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The Plant Journal (1998) 13(5), 717–721 TECHNICAL ADVANCE A general method for gene isolation in tagging approaches: amplification of insertion mutagenised sites (AIMS) Monika Frey*, Cornelia Stettner and Alfons Gierl Institut und Lehrstuhl für Genetik, Technische Universität München, Lichtenbergstraße 4, D-85747 Garching, Germany Summary A polymerase chain reaction (PCR) based procedure for the isolation of genes in transposon or T-DNA tagging approaches has been developed. The method can be generally applied and allows the rapid isolation of putative gene sequences even in the presence of high numbers of insertion sequences. The technique has been used successfully for the isolation of the maize Bx1 gene tagged by a Mutator element. Introduction In plants, T-DNA (Feldman, 1991) and transposon tagging (Gierl and Saedler, 1992) have been used widely for the isolation of genes. No prior knowledge concerning the nature of the product of the tagged gene is required as the method relies solely on the detection of a mutant phenotype. As such, genes involved in development, pathogen resistance and biochemical pathways have been cloned in this way. First, the mutation caused putatively by the insertion sequence has to be identified through phenotypic screening. Second, cosegregation of an insertion sequence and the mutant phenotype has to be verified. Subsequently, the gene can be isolated molecularly by cloning the DNA sequences flanking the transposable element insertion. Transposable element systems have been used as tags in their host plants (Ac/Ds, En/Spm, Mu in maize; Tam elements in Antirrhinum majus), and in transgenic plant systems (see Kunze et al., 1997 for a recent review). Insertion of a sequence into a particular gene is a statistical event and the probability increases with the number of random insertions in a given genome. Highly efficient systems, like the Mu-tagging populations of maize, are distinguished by the presence of a large number (about 100 copies) of distinct element insertions within the Received 16 July 1997; revised 3 November 1997; accepted 7 November 1997. *For correspondence: (fax 149 89289 12892; e-mail [email protected]). © 1998 Blackwell Science Ltd individual genome. This, however, makes it difficult to identify a particular insertion sequence as the causal agent of the observed mutant phenotype by conventional Southern-based methods. Traditionally, the number of insertion sequences per plant was reduced by time-consuming outcrossing to lines with low numbers of insertion elements. To circumvent this problem Souer et al. (1995) combined inverse PCR and differential screening to clone tagged genes and TAIL-PCR has been used for the isolation of Ds elements in transgenic Arabidopsis (Smith et al., 1996). We have established a rapid method that should allow for the direct identification of insertion sequences cosegregating with the mutant phenotype, in the presence of a multitude of insertion elements. The technique is based on the reduction of band complexity by specific PCR amplification of insertion mutagenised sites (AIMS). The second advantage of the procedure is that flanking sequences representing part of the gene of interest are amplified during the segregation analysis and can readily be used for the screening of cDNA or genomic libraries. This method has been used for the isolation of the Bx1 gene of maize from a Mu-tagging approach (Frey et al., 1997). The BX1 protein catalyses the committing step in the biosynthesis of the secondary metabolite DIMBOA (2,4dihydroxy-7-methoxy-1,4-benzoxazin-3-one). This benzoxazinone is a main component of the chemical defence mechanism of the maize seedling against a wide variety of pests. Results and discussion Genetic requirements for the application of AIMS Within a tagging population, tagged genes are present in heterozygous constitution. No phenotype is displayed in the common case of a recessive mutation caused by DNA insertion. In a non-targeted tagging experiment the recessive allele is uncovered by selfing of the individuals of the population. One-fourth of the progeny of a tagged plant has a mutant phenotype. In these individuals the DNA insertion is present in a homozygous condition, whilst in one-third of the phenotypically wild-type siblings this particular insertion sequence is not present. Consequently, if an insertion sequence tags a gene, it is present in all mutants and absent in one-third of the phenotypically wild-types. In targeted tagging approaches an insertion-induced 717 718 Monika Frey et al. mutant allele is revealed by combining it with a recessive reference allele by crossing. We used targeted tagging for the isolation of the Bx1 gene. Hamilton (1964) detected a mutant (bx1, bx1) in the DIMBOA biosynthetic pathway that does not accumulate this benzoxazinone. The presence or absence of DIMBOA can easily be recognised by a staining assay (Simcox and Weber, 1985). The recessive standard bx1 mutant was used to pollinate a Mu female line and about 150.000 progeny of this cross were screened. Seventeen putative Mu induced mutant alleles were identified and the respective lines were crossed to a wild-type line (Bx1/Bx1; H99) to separate the Mu induced (bx1::Mu) and reference mutant (bx1) allele. The resulting F1 progeny was analysed with a closely linked CAPS marker to Bx1 (Konieczny and Ausubel, 1993) for the 1:1 transmission of the two types of alleles. One putative Mu insertion mutant showed the expected segregation of bx1::Mu/Bx1 and bx1/ Bx1 plants. These F1 plants were selfed and the respective homozygous mutants of the resulting F2 generation were selected phenotypically. These plants were used for AIMS analysis and gene isolation. The Mu element inserted in the Bx1 gene has to be present without exception in the bx1::Mu mutant plants because it is the causal agent for the mutant phenotype, but it must be absent in every bx1 standard mutant individual. Insertion sequence specific amplification DNA of individual plants is digested by a restriction endonuclease that has a four base pair recognition site and generates two base pair sticky ends (e.g. MseI or BfaI). The restriction enzyme is chosen such that the resulting restriction fragments still allow the positioning of two nonoverlapping primers within the termini of the insertion element (Figure 1a, c and e). After digestion an adapter sequence is ligated in the way described for amplified fragment length polymorphism (AFLP) (Vos et al., 1995; Figure 1(b). Fragments including insertion sequences are amplified by linear PCR using a 59-end biotinylated primer complementary to the insertion sequence termini (Figure 1c). In a Mu tagging approach it is possible to use one primer complementary to both termini due to the longterminal inverted repeats of this transposable element. To minimise PCR artefacts, only 12 cycles of linear DNA synthesis are performed. The PCR products are purified by biotin–streptavidin interaction (Figure 1d). By this process DNA fragments are isolated that include one end of the insertion sequence, flanking DNA and the adapter sequence (tag border fragments). Display of tag border sequences In the next step a nested primer specific for the insertion element is used. Therefore, DNA fragments that do not Figure 1. Principle of the AIMS analysis. (a) Genomic plant DNA (black line) is digested with a restriction enzyme. The (4 bp) enzyme recognition site is indicated by the hooked line. In the following sections only a DNA fragment including a terminal part of an insertion sequence (grey line) is displayed. (b) The adapter (striped line) is ligated in the presence of the restriction enzyme. Ligation of the adapter to genomic fragments does not restore the restriction site (see Experimental procedures). (c) A primer (grey arrow) complementary to the terminal region of the insertion sequence is used for linear PCR. The primer is 59-labelled with biotin (d). PCR results in single-stranded DNA comprising a terminal part of the insertion sequence, flanking genomic DNA and the adapter sequence. (d) The PCR product is isolated by biotin-streptavidin binding. (e) The purified single stranded DNA is used as a template in an exponential PCR. A nested insertion sequence specific primer (grey arrow) and a primer complementary to the adapter (striped arrow) are employed. The adapter primer can be extended at its 39-end by one nucleotide located in the genomic plant DNA (e.g. ‘G’). Therefore, the complexity of the amplified fragments is reduced to one-fourth compared to (d). To amplify all fragments isolated in (d), adapter primers with G/A/C/T extensions, respectively, have to be used. The nested primer is labelled at the 59-end (star) for visualisation of the PCR products. (f) The products of the procedure are labelled double-stranded DNA fragments that represent the flanks of insertion sequences. harbour insertion sequences but were amplified in the first PCR due to accidental complementarity to the biotinylated