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Journal of Biotechnology 78 (2000) 301 – 312
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Target selected insertional mutagenesis on chromosome IV
of Arabidopsis using the En–I transposon system
Elly Speulman, Ronald van Asperen, Jessica van der Laak,
Willem J. Stiekema, Andy Pereira *
CPRO, Department of Molecular Biology, PO Box 16, 6700 AA Wageningen, The Netherlands
Received 9 February 1999; received in revised form 29 November 1999; accepted 3 December 1999
Abstract
Reverse genetics using insertional mutagenesis is an efficient experimental strategy for assessing gene functions. The
maize Enhancer–Inhibitor (En–I) transposable element system was used to develop an effective reverse genetics
strategy in Arabidopsis based on transposons. To generate insertion mutations in a specific chromosomal region we
developed a strategy for local transposition mutagenesis. A small population of 960 plants, containing independent
I transpositions was used to study local mutagenesis on chromosome IV of Arabidopsis. A total of 15 genes, located
on chromosome IV, were tested for I insertions and included genes identified by the European ESSA I sequencing
programme. These genes were of particular interest since homologies to other genes and gene families were identified,
but their exact functions were unknown. Somatic insertions were identified for all genes tested in a few specific plants.
Analysis of these progeny plants over several generations revealed that the ability to generate somatic insertions in the
target gene were heritable. These genotypes that show high levels of somatic insertions can be used to identify
germinal insertions in the progeny. © 2000 Elsevier Science B.V. All rights reserved.
Keywords: Reverse genetics; En-I; Transposon; Pectinesterase; Arabidopsis
1. Introduction
Arabidopsis thaliana is used as a model in plant
science to study many aspects of plant biology. Due
to its small genome and its well-studied genetics,
Arabidopsis was the first plant species chosen to
generate a complete physical map of the genome
(Choi et al., 1995; Liu et al., 1995; Schmidt et al.,
* Corresponding author. Tel.: +31-31-777114; fax: + 3131-7418094.
E-mail address: [email protected] (A. Pereira)
1996, 1997; Zachgo et al., 1996; Camilleri et al.,
1998; Kaneko et al., 1998; Mozo et al., 1998). This
effort was followed by the multinational co-ordinated sequencing of the Arabidopsis genome using
bacterial artificial chromosomes (BACs) and P1
clones which had been mapped to defined regions
of the genome (Goodman et al., 1995; Bevan et al.,
1998). However, while the complete nucleotide
sequence of the Arabidopsis genome is expected to
become available at the end of the year 2000
(Meinke et al., 1998) the function of most of the
unravelled gene sequences remains unclear.
0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 2 0 3 - 0
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E. Speulman et al. / Journal of Biotechnology 78 (2000) 301–312
Information on the function of a particular
gene might be obtained from the analysis of mutants in which the function of the gene is abolished. Insertional mutagenesis is a convenient
method to create knockout mutants to study gene
function. Transposon or T-DNA insertions can
result in complete inactivation of the target gene
and have been used for the isolation and identification of genes (Feldmann, 1991; Koncz et al.,
1992; Aarts et al., 1993; Bancroft et al., 1993;
Jones et al., 1994; Okuley et al., 1994; AzpirozLeehan and Feldmann, 1997). Insertional mutagenesis can also be used to identify insertions in
specific target genes with known sequence but
unknown function. This reverse genetics procedure termed target selected insertional mutagenesis uses T-DNA or transposable elements to
mutagenize the entire genome with insertions at
random positions in the genome. This is followed
by the identification of an insertion in the gene of
interest making use of the sensitivity of the polymerase chain reaction (PCR). A primer, specific
for the gene of interest is used in combination
with primers specific for the insertion sequence
and amplification of a gene-specific product occurs when an insert is present in the gene. The
efficiency of these screens can be improved by
dividing a population of mutagenized individuals
into pools and using specific pooling strategies.
Individual plants identified in such a screen are
subsequently analysed phenotypically to determine the function of the disrupted gene. This
technique has been shown to work in Drosophila,
Caenorhabditis, maize and petunia populations
saturated with transposable elements in which
knockout mutations in specific genes were identified (Ballinger and Benzer, 1989; Kaiser and
Goodwin, 1990; Rushforth et al., 1993; Zwaal et
al., 1993; Das and Martienssen, 1995; Koes et al.,
1995; Mena et al., 1996).
For reverse genetics purposes in Arabidopsis,
populations of T-DNA insertions have been used
successfully (McKinney et al., 1995; Krysan et al.,
1996; Azpiroz-Leehan and Feldmann, 1997;
Bouchez and Höfte, 1998; Winkler et al., 1998).
Although T-DNA insertions are in general single
or low copy and stable, an advantage of using
transposon- over T-DNA insertions is their ability
to transpose. Transposons can excise from the
gene of interest and the resulting reversion of a
mutation can be used to confirm that it was
caused by the transposon. Also, characteristic of
the maize Ac/Ds and En/Spm transposable elements is their preference to transpose to linked
sites and this property can be advantageously
used in a local mutagenesis strategy to tag genes
located in the vicinity of the donor site (Peterson,
1970; Bancroft and Dean, 1993; Cardon et al.,
1993; Aarts et al., 1995; James et al., 1995).
In order to further establish a system that could
be used in reverse genetics screens in Arabidopsis,
to complement the already available T-DNA populations, we used a modified two component
maize Enhancer– Inhibitor (En– I or Spm– dSpm)
transposable element system. A stable transposase
locus T-En5, was shown to mediate frequent
transposition of I elements which occurred continuously throughout plant development (Aarts et
al., 1993, 1995). The high levels of activity could
result in rapid amplification and proliferation of
insertions to linked and unlinked sites. These
characteristics were utilised for the generation of a
small population of 960 lines containing multiple
I elements per line. This population was in particular established to study local transposition of the
En– I system by using an I element (I-201 ), which
had previously been mapped on chromosome IV,
as a ‘launch pad’ for further transposition events.
The genes tested for I insertions were therefore
located at relatively near distances (0–15 cM)
from I-201 and included genes identified by the
ESSA I (European Scientists Sequencing Arabidopsis) programme. These genes were of particular interest since they are homologous to other
genes and gene families but their function remains
to be analysed. Two other well-characterised
genes, GA1 and APETALA 2 (AP2 ) located at
much further distances (31–55 cM) from I-201,
were used to test the long distance mutagenesis
potential of this transposon system. Insertions
were identified for all genes tested, and transpositions of I-201 had therefore occurred across the
chromosome. However, all insertions were identified as somatic insertions and the tendency to
insert somatically was inherited in some plants
and their progeny. Insertion rates and insertion
E. Speulman et al. / Journal of Biotechnology 78 (2000) 301–312
stability are influenced by many factors and include the proximity of the transposon to the
target, the timing and level of transposase expression and other element-encoded gene products,
and also host-encoded products and regulation
mechanisms (Fedoroff and Banks, 1988; Donlin et
al., 1995; Eisses et al., 1997). Somatic insertions
will be transmitted germinally to the progeny at a
lower frequency and can be identified when more
progeny of plants are screened and selected for
inheritance.
2. Material and methods
2.1. Plant material
In earlier experiments described by Aarts et al.
(1995), the ‘in cis two-element En – I’ transposon
system was introduced into the A. thaliana ecotype Landsberg erecta using a T-DNA construct
(cwEnN::I), which contained an unmarked 2.2 kb
I element inserted in the open reading frame of an
NPTII marker gene. This construct also harboured an En element as stable transposase
source, from which the 5% and 3% ends were removed and replaced by the cauliflower mosaic
virus (CaMV) 35S promoter (5%) and terminator
(3%) sequences. A primary transformant displaying
active transposition was used as source of the
transposon population. This transformant contained two T-DNA loci harbouring the transposase T-En5 on chromosome II, and T-En2 on
chromosome I, conferring 10 – 30% and 2 – 5%
transposition, respectively. Plants containing the
T-En5 locus were selfed over several generations
resulting in lines in which new transpositions of
the original I element(s) had taken place. The
positions of I elements were determined for several of such lines by using inverse PCR to isolate
flanking sequences of I elements and subsequently
mapping these products on the genome using
recombinant inbred lines (RILs) (Lister and
Dean, 1993; Aarts et al., 1995). One line harboured I-201 on chromosome IV and was chosen
for further experiments. A F2 segregating population was obtained after a cross with wildtype
Landsberg erecta. One plant homozygous for I-
303
201, and hemizygous for T-En5 was selected, after
probing genomic Southern blots with an I-201
and T-En5 flanking probe. A total of 50 hygromycin resistant progeny plants were transferred to the greenhouse and used as pollen
donors in crosses with a marker stock (F2 of
ecotype Columbia X Landsberg erecta; phenotype
erecta, cer2, ms1 ). Around 100 crosses were made
with every pollen donor plant, resulting in a total
of 5000 siliques with different genetic backgrounds. Seed (960), from these crosses was chosen randomly and grown in the greenhouse as a
F1 population and planted in ten trays/blocks,
and young inflorescence material was harvested in
a three dimensional pooling strategy (as outlined
in Results and Discussion (Section 3)). As the
pollen parent was a hygromycin resistant selfed
progeny of a T-En5 hemizygous plant, the resultant F1 population consisted of one third T-En5
transposase free stable plants and 2/3 hemizygous
for the T-En5 locus. The CIC-library (Creusot et
al., 1995) was later used to fine map I-201 on
CIC1H1 and CIC11C1 at position 62 cM on
chromosome IV to establish a more precise correlation with the genes and sequences which were
tested for insertions and which had also been
anchored to CICs on chromosome IV (Schmidt et
al., 1996).
2.2. Isolation of genomic DNA
Inflorescence or leaf material of a single plant
was used for DNA extraction. Extractions were
performed in Eppendorf tubes using liquid nitrogen and twice a volume of 250 ml of DNA extraction buffer (0.3 M NaCl, 50 mM Tris pH 7.5, 20
mM EDTA, 2% sarkosyl (v/v), 0.5% SDS (w/v),
5% phenol (v/v), 5% H2O (v/v)) as described by
Pereira and Aarts (1998). Pools of inflorescence
material were ground with a mortar and pestle in
liquid nitrogen and twice a volume of 2.5 ml of
DNA extraction buffer. After phenol/chloroform
extraction and centrifugation at 4000 rpm for 15
min, the DNA was precipitated after adding 0.8
volumes of isopropanol to the supernatant, incubation at RT for 10 min and centrifugation at
4000 rpm for 10 min. The pellet was washed twice
with 70% ethanol and dissolved in 1 ml TE pH
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E. Speulman et al. / Journal of Biotechnology 78 (2000) 301–312
8.0 and 10 mg ml − 1 RNase. After a second round
of phenol/chloroform extractions, the pellet was
dissolved in 200–400 ml TE pH 8.0 to a final
concentration of 50 – 100 ng − 1 ml of DNA, depending upon the size of the pool.
2.3. Detection and analysis of I insertions
To detect I insertions in the genes tested, two I
specific primers were used; itir2 (CTT TGA CGT
TTT CTT GTA GTG) and itir3 (CTT GCC TTT
TTT CTT GTA GTG) (Life Technologies), complementary to the 5% and 3% terminal inverted
repeats of I. Both primers were used in one PCR
reaction together with one gene-specific primer.
Gene-specific primers were designed complementary to 5% and 3% regions of the coding sequence, in
such a way that putative insertions could be identified within the gene. The gene-specific primers
used were: Ag-6 (ACTCCA GGC CAT TTC CTT
CAG) for AGAMOUS, primer 1010 (CGT CCC
AAA AAT GCT CTG TTC) and primer 1011
(TGC TAC ACG TCA TGT TCG TTG) for
pectinesterase (accession number: Z97340g2244956), primer 1014 (CTA CCA AGA GAC
AAT ATC ATG) and primer 1015 (TCC GGC
CAC ACG TTC ACA CGA) for HAT-1 (access.
no: Z97343-g2245105), primer 1016 (GGG TGA
CTC CTT TAG TGT AGC) and primer 1017
(GTC TCC GTA GAT GAG TCC AGC) for
membrane channel protein (access. no: Z97343g2245093), primer 1030 (GCA GTC GGA TGT
GAT GAG GTT) and 1031 (CAA CCC TAA
GGC CTC CCA GTT) for glycerol-3-phosphate
permease (access. no: Z97343-g2245113), primer
1022 (CGGCAG GCC AGG AAC GTT TCA)
and primer 1023 (TCG GAT CTG GAC CGT
TGG TGG) for GTP-binding protein (access. no:
Z97343-g2245111), primer 1040 (CTT CAC CAA
GTA AAC GGG TTG G) and primer 1041
(GTG ATG ATGAGG AGA GAA TCC) for
AP-2, primer 1052 (CGCCGA CGG AAC TCG
AAG GGG) and primer 1053 (CAA GAT AAA
CCT CGC CGG GTG) for CH42, primer 1050
(TCA TCT GGC CTG ACG ACG AAA) and
primer 1051 (GCC CGA GAA GCT CCA TGA
TCT) for GA5, primer 1032 (TGT CGC TAG
AGA CAA ATC CAG) and primer 1034 (GTG
AGT TTG GAG ATG ATC GCC) for GA1 and
primer 1048 (TGG AGG GAA GCC CAG TGA
CCA) and primer 1049 (TCC GAC CCC ATG
AAT CGT GTA) for Cer-2. Primers for PRHA,
Mek1 and DD-1 were sent by collaborators for
insertion screens. PCR reactions were performed
in a total volume of 50 ml (0.5 mg of each primer,
250 mM of each nucleotide, 1× Supertaq buffer),
containing 50–100 ng of DNA and 1U Taq DNA
polymerase (Supertaq, HT Biotechnology Ltd,
UK). The reactions were performed in a PTC-200
Peltier thermal cycler (MJ Research), using 1 cycle
at 94°C for 4 min and 31 cycles of 45 s at 94°C,
45 s at 60°C, 3 min at 72°C, followed by a 7 min
extension step at 72°C. Amplification products
were size-separated in 1% TBE agarose gels containing ethidium-bromide, alkali treated, then
vacuum transferred onto Hybond-N+ membranes
and hybridised to a gene-specific probe. Primers
corresponding to both 5% and 3% regions of the
coding sequence, as mentioned above, were used
in combination to amplify genomic DNA products, which were used as probes. For AGAMOUS,
its corresponding cDNA clone was used as a
probe. After gel-electrophoresis and isolation of
the DNA with a DNA gel-purification kit (QIAGEN), the DNA was used as a probe. Membranes were pre-hybridised at 65°C in 1 M NaCl,
1% SDS, 10% dextran sulphate for 1–4 h and
subsequently hybridised with a 32P labelled probe
at 65°C, o/n in the same solution. The membranes
were washed at room temperature in 2× SSC for
5 min and at 65°C in 2× SSC, 1% SDS for 1 h.
After identification of a positive three-dimensional insertion address, 12 seeds of each three-dimensional address were sown as individuals in the
greenhouse and one leaf was isolated from all 12
individuals and pooled. After reconfirmation of
an insertion in a pool of a putative three-dimensional address by DNA extraction, PCR, Southern blotting and hybridisation; inflorescences of
individual plants were subsequently analysed
separately.
Amplification products from DNA of individual plants, obtained with one gene-specific primer
and both I primers were gel-purified (QIAGEN)
and subjected to another round of PCR amplifications. The purified fragments were cloned into the
E. Speulman et al. / Journal of Biotechnology 78 (2000) 301–312
PGEM-T-vector (Promega), sequenced via an automated sequencing system (Applied Biosystems
373 DNA Sequencer), and analysed with the
BLAST algorithm at the National Centre for
Biological Information (NCBI).
To determine if individual plants were either
stable or homo- or hemizygous for the T-En5
locus, primers were designed which were complementary to the flanking sequences of T-En5 using
primers Ten3 (GGA CCA CAA AAC ATG GAA
CAT G) and Ten5 (CAC AGC ACA TGT ACA
CAA CTG GCG) and a third primer TEn (CAC
CAG TTG CTA TCA TTG CGG CC) complementary to sequences within T-En5. PCR conditions were as described above with three primers
in one reaction, using 1 cycle at 94°C for 1 min
and 35 cycles of 30 s at 94°C, 30 s at 60°C and 3
min at 68°C.
305
3. Results and discussion
3.1. Three-dimensional pooling and PCR
strategies
To identify insertions in genes of interest using
PCR-based reverse genetics screens, the population of 960 plants was divided into three-dimensional pools in order to minimise the amount of
PCRs necessary to identify an insertion. In a
three-dimensional pooling strategy, one or more
plants carrying an insertion in the gene of interest
can be identified directly in a large population by
using just one round of PCRs on all the pools
representing the population. The population was
divided over ten blocks of 96 plants each (8
rows×12 columns) shown in Fig. 1A. A total of
six flower buds were taken from the main inflorescence of each plant and divided over three pools.
