Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Abstract

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic β-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.

This is a preview of subscription content, log in to check access.

References

  1. Araki H, Jearnpipatkul A, Tatsumi H, Sakurai T, Ushio K, Muta T, Oshima Y (1985) Molecular and functional organization of yeast plasmid pSR1. J MolBiol 182:191–203

  2. Araki H, Nakanishi N, Evans BR, Matsuzaki H, Jayaram M, Oshima Y (1992) Site-specific recombinase, R, encoded by yeast plasmid pSR1. J Mol Biol 225:25–37

  3. Bancroft I, Dean C (1993) Transposition pattern of the maize element Ds in Arabidopsis thaliana. Genetics 134:1221–1229

  4. Bayley CC, Morgan M, Dale EC, Ow DW (1992) Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system. Plant Mol Biol 18:353–361

  5. Benfey PN, Ren L, Chua NH (1989) The CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J 8:2195–2202

  6. Broach JR (1981) In: Strathern JN, Jones EW, Broach JR (eds) The molecular biology of the yeast Saccharomyces: life cycle and inheritance. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York pp 445–470

  7. Cardon GH, Frey M, Saedler H, Gierl A (1993a) Definition and characterization of an artificial En/Spm-based transposon tagging system in transgenic tobacco. Plant Mol Biol 23, 157–178

  8. Cardon GH, Frey M, Saedler H, Gierl A (1993b) Mobility of the maize transposable element En/Spm in Arabidopsis thaliana. Plant J 3:773–784

  9. Dale EC, Ow DW (1990) Intra- and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase. Gene 91:79–85

  10. Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 88:10558–10562

  11. Dooner HK, Belachew A (1989) Transposition pattern of the maize element Ac from the bz-m2 (Ac) allele. Genetics 122:447–457

  12. Dooner HK, Keller J, Harper E, Ralston E (1991) Variable patterns of transposition of the maize element Activator in tobacco. Plant Cell 3:473–482

  13. Furner IJ, Pumfrey JE (1992) Cell fate in the shoot apical meristem of Arabidopsis thaliana. Development 115:755–764

  14. Golic KG, Lindquist S (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59:499–509

  15. Greenblatt IM (1984) A chromosome replication pattern deduced from pericarp phenotypes resulting from movements of the transposable element, Modulator, in maize. Genetics 108:471–485

  16. Greenblatt IM, Brink RA (1962) Twin mutations in medium variegated pericarp maize. Genetics 47:489–501

  17. van Haaren MJJ, Ow DW (1993) Prospects of applying a combination of DNA transposition and site-specific recombination in plants: a strategy for gene identification and cloning. Plant Mol Biol 23, 525–533

  18. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hyper-virulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301

  19. Irish VF, Sussex IM (1992) A fate map of the Arabidopsis embryonic shoot apical meristem. Development 115:745–753

  20. Jayaram M (1985) Two-micrometer circle site-specific recombination: the minimal substrate and the possible role of flanking sequences. Proc Natl Acad Sci USA 82:5875–5879

  21. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907

  22. Jones JDG, Carland F, Lim E, Ralston E, Dooner H (1990) Preferential transposition of the Maize element Activator to linked chromosomal locations in tobacco. Plant Cell 2:701–707

  23. Jones JDG, Carland FM, Maliga P, Dooner HK (1989) Visual detection of transposition of the maize element Activator (Ac) in tobacco seedlings. Science 244:204–207

  24. Kawasaki H, Matsuzaki H, Nakajima R, Oshima Y (1991) The PHO80/TUP7 locus in Saccharomyces cerevisiae is on the left arm of chromosome XV: mapping by chromosome engineering. Yeast 7:859–865

  25. Kilby NJ, Snaith MR, Murray JAH (1993) Site-specific recombinases: tools for genome engineering. Trends Genet 9:413–421

  26. Lloyd AM, Davis RW (1994) Functional expression of the yeast FLP/FRT site-specific recombination system in Nicotiana tabacum. Mol Gen Genet 242:653–657

  27. Machida C, Onouchi H, Machida Y (1993) Analysis of plant genes using T-DNA transfer from Agrobacterium. Nippon Nougeikagaku Kaishi 67:711–715

  28. Matsuzaki H, Araki H, Oshima Y (1988) Gene conversion associated with site-specific recombination in yeast plasmid pSR1. Mol Cell Biol 8:955–962

  29. Matsuzaki H, Nakajima R, Nishiyama J, Araki H, Oshima Y (1990) Chromosome engineering in Saccharomyces cerevisiae by using a site-specific recombination system of a yeast plasmid. J Bacteriol 172:610–618

  30. Medberry SL, Dale E, Qin M, Ow DW (1995) Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination. Nucleic Acids Res (in press)

  31. Moreno MA, Chen J, Greenblatt I, Dellaporta SL (1992) Reconstitutional mutagenesis of the maize P gene by short-range Ac transpositions. Genetics 131:939–956

  32. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325

  33. Odell J, Caimi P, Sauer B, Russell S (1990) Site-directed recombination in the genome of transgenic tobacco. Mol Gen Genet 223:369–378

  34. O'Gorman S, Fox DT, Wahl GM (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251:1351–1355

  35. Onouchi H, Yokoi K, Machida C, Matsuzaki H, Oshima Y, Matsuoka K, Nakamura K, Machida Y (1991) Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. Nucleic Acids Res 19:6373–6378

  36. Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7:4021–4026

  37. Qin M, Bayley C, Stockton T, Ow DW (1994) Cre recombinase-mediated site-specific recombination between plant chromosomes. Proc Natl Acad Sci USA 91:1706–1710

  38. Russell SH, Hoopes JL, Odell JT (1992) Directed excision of a transgene from the plant genome. Mol Gen Genet 234:49–59

  39. Sauer B (1987) Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol 7:2087–2096

  40. Sauer B, Henderson N (1988) Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1. Proc Natl Acad Sci USA 85:5166–5170

  41. van Schaik NW, Brink RA (1959) Transposition of Modulator, a component of the variegated pericarp allele in maize. Genetics 44:725–738

  42. Scheres B, Wolkenfelt H, Willemsen V, Terlouw M, Lawson E, Dean C (1994) Embryonic origin of the Arabidopsis primary root and root meristem initials. Development 120:2475–2487

  43. Swoboda P, Hohn B, Gal S (1993) Somatic homologous recombination in planta: the recombination frequency is dependent on the allelic state of recombining sequences and may be influenced by genomic position effects. Mol Gen Genet 237:33–40

  44. Swoboda P, Gal S, Hohn B, Puchta H (1994) Intrachromosomal homologous recombination in whole plants. EMBO J 13:484–489

  45. Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512

  46. Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Agrobacterium tumefaciens-mediated transformation of Arabodopsis thaliana root explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5540

Download references

Author information

Correspondence to Yasunori Machida or Chiyoko Machida.

Additional information

Communicated by M. Sekiguchi

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Onouchi, H., Nishihama, R., Kudo, M. et al. Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana . Molec. Gen. Genet. 247, 653–660 (1995). https://doi.org/10.1007/BF00290396

Download citation

Key words

  • Deletion
  • Arabidopsis thaliana
  • Site-specific recombination
  • R-RS recombination system of Zygosaccharomyces rouxii
  • β-Glucuronidase