed] [Google Scholar]
  15. Kelner M. J., Stokely M. N., Stovall N. E., Montoya M. A. Structural organization of the human microsomal glutathione S-transferase gene (GST12). Genomics. 1996 Aug 15;36(1):100–103. doi: 10.1006/geno.1996.0429. [DOI] [PubMed] [Google Scholar]
  16. Lerosey I., Chardin P., de Gunzburg J., Tavitian A. The product of the rap2 gene, member of the ras superfamily. Biochemical characterization and site-directed mutagenesis. J Biol Chem. 1991 Mar 5;266(7):4315–4321. [PubMed] [Google Scholar]
  17. Pizon V., Chardin P., Lerosey I., Olofsson B., Tavitian A. Human cDNAs rap1 and rap2 homologous to the Drosophila gene Dras3 encode proteins closely related to ras in the 'effector' region. Oncogene. 1988 Aug;3(2):201–204. [PubMed] [Google Scholar]
  18. Robertson H. M., Preston C. R., Phillis R. W., Johnson-Schlitz D. M., Benz W. K., Engels W. R. A stable genomic source of P element transposase in Drosophila melanogaster. Genetics. 1988 Mar;118(3):461–470. doi: 10.1093/genetics/118.3.461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Rørth P. A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc Natl Acad Sci U S A. 1996 Oct 29;93(22):12418–12422. doi: 10.1073/pnas.93.22.12418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Salzberg A., Prokopenko S. N., He Y., Tsai P., Pál M., Maróy P., Glover D. M., Deák P., Bellen H. J. P-element insertion alleles of essential genes on the third chromosome of Drosophila melanogaster: mutations affecting embryonic PNS development. Genetics. 1997 Dec;147(4):1723–1741. doi: 10.1093/genetics/147.4.1723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sidow A., Thomas W. K. A molecular evolutionary framework for eukaryotic model organisms. Curr Biol. 1994 Jul 1;4(7):596–603. doi: 10.1016/s0960-9822(00)00131-7. [DOI] [PubMed] [Google Scholar]
  22. Spradling A. C., Stern D. M., Kiss I., Roote J., Laverty T., Rubin G. M. Gene disruptions using P transposable elements: an integral component of the Drosophila genome project. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10824–10830. doi: 10.1073/pnas.92.24.10824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Staehling-Hampton K., Jackson P. D., Clark M. J., Brand A. H., Hoffmann F. M. Specificity of bone morphogenetic protein-related factors: cell fate and gene expression changes in Drosophila embryos induced by decapentaplegic but not 60A. Cell Growth Differ. 1994 Jun;5(6):585–593. [PubMed] [Google Scholar]
  24. Thummel C. S., Boulet A. M., Lipshitz H. D. Vectors for Drosophila P-element-mediated transformation and tissue culture transfection. Gene. 1988 Dec 30;74(2):445–456. doi: 10.1016/0378-1119(88)90177-1. [DOI] [PubMed] [Google Scholar]
  25. Tower J., Karpen G. H., Craig N., Spradling A. C. Preferential transposition of Drosophila P elements to nearby chromosomal sites. Genetics. 1993 Feb;133(2):347–359. doi: 10.1093/genetics/133.2.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Xu T., Rubin G. M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development. 1993 Apr;117(4):1223–1237. doi: 10.1242/dev.117.4.1223. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES