Download A general method for gene isolation in tagging approaches

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
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