Summary
The excision of a Ds-like transposable element (AcΔ) is mediated in trans by the transposable element Ac or its derivatives in Petunia protoplasts cotransfected with two plasmid DNAs. Excision restores the activity of the β-glucuronidase (GUS) gene that is otherwise shut off by the presence of AcΔ in its leader sequence. A transient expression assay (histochemical test) is used to detect the β-glucuronidase activity at the protoplast level. The number of blue-stained protoplasts is a measure of the excision frequency. With AcΔ alone a near-zero background of GUS activity is detected, which is weakly enhanced by the presence, in trans, of either the wild-type Ac or the coding region (ORFa) transcribed from the 2′ promoter of Agrobacterium tumefaciens TR-DNA. A strong enhancement is observed when a truncated Ac coding region, also under the control of the 2′ promoter, is supplied in trans. The truncated version has ATG10 at codon 103 in frame with ORFa and is preceded by 7 out-of-frame ATGs. The assay is quick and well suited for detection of excision frequencies above the value obtained with the wild-type Ac. The presence of empty donor sites following excision can be demonstrated by PCR amplification and direct sequencing of the appropriate DNA fragment.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Baker B, Schell J, Lörz H, Fedoroff N (1986) Transposition of the maize controlling element Activator in tobacco. Proc Natl Acad Sci USA 83:4844–4848
Baker B, Coupland G, Fedoroff N, Starlinger P, Schell J (1987) Phenotypic assay for excision of the maize controlling element Ac in tobacco. EMBO J 6:1547–1554
Bohmann D, Tjian R (1989) Biochemical analysis of transcriptional activation by Jun: differential activity of c- and v-Jun. Cell 59:709–717
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Coupland G, Baker B, Schell J, Starlinger P (1988) Characterization of the maize transposable element Ac by internal deletions. EMBO J 7:3653–3659
Coupland G, Plum C, Chatterjee S, Post A, Starlinger P (1989) Sequences near the termini are required for transposition of the maize transposon Ac in transgenic tobacco plants. Proc Natl Acad Sci USA 86:9385–9388
English J, Ralston E, Dooner HK (1987) Corrections in the nucleotide sequence of Activator (Ac). Maize Genet Coop Newslett 61:81
Finnegan CEJ, Taylor BH, Dennis ES, Peacock WJ (1988) Transcription of the maize transposable element Ac in maize seedlings and in transgenic tobacco. Mol Gen Genet 212:505–509
Greenblatt IM (1984) A chromosomal replication pattern deduced from pericarp phenotypes resulting from movements of the transposable element, modulator, in maize. Genetics 108:471–485
Harpster MH, Townsend JA, Jones JDG, Bedbrook J, Dunsmuir P (1988) Relative strengths of the 35S cauliflower mosaic virus, 1′, 2′ and nopaline synthase promoters in transformed tobacco, sugarbeet and oilseed rape callus tissue. Mol Gen Genet 212:182–190
Hauser C, Fusswinkel H, Li J, Oellig C, Kunze R, Müller-Neumann M, Heinlein M, Starlinger P, Doerfler W (1988) Overproduction of the protein encoded by the maize transposable element Ac in insect cells by a baculovirus vector. Mol Gen Genet 214:373–378
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
Koncz C, Olsson O, Landridge WHR, Schell J, Szalay AA (1987) Expression and assembly of functional bacterial luciferase in plants. Proc Natl Acad Sci USA 84:131–135
Kozak M (1987 a) An analysis of 5′-noncoding sequences from 699 vertebrate messeger RNAs. Nucleic Acids Res 15:8125–8148
Kozak M (1987 b) Effects of intercistronic length on the efficiency of reinitiation by eucaryotic ribosomes. Mol Cell Biol 7:3438–3445
Kunze R, Stochaj U, Laufs J, Starlinger P (1987) Transcription of the transposable element Activator (Ac) of Zea mays L. EMBO J 6:1555–1563
Li Min-G, Starlinger P (1990) Mutational analysis of the N-terminus of the protein of maize transposable element Ac. Proc Natl Acad Sci USA 87:6044–6048
Maniatis T, Fritsch EF, Sambrock J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Masson P, Fedoroff NV (1989) Mobility of the maize Suppressor-mutator element in transgenic tobacco cells. Proc Natl Acad Sci USA 86:2219–2223
McClintock B (1951) Chromosome organization and genetic expression. Cold Spring Harbor Symp Quant Biol 16:13–47
Meyer P, Heidmann I, Forkmann G, Saedler H (1987) A new petunia flower colour generated by transformation of a mutant with a maize gene. Nature 330:677–678
Mueller P, Hinnebusch A (1986) Multiple upstream AUG codons mediate translational control of GCN4. Cell 45:201–207
Müller-Neumann M, Yoder JI, Starlinger P (1984) The DNA sequence of the transposable element Ac of Zea mays L. Mol Gen Genet 198:19–24
Müller-Neumann M, Fusswinkel H, Starlinger P (1986) Studies on the expression of the transposable element Activator at the protein level. Maize Genet Coop Newslett 60:39
Nagy JI, Maliga P (1976) Callus induction and plant regeneration from mesophyll protoplasts of Nicotiana sylvestris. Z Pflanzenphysiol 78:453–455
Negrutiu I, Shillito R, Potrykus I, Biasini G, Sala F (1987) Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8:363–373
Nevers P, Shepherd N, Saedler H (1985) Plant transposable elements. Adv Bot Res 12:102–203
Ow DW, Wood KV, DeLuca M, de Wet JR, Helinski DR, Howell SH (1986) Transient and stable expression of the firefly luciferase gene in plant cells and transgenic plants. Science 856–859
Peacock WJ, Dennis ES, Gerlach WL, Sachs MM, Schwartz D (1984) Insertion and excision of Ds controlling elements in maize. Cold Spring Harbor Symp Quant Biol 49:347–354
Pohlman R, Fedoroff N, Messing J (1984) The nucleotide sequence of the maize controlling element Activator. Cell 37:635–643
Potrykus I, Shillito RD (1986) Protoplasts: isolation, culture, plant regeneration. Methods Enzymol 118:549–578
Rubin GM, Spradling AC (1982) Genetic transformation of Drosophila with transposable element vectors. Science 218:348–353
Saiki RK, Scharf S, Faloona F, Mussil KB, Horn GT, Erlich HA, Arnheim N (1985) Enzymatic amplification of β-globin sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350–1354
Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Stanton VP, Nichols DW, Laudano AP, Cooper GM (1989) Definition of the human raf amino-terminal regulatory region by deletion mutagenesis. Mol Cell Biol 9:639–647
Werr W, Lörz H (1986) Transient gene expression in a Gramineae cell line. A rapid procedure for studying plant promoters. Mol Gen Genet 202:471–475
Author information
Authors and Affiliations
Additional information
Communicated by H. Saedler
Rights and permissions
About this article
Cite this article
Houba-Hérin, N., Becker, D., Post, A. et al. Excision of a Ds-like maize transposable element (AcΔ) in a transient assay in Petunia is enhanced by a truncated coding region of the transposable element Ac . Molec. Gen. Genet. 224, 17–23 (1990). https://doi.org/10.1007/BF00259446
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00259446