primer are excluded. This primer is radioactively labelled at the 59-end for visualisation and used in combination with an adapter complementary primer in an exponential PCR (Figure 1e). If necessary, the complexity of the amplified fragments can be reduced at will by extension of the adapter and/or nested primer by one or more basepairs. The PCR products are separated on standard DNA sequencing gels. The high resolution capacity of a sequencing gel is used for a reliable identification of fragments. © Blackwell Science Ltd, The Plant Journal, (1998), 13, 717–721 Gene isolation in tagging approaches 719 In the Bx1 tagging experiment the copy number of Mu elements in the F2 plants was too high to allow the identification of a cosegregating transposable element by Southern analysis (data not shown). However, in the AIMS assay it was sufficient to lower the band complexity by extending the adapter primer by one nucleotide to get a clear picture (Figure 2). Due to this extension, four sets of PCR reactions were required for the complete analysis. One single band was displayed in all bx1::Mu mutants and in none of the bx1 mutants. This band, fulfilling the criterion of a putative Bx1 tag border fragment, had the size of 197 bp in the BfaI assay and of 290 bp in the MseI experiment. Further analysis revealed that the BfaI and MseI fragments represent the same tag border (Figure 3). Cloning of specific tag border sequences After identification the relevant tag border fragment can readily be cloned. The band is cut out from the gel, eluted and re-amplified (see Experimental procedures). However, contaminating fragments might be present in the eluted DNA due to inaccurate excision or by accidental amplification of unrelated fragments of exactly the same size. It is recommended, therefore, to analyse several plasmid clones. For confirmation, the cloned fragment is amplified by PCR with the primer pair and conditions used for its identification, followed by comparison with the respective plant-derived band on a sequencing gel. It might be necessary to cut plasmid derived and plant derived fragments prior to electrophoresis to distinguish between correct and contaminating fragments of exactly the same size. The cloned tag border fragment is used to isolate the gene from genomic and cDNA libraries. Verification of gene isolation The AIMS procedure delivers candidates for the gene of interest. The isolation of the gene has to be verified by an independent method. This can be accomplished by complementation of the mutant with the isolated gene and by isolation and examination of independent mutant alleles. To confirm the isolation of the Bx1 gene, the 290 bp MseI tag border fragment was used for the isolation of the genomic and full-size cDNA clones of wild-type and standard mutant maize lines. Isolation of the genomic clone showed that the reference bx1 allele harbours a 924 bp deletion comprising 59 upstream sequences, the first exon and part of the second exon (Figure 3). The Mu insertion is located in the fourth exon of the gene. The fact that independent Bx1 mutants carry substantial mutations in the same gene gave conclusive evidence for the isolation of the Bx1 gene. Functional analysis verified the isolation of the Bx1 gene and demonstrated that the encoded protein © Blackwell Science Ltd, The Plant Journal, (1998), 13, 717–721 Figure 2. Display of tag border fragments on sequencing gels DNA fragments amplified using BfaI digested DNA and the BfaI selective primer T are shown. The arrow head indicates the tag border fragment of 197 bp that is present in all bx1::Mu mutants and lacking in all standard bx1 mutants. Fragments displayed at the bottom have a size of ~ 80 bp. is responsible for the synthesis of free indole, the committing step in DIMBOA biosynthesis (Frey et al., 1997). Limitations of the method In our hands the generation of tag border fragments is reliable and reproducible. However, the interpretation of 720 Monika Frey et al. teriophage lambda NM1149 was used for cDNA cloning and the isolation of the genomic bx1 mutant allele. Wild-type sequences were isolated from libraries described in Frey et al. (1995). Primer and adapter sequences (59-39 orientation) Figure 3. Structure of the wild-type Bx1 gene and the standard bx1 mutant allele. The position of the Mu element in bx1::Mu and of the MseI and BfaI derived tag border fragments isolated in the AIMS assay are indicated. The position of the deletion is given with respect to the first nucleotide (11) of