Fig. 1. Schematic representation of three-dimensional pooling and PCR strategies. A small population of 960 I element containing
Arabidopsis lines was divided over 10 blocks (trays) and each block contained 96 plants (8 rows and 12 columns). Inflorescence
material of every plant in the population was harvested in three dimensions. Two flower buds of one particular plant in the
population were taken for the pool of its block; another two were taken for the pool of its row and two more were used for the
pool of its column. A. This resulted in 30 DNA pools (12 columns + 8 rows + 10 blocks). For the identification of an I insertion
in a gene of interest, one gene-specific and two I specific primers were used in 30 PCR reactions. B. After gel-electrophoresis, blotting
and hybridisation with a gene-specific probe, three-dimensional bands can be observed. The autoradiogram shows the result of a
PCR reaction with a primer (1010) specific for pectinesterase. Putative three-dimensional insertions were identified, but numbered
are only the three-dimensional insertions of 800 bp identified in column 9 and 10, block IV and VI and row B and F (L represents
the negative control Landsberg erecta). These positives correspond to plants IV-9-F, IV-9-B, IV-10-F, IV-10-B, VI-9-F, VI-9-B,
VI-10-F and VI-10-B in the population. C. PCR analysis of progeny plants (F2) of putative positives IV-9-F, VI-10-F and VI-10-B
(plant 3 of IV-9-F, and plant 12 of VI-10-B did not germinate).
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E. Speulman et al. / Journal of Biotechnology 78 (2000) 301–312
Two flower buds were used to represent the pool
of its block (96 plants), another two were taken
for the pool of its row (10 ×12 =120 plants) and
the last two for the pool of its column (8 ×10 =
80 plants). In this way material from each plant is
represented in a unique combination of one block,
one row and one column. A total of 30 pools were
obtained after DNA extraction of all pooled material. To further minimise the effort needed to
identify an insertion in a gene of interest we used
two I element and one gene-specific primer in one
reaction. The I primers were complementary to
the left and right terminal inverted repeat ends of
the I element. After gel-electrophoresis and blotting, the membrane was hybridised with a genespecific probe (Fig. 1B). Hybridising bands are the
result of an amplification reaction between one of
the I-element primers and the gene specific primer
and indicate the number of different insertion
positions in the target gene. However, only those
bands which appear to be of equal size in a
column, a block as well as a row can be considered as a three-dimensional insertion and correspond to one particular plant in the population.
3.2. Identification and analyses of insertion sites
To identify insertions in all genes of interest on
chromosome IV, we used two gene-specific
primers that were complementary to 5% and 3%
regions of the coding sequence. Insertions were
found in all genes tested and a relatively high
number of independent three-dimensional insertions (hybridising bands of independent sizes)
were obtained as displayed in Table 1. Given the
nature of the population used, we did not expect
to find any more than one three-dimensional insertion per gene tested. Putative three-dimensional
insertions were usually identified in a number of
blocks, columns and row pools. Consequently, all
possible combinations of one block, one row and
one column (=one three-dimensional address
and one plant in the population) had to be considered and this resulted on average a total of 15
plants with a putative insertion in the target gene
(Table 1, c total insertions). When positives
were identified in e.g. three different blocks, one
row and one column, three different combinations
of one block, one row and one column had to be
analysed, although only one such address could
have been positive, unless multiple insertions were
present in these pools. Because of the high number of putative positives found in these screens, 12
progeny plants of each address were grown in the
greenhouse the leaves harvested of all plants and
analysed as a pool. Pools of addresses that were
positive after a new round of PCR reactions and
Southern analysis were subsequently chosen for
further individual analysis. Insertions were identified in progenies of 90% of all genes tested.
These insertions usually differed in size from the
ones identified in the three-dimensional screen.
Multiple putative three-dimensional insertions
were also identified in a screen with a gene-specific
primer (see Material and Methods) for the putative pectinesterase enzyme (Fig. 1B). The results
obtained with this gene will be used further to
describe this population.
One independent insertion was identified in a
number of three-dimensional pools and was located at a distance of 800 bp from the gene-specific
primer.
Pectinesterases
and
pectin
methylesterases exist as multi-gene families in the
genome (Richard et al., 1996) and the multiple
insertions found for this pectinesterase could
therefore also result from insertions in close family members. However, genomic Southern analysis
showed that this gene was not a member of a
multi-gene family. Homology searches in the
databases also did not identify DNA sequence
homologies of any significance to other known
pectinesterases although amino acid homologies
did exist and were as high as e − 54 over 165 amino
acids for a pectinesterase on chromosome IV
(BAC T24M8). A maximum of two homologous
genes is present in the genome according to
Southern analysis, and insertions are therefore
most likely insertions in the pectinesterase gene
that was tested.
