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.
Similar content being viewed by others
References
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
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
Bancroft I, Dean C (1993) Transposition pattern of the maize element Ds in Arabidopsis thaliana. Genetics 134:1221–1229
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
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
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
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
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
Dale EC, Ow DW (1990) Intra- and intermolecular site-specific recombination in plant cells mediated by bacteriophage P1 recombinase. Gene 91:79–85
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
Dooner HK, Belachew A (1989) Transposition pattern of the maize element Ac from the bz-m2 (Ac) allele. Genetics 122:447–457
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
Furner IJ, Pumfrey JE (1992) Cell fate in the shoot apical meristem of Arabidopsis thaliana. Development 115:755–764
Golic KG, Lindquist S (1989) The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome. Cell 59:499–509
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
Greenblatt IM, Brink RA (1962) Twin mutations in medium variegated pericarp maize. Genetics 47:489–501
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
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
Irish VF, Sussex IM (1992) A fate map of the Arabidopsis embryonic shoot apical meristem. Development 115:745–753
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
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
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
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
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
Kilby NJ, Snaith MR, Murray JAH (1993) Site-specific recombinases: tools for genome engineering. Trends Genet 9:413–421
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
Machida C, Onouchi H, Machida Y (1993) Analysis of plant genes using T-DNA transfer from Agrobacterium. Nippon Nougeikagaku Kaishi 67:711–715
Matsuzaki H, Araki H, Oshima Y (1988) Gene conversion associated with site-specific recombination in yeast plasmid pSR1. Mol Cell Biol 8:955–962
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
Medberry SL, Dale E, Qin M, Ow DW (1995) Intra-chromosomal rearrangements generated by Cre-lox site-specific recombination. Nucleic Acids Res (in press)
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
Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325
Odell J, Caimi P, Sauer B, Russell S (1990) Site-directed recombination in the genome of transgenic tobacco. Mol Gen Genet 223:369–378
O'Gorman S, Fox DT, Wahl GM (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251:1351–1355
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
Paszkowski J, Baur M, Bogucki A, Potrykus I (1988) Gene targeting in plants. EMBO J 7:4021–4026
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
Russell SH, Hoopes JL, Odell JT (1992) Directed excision of a transgene from the plant genome. Mol Gen Genet 234:49–59
Sauer B (1987) Functional expression of the cre-lox site-specific recombination system in the yeast Saccharomyces cerevisiae. Mol Cell Biol 7:2087–2096
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
van Schaik NW, Brink RA (1959) Transposition of Modulator, a component of the variegated pericarp allele in maize. Genetics 44:725–738
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
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
Swoboda P, Gal S, Hohn B, Puchta H (1994) Intrachromosomal homologous recombination in whole plants. EMBO J 13:484–489
Thomas KR, Capecchi MR (1987) Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51:503–512
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
Author information
Authors and Affiliations
Corresponding authors
Additional information
Communicated by M. Sekiguchi
Rights 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
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00290396