the transcript. fragments larger than 500 base pairs is difficult since artefacts of uncut DNA might be displayed, or the fragments might not always be amplified thoroughly by the Taq polymerase. Premature termination of DNA synthesis leads to an accumulation of small-sized bands (less than 80 bp) which also makes their analysis difficult. This might be the reason why in the Bx1-tagging study presented here only the right tag border fragment was detected, although the biotinylated and nested primers used for amplification were complementary to both Mu element ends. The left flanking restriction site was 40 bp (BfaI) and 75 bp (MseI) apart from the Mu integration site, respectively. We would suggest using additional restriction enzymes if no cosegregating bands are detected in the size range (100– 400 bp). It might also be helpful to optimise the PCR conditions with respect to magnesium and dimethyl-sulfoxide concentrations according to standard protocols. The Mu selective primer has been end-labelled with the radioactive isotope 32P, resulting in strong signals which facilitate the detection by autoradiography and elution of the DNA from the gel. Alternatively, the isotope 33P might be used without changing the protocol. Labelling of the nested Mu-specific primer at the 59-end with digoxigenin or fluorescent dyes would allow the adaptation of standard non-radioactive detection protocols for visualisation of the bands. Experimental procedures Materials Enzymes were purchased from Boehringer Mannheim, New England Biolabs, and Pharmacia. Gamma 32P ATP (~ 110 TBq/ mmol) was purchased from Amersham. Streptavidin-coated magnetic beads were obtained from Dynal (Hamburg), and Qiagen (Hilden) QIA-quickspin columns were used. PCR reactions were performed with a UNO cycler from Biometra (Göttingen). Oligonucleotides were synthesised by MWG (Ebersberg). Plasmid Bluescript KS 1 (Stratagene) was used as a cloning vector, bac- MseI/BfaI Adapter: TACTCAGGACTCAT and GACGATGAGTCCTGAG. Mu specific biotinylated primer: AGAGAAGCCAACGCCA (A/T)CGCCTCCATT. Mu specific nested primer: TCTATAATGGCAATTATCTC. MseI selective primer A(G/C/T): GATGAGTCCTGAGTAA/A (G/C/T). BfaI selective primer A (G/C/T): GATGAGTCCTGAGTAG/A (G/C/T). Plant material The Mu active tagging population is described in Chomet (1994); the bx1 standard mutant line was provided by D. Weber, Illinois State University. All wild-type sequences are derived from the inbred line CI31A. The CAPS marker is derived from Bx4 which is located 6 centiMorgan apart from Bx1 (Frey et al., 1997). All standard techniques of DNA isolation, analysis and cloning are as described by Frey et al., 1995. Amplification of insertion mutagenised sites The protocol is based on the AFLP method developed by Keygene (Zabeau and Voss, European Patent Application 92402629). Restriction/ligation of genomic DNA: 500 ng of genomic DNA are digested with 5 units of restriction enzyme in a volume of 40 µl in 13 RL buffer (Pharmacia) for 1 h at 37°C. Then 1 µl of 50 µM adapter DNA, 1 µl 103 ligation buffer (Boehringer Mannheim), 1 U ligase is added and the volume increased to 50 µl. Incubation is for 1 h at 20°C and 2 h at 37°C. The adapter is designed such that its ligation does not restore the restriction site (e.g. the BfaI recognition site CTAG is transformed into CTAC after ligation), hence linker ligation to genomic DNA is optimised by synchronous ligation and restriction. DNA is precipitated and redissolved in 27.5 µl H2O. Mu specific amplification: 12 cycles of DNA synthesis are performed with 27.5 µl DNA solution and 2.5 µl Mu Biotin primer (12 µM), 10 µl 2.5 mM dNTPs and 1 unit Taq polymerase in a volume of 50 µl in 13 PCR buffer. DNA is denaturated for 2 min at 94°C. In the following cycles denaturation is for 1 min at 94°C, annealing at 65°C for 30 sec, extension for 60 sec at 72°C. A final elongation step of 3 min is included. Excess Mu Biotin primer is removed with a QIA-quickspin column according to the manufacturer and 50 µl of eluate are mixed with 50 µl 4 M NaCl. Binding to Streptavidin beads: 10 µl Streptavidin Dynabeads are prepared and 100 µl PCR solution in 2 M NaCl are applied according to the supplier’s manual. End-labelling of Mu nested primer: For 20 PCR reactions 2.5 µl primer (10 µM) are labelled with 1.85 MBq Gamma ATP and 1–2.5 units T4 polynucletide kinase in a volume of 12.5 µl for 30 min 37°C. Exponential PCR with 32P labelled primer: 5 µl of DNA loaded Dynabeads and 0.5 µl 32P-labelled Mu Sel Primer, 0.6 µl MseI selective primer/N or BfaI selective primer/N (10 µM), 4.0 µl 2.5 mM dNTPs, in a final volume of 17 µl in 13 PCR buffer are covered with paraffin. 