Abstract
FLP/FRT-mediated site-specific recombination was studied with a recombination-reporter gene system which allows visualization of β-glucuronidase (GUS) expression after site-specific excisional activation of a silent gusA gene. This system was used for characterization of the functional activity of the Saccharomyces cerevisiae native FLP recombinase driven by the cauliflower mosaic virus (CaMV) 35s promoter [linked to the tobacco mosaic virus (TMV) omega translational leader] in mediating site-specific recombination of chromosomal FRT sites in tobacco FLP x FRT-reporter hybrids. Six hybrids were generated from crosses of lines containing either a stably integrated recombination-reporter or a FLP-expression construct. The activated gusA phenotype was specific to hybrid progenies and was not observed in either parental plants or their selfed progenies. Recombination efficiency in whole seedlings was estimated by the percent of radioactivity on a Southern blot which was incorporated into the recombined DNA product. Estimated efficiency mean values for the six crosses ranged from 5.2 to 52.0%. Histochemical analysis in hybrid plants visualized GUS activity with variable chimeric patterns and intensities. Recombination efficiency and GUS expression varied both among and within crosses, while higher recombination efficiency coincided with larger and more intense patterns of GUS activity. These data suggest that recombination is induced randomly during somatic developmental stages and that the pattern and intensity generated in a given plant are affected by factors imposing varibility not only between but also within crosses. Additionally, while recombination in a population of FLP/FRT hybrids may occur in all plants, recombination efficiency may still be low in any given plant. The activity of the native, as compared to a modified, FLP (Kilby et al. 1995) in the activation of transgenic traits in tobacco is discussed.
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An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. In: Gelvin SB, Schilperoot RA, Vernma DPS (eds) Plant molecular biology manual, A3. Kluwer Academic Publishers, Dordrecht, pp 1–19
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
Beeckman T, Engler G (1994) An easy technique for the clearing of histochemically stained plant tissue. Plant Mol Biol Rep 12:37–42
Craig N (1988) The mechanism of conservative site-specific recombination. Annu Rev Genet 22:77–105
Dale EC, Ow DW (1990) Intra- and inter-molecular site-specific recombination in plant cells mediated by bacteriophage Pl 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 88:10558–10565
Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15
Gallie DR, William JL, Walbot V (1989) Visualizing mRNA expression in plant protoplasts: factors influencing efficient mRNA uptake and translation. Plant Cell 1:301–311
Haaren MJJ van, 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
Horsch RB, Fry JE, Hoffmann NL, Eichholz D, Rogers SG, Fraley RT (1985) A simple and general method for transferring genes into plants. Science 227:1229–1231
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
Kilby NJ, Snaith MR, Murray JAH (1993) Site-specific recombinase: tools for genome engineering. Trends Genet 9:413–421
Kilby NJ, Davies GJ, Snaith MR, Murray JAH (1995) FLP recombinase in transgenic plants: constitutive activity in stably transformed tobacco and generation of marked cell clones in Arabidopsis. Plant J 8:637–652
Koncz C, Schell J (1986) The promoter T1-DNA gene 5 controls the tissue-specific expression of chimeric genes carried by a novel type of Agrobacterium binary vector. Mol Gen Genet 204:383–396
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
Lyznik LA, Mitchell JC, Hirayama L, Hodges TK (1993) Activity of yeast FLP recombinase in maize and rice protoplasts. Nucleic Acids Res 21:969–975
Lyznik LA, Hirayama L, Rao KA, Abad A, Hodges TK (1995) Heat-inducible expression of the FLP gene in maize cells. Plant J 8:177–186
Maeser S, Kahmann R (1991) The Gin recombinase of phage Mu can catalyse site-specific recombination in plant protoplasts. Mol Gen Genet 230:170–176
Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
Odell JT, Russell SH (1994) Use of site-specific recombination systems in plants. In: Paszkowski J (ed) Homologous recombination in plants. Kluwer Academic Publishers, Dordrecht, pp 219–270
Odell JT, Caimi PG, Sauer B, Russell SH (1990) Site-directed recombination in the genome of transgenic tobacco. Mol Gen Genet 223:369–378
Odell JT, Hoopes JL, Vermerris W (1994) Seed-specific gene activation mediated by the Cre-lox site-specific recombination system. Plant Physiol 106:447–458
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. Nuclei Acids Res 19:6373–6378
Onouchi H, Nishihama R, Kudo M, Machida Y, Machida C (1995) Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana. Mol Gen Genet 247:653–660
Ow DW, Medberry SL (1995) Genome manipulation through site-specific recombination. Crit Rev Plant Sci 14:239–261
Russell SH, Hoopes JL, Odell JT (1992) Directed excision of a trasgene from the plant genome. Mol Gen Genet 234:49–59
SAS Institute Inc (1989) SAS/STAT user's guide, version 6, 4th edn. SAS Institute Inc, North Carolina
Sauer B (1994) Site-specific recombination: development and applications. Curr Biol 5:521–527
Sonti RV, Tissier AF, Wong D, Viret J-F, Signer ER (1995) Activity of the yeast FLP recombinase in Arabidopsis. Plant Mol Biol 28:1127–1132
Volkert FC, Broach JR (1986) Site-specific recombination promotes plasmid amplification in yeast. Cell 46:541–550
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Bar, M., Leshem, B., Gilboa, N. et al. Visual characterization of recombination at FRT-gusA loci in transgenic tobacco mediated by constitutive expression of the native FLP recombinase. Theoret. Appl. Genetics 93, 407–413 (1996). https://doi.org/10.1007/BF00223183
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DOI: https://doi.org/10.1007/BF00223183