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Molecular and General Genetics MGG

, Volume 245, Issue 6, pp 734–740 | Cite as

Facilitated isolation of rare recombinants by ligase chain reaction: selection for intragenic crossover events in the Drosophila optomotor-blind gene

  • Jürgen Balles
  • Gert O. Pflugfelder
Original Paper

Abstract

Ligase chain reaction (LCR) was evaluated as a tool for the detection of point mutations. For the mutation studied, the specificity of the method is sufficient to detect the mutant allele in the presence of a 200-fold molar excess of the wild-type sequence. LCR was therefore employed in a genetic recombination experiment as a probe for a recessive lethal point mutation. LCR greatly facilitated the isolation of a rare recombinant originating from a crossover event in the 40 kb interval separating the lethal mutation and an enhancer trap insertion in the optomotor-blind locus. The recombinant will allow the study of gene control in situ, in a largely unperturbed regulatory environment.

Key words

Ligase chain reaction Intragenic recombination Gene regulation Point mutation Drosophila melanogaster 

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References

  1. Ballinger DG, Benzer S (1989) Targeted gene mutations in Drosophila. Proc Natl Acad Sci USA 86:9402–9406Google Scholar
  2. Barany F (1991a) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc Natl Acad Sci USA 88:189–193Google Scholar
  3. Barany F (1991b) The ligase chain reaction in a PCR world. PCR Methods Applic 1:5–16Google Scholar
  4. Bellen HJ, O'Kane CJ, Wilson C, Grossniklaus U, Pearson RK, Gehring WJ (1989) P-element-mediated enhancer detection: a versatile method to study development in Drosophila. Genes Dev 3:1288–1300Google Scholar
  5. Bier E, Vaessin H, Shepherd S, Lee K, McCall K, Barbel S, Ackerman L, Carretto R, Uemura T, Grell E, Jan LY, Jan YN (1989) Searching for pattern and mutation in the Drosophila genome with a P-lacZ vector. Genes Dev 3:1273–1287Google Scholar
  6. Champlin DT, Frasch M, Saumweber H, Lis JT (1991) Characterization of a Drosophila protein associated with boundaries of transcriptionally active chromatin. Genes Dev 5:1611–1621Google Scholar
  7. Chou Q, Russell M, Birch DE, Raymond J, Bloch W (1992) Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Res 20:1717–1723Google Scholar
  8. Chung JH, Whiteley M, Felsenfeld G (1993) A 5' element of the chicken β-globin domain serves as an insulator in human erythroid cells and protects against position effects in Drosophila. Cell 74:505–514Google Scholar
  9. Freeman M, Kimmel BE, Rubin GM (1992) Identifying targets of the rough homeobox gene of Drosophila: evidence that rhomboid functions in eye development. Development 116:335–346Google Scholar
  10. Haenlin M, Steller H, Pirrotta V, Mohier E (1985) A 43 kilobase cosmid P transposon rescues the fs(1)K10 morphogenetic locus and three adjacent Drosophila developmental mutants. Cell 40:827–837Google Scholar
  11. Heisenberg M, Wonneberger R, Wolf R (1978) Optomotor-blind H31 — a Drosophila mutant of the lobula plate giant neurons. J Comp Physiol 124:287–296Google Scholar
  12. Hooper JE, Pérez-Alonso M, Bermingham JR, Prout M, Rocklein BA, Wagenbach M, Edstrom J-E, de Frutos R, Scott MP (1992) Comparative studies of the Drosophila Antennapedia genes. Genetics 132:453–469Google Scholar
  13. Jupe ER, Sinden RR, Cartwright IL (1993) Stably maintained microdomain of localized unrestrained supercoiling at a Drosophila heat shock gene locus. EMBO J 12:1067–1075Google Scholar
  14. Kaiser K, Goodwin SF (1990) “Site-selected” transposon mutagenesis of Drosophila. Proc Natl Acad Sci USA 87:1686–1690Google Scholar
  15. Kitamoto T, Salvaterra PM (1993) Developmental regulatory elements in the 5' flanking DNA of the Drosophila choline acetyltransferase gene. Roux's Arch Dev Biol 202:159–169Google Scholar
  16. Marsh E, Schoetllin W, Cline J, Callen W, Kretz K, Mathur E (1992) Pyrococcus furiosus DNA ligase and the ligase chain reaction. Strategies in Molecular Biology (Stratagene) 5:73–76Google Scholar
  17. Martín-Bermudo MD, González F, Dominguez M, Rodríguez I, Ruiz-Gómez M, Romani S, Modolell J, Jiménez F (1993) Molecular characterization of the lethal of scute genetic function. Development 118:1003–1012Google Scholar
  18. Mirkovitch J, Gasser SM, Laemmli UK (1988) Scaffold attachment of DNA loops in metaphase chromosomes. J Mol Biol 200:101–109Google Scholar
  19. Muralidhar MG, Callahan CA, Thomas JB (1993) Single-minded regulation of genes in the embryonic midline of the Drosophila central nervous system. Mech Dev 41:129–138Google Scholar
  20. O'Kane CJ, Gehring WJ (1987) Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci USA 84:9123–9127Google Scholar
  21. Pflugfelder GO, Roth H, Poeck B, Kerscher S, Schwarz S, Jonschker B, Heisenberg M (1992a) The l(1)optomotor-blind gene of Drosophila melanogaster is a major organizer of optic lobe development: isolation and characterization of the gene. Proc Natl Acad Sci USA 89:1199–1203Google Scholar
  22. Pflugfelder GO, Roth H Poeck B (1992b) A homology domain shared between Drosophila optomotor-blind and mouse Brachyury is involved in DNA binding. Biochem Biophys Res Commun 186:918–925Google Scholar
  23. Poeck B (1992) Untersuchungen zur Genetik und Expression des optomotor-blind locus von Drosophila melanogaster. Thesis, University of WürzburgGoogle Scholar
  24. Poeck B, Hofbauer A, Pflugfelder GO (1993a) Expression of the Drosophila optomotor-blind gene transcript in neuronal and glial cells of the developing nervous system. Development 117:1017–1029Google Scholar
  25. Poeck B, Balles J, Pflugfelder GO (1993b) Transcript identification in the optomotor-blind locus of Drosophila melanogaster by intragenic recombination mapping and PCR-aided sequence analysis of lethal point mutations. Mol Gen Genet 238:325–332Google Scholar
  26. Ramos RGP, Grimwade BG, Wharton KA, Scottgale TN, Artavanis-Tsakonas S (1989) Physical and functional definition of the Drosophila Notch locus by P element transformation. Genetics 123:337–348Google Scholar
  27. Rothe M, Pehl M, Taubert H, Jäckle H (1992) Loss of gene function through rapid mitotic cycles. Nature 359:156–159Google Scholar
  28. Rychlik W, Rhoads RE (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amplification of DNA. Nucleic Acids Res 17:8543–8551Google Scholar
  29. Wagner-Bernholz JT, Wilson C, Gibson G, Schuh R, Gehring WJ (1991) Identification of target genes of the homeotic gene Antennapedia by enhancer detection. Genes Dev 5:2467–2480Google Scholar
  30. Walter N (1992) The development of a new method to introduce mutations into cloned genes. Thesis, University of ZürichGoogle Scholar
  31. Weigel D, Seifert E, Reuter D, Jäckle H (1990) Regulatory elements controlling expression of the Drosophila homeotic gene fork head. EMBO J 9:1199–1207Google Scholar
  32. Zavortink M, Sakonju S (1989) The morphogenetic and regulatory functions of the Drosophila Abdominal-B gene are encoded in overlapping RNAs transcribed from separate promoters. Genes Dev 3:1969–1981Google Scholar

Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Jürgen Balles
    • 1
  • Gert O. Pflugfelder
    • 1
  1. 1.Theodor-Boveri-Institut, Biozentrum, Lehrstuhl für GenetikUniversität WürzburgWürzburgGermany

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