1 unit Taq polymerase in 3 µl is added after 2 min DNA denaturation at 94°C. In all following cycles denaturation is for 1 min at 94°C, annealing is for 30 sec and extension is for 1 min at 72°C. The annealing temperature is 65°C for the first cycle and the temperature is decreased by 0.7°C in 18 consecutive © Blackwell Science Ltd, The Plant Journal, (1998), 13, 717–721 Gene isolation in tagging approaches 721 cycles. Twenty-seven additional cycles at an annealing temperature of 52°C are performed. Analysis on sequencing gels: The PCR reaction is mixed with an equal volume of 0.3% each bromphenol blue and xylene cyanol FF; 10 mM EDTA pH 7.5; 97.5% deionized formamid and denatured at 80°C for 3 min. 1–2 µl are applied per slot on a 6% standard (acrylamide:bis-acrylamide 19:1, 8 M urea) 40 cm denaturating sequencing gel and electrophoresis is done until the xylene cyanole dye has reached 25 cm. DNA fragments from 60 bp to 400 bp are reasonably separated. Fixation is by incubation of the gel in 10% acetic acid for 30 min at room temperature, the gel is dried for 60 min at 80°C and exposed overnight. Kodak X-ray film BIOMAX MR is used for autoradiography. Elution of a PCR band: One PCR reaction is chosen in which the band in question is nicely separated from neighbouring bands. The reaction is loaded in several adjacent slots on a 6% sequencing gel. In contrast to the analytical assay the gel is not polymerised to the glass plate nor fixed and dried. The gel is covered with saran wrap, radioactive marker points are added for orientation and exposure is overnight. The gel slice with the desired band is cut using the X-ray film as a stencil. DNA elution is as described by Sambrook et al. (1989). DNA is dissolved in 10 µl TE per 4 mm slot. 1–5 µl of the DNA solution are used for PCR amplification as described for the exponential amplification with the 32P labelled primer, but in a volume of 50 µl and with 5 µl of unlabelled nested and respective adapter primer (10 µM each). The amplified sequence is treated with T4 DNA polymerase and cloned into a plasmid vector. Acknowledgements We thank Georg Haberer and Alexander Yephremov for valuable suggestions, Regine Hüttl for excellent technical assistance, and Timothy Golds and Ramon Torres-Ruiz for critical reading of the manuscript. This work was supported by Deutsche Forschungsgemeinschaft (SFB 369) and the Fonds der Chemischen Industrie. © Blackwell Science Ltd, The Plant Journal, (1998), 13, 717–721 References Chomet, P. (1994) Transposon tagging with Mutator. In The Maize Handbook (Freeling, M. & Walbot, V., eds). New York: Springer Verlag, pp. 234–248. Feldman, K.A. (1991) T-DNA insertion mutagenesis in Arabidopsis: Mutational spectrum. Plant J. 1, 71–82. Frey, M., Chomet, P., Glawischnig, E. et al. (1997) Analysis of a chemical defense mechanism in grasses. Science, 277, 696–699. Frey, M., Kliem, R., Saedler, H. and Gierl, A. (1995) Expression of a cytochrome P450 gene family in maize. Mol. Gen. Genet. 248, 100–109. Gierl, A. and Saedler, H. (1992) Plant-transposable elements and gene tagging. Plant Mol. Biol. 19, 39–49. Hamilton, R.H. (1964) A corn mutant deficient in 2,4-dihydroxy7-methoxy-1,4-benzoxazin-3-one with an altered tolerance of atrazine. Weeds, 12, 27–30. Konieczny, A. and Ausubel, F.M. (1993) A procedure for mapping Arabidopsis mutants using co-dominant ecotype-specific PCRbased markers. Plant J.,4, 403–410. Kunze, R., Saedler, H. and Lönning, W.-E. (1997) Plant transposable elements. Adv. in Bot. Res. 27, 321–470. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual. 2nd edn. New York: Cold Spring Harbor Academic Press. Simcox, K.D. and Weber, D.F. (1985) Location of the benzoxazinless (bx) locus in maize by monosomic and A-B translocation analysis. Crop Sci. 25, 827–830. Smith, D., Yanai, Y., Liu, Y.-G., Ishiguro, S., Okada, K., Shibata, D., Whittier, R.F. and Fedoroff, N.V. (1996) Characterisation and mapping of Ds-Gus-T-DNA lines for targeted insertional mutagenesis. Plant J. 10, 721–732. Souer, E., Quattrocchio, F., de Vetten, N., Mol, J. and Koes, R. (1995) A general method to isolate genes tagged by a high copy number transposable element. Plant J. 7, 677–685. Vos, P., Hogers, R., Bleeker, M., van Reijans, M., de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J. and Kuiper, M. (1995) AFLP: a new technique for DNA fingerprinting. Nucl Acids Res. 23, 4407–4414.