A total of 25 possible three-dimensional addresses were tested for insertions in pectinesterase,
which corresponds to an initial insertion frequency of 2.6% (25 addresses/960 lines). After
PCR and Southern analysis 11 progeny pools
were positive and this corresponds to a frequency
of 44% (11 positive pools/25 putative positive
E. Speulman et al. / Journal of Biotechnology 78 (2000) 301–312
307
Table 1
Genes tested for insertionsa
Gene
c Insertions
c Total insertions
Insertions in
progeny
Function
References
AGAMOUS
2
19
No
Yanofsky et al.
(1990)
Pectinesterase (ESSA I)
HAT-1 (ESSA I)
2
1
25
12
Yes
Yes
Palmitoyl protein thioesterase
(ESSA I)
Membrane channel protein
(ESSA I)
Glycerol-3-phosphate permease
(ESSA I)
GTP-binding protein (ESSA I)
DD1
Mek1
PRHA (a)
3
3
Nt
Flower development
Unknown
Plant development
Unknown
4
12
Yes
Unknown
2
15
Yes
Unknown
2
4
5
3
28
13
\30
\30
Yes
Yes
Yes
Yes
Unknown
Unknown
MAP-kinase
Unknown
PRHA (b)
Ap-2 (3%)
3
2
15
9
Yes
Yes
Ap-2 (5%)
CH42
1
2
1
4
Yes
Yes
GA5
1
4
Nt
GA1
1
1
Yes
Cer-2
2
\30
Yes
Schena and Davis
(1992)
Morris et al. (1997)
Korfhage et al.
(1994)
Flower development
Jofuku et al.
(1994)
Chloroplast
protein
Gibberellin
biosynthesis
Gibberellin
biosynthesis
Lipid synthesis
Koncz et al. (1990)
Xu et al. (1995)
Sun and Kamiya
(1994)
Xia et al. (1997)
a
The total number of three-dimensional insertions of distinct sizes obtained ( c insertions), and the total number of
corresponding putative three-dimensional combinations ( c total insertions) are indicated. Progenies of three-dimensional insertions
were tested and it is indicated when insertions were identified.
pools). However, the previously obtained 800 bp
band was identified in only 10% of the progeny
pools, while bands of different sizes and thus
different insertion sites, were identified in the
other positive pools (results not shown). In this
way 14 addresses could be discarded. Of the 11
positive pools, five that showed strong hybridisation signals were further analysed on individual
plant level and PCR progeny analyses of addresses IV-9-F, VI-10-F and VI-10-B (F2 generation) are shown in Fig. 1C. Insertions were
detected in the progenies of all five positive pools
and this corresponds to a frequency of 100%
(5/5). Insertions were confirmed in 25 – 50% of the
individuals at these addresses. However, as was
found for the pools, most of these insertions were
present at other positions in the gene. Bands of
around 800 bp were identified in 10–67% of positive individuals of an address. The frequencies
with which insertions in pectinesterase recur in the
progenies suggest that the initial three-dimensional insertion was somatic and that the property
to insert somatically was heritable. These were
insertion events that had occurred in at least
several cells of the flower and hence insertions
could be visualised only after hybridisation with a
gene-specific probe. To assess whether the insertions were somatic, we analysed the individual
progeny plants by genomic Southern analysis.
Insertions that are transmitted through the
gametes to one or more of the progeny are considered germinal events and can be identified as
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E. Speulman et al. / Journal of Biotechnology 78 (2000) 301–312
band shifts of the wild-type allele (the size of an I
element is 2.2 kb) when restriction enzymes are
used to digest the genomic DNA that do not cut
within the I element. No band-shifts were identified for any of the progeny plants and this
confirms the somatic but heritable nature of these
insertions.
Progeny of plant IV-9-F-4 (F3 generation), that
was homozygous for the T-En5 locus, were also
analysed (results not shown) and also displayed a
similar pattern of somatic insertions. The F2 generation segregated as 1:2:1 for the T-En5 locus. It
was found that transposase free plants did not
display somatic or stable insertions; thus the somatic insertions are a characteristic of transposase
containing families.
To identify the position of one such insertion in
pectinesterase and also to confirm that this insertion was not present in a homologue, a weak band
of 400 bp was isolated from the gel and after
purification re-amplified using one gene-specific
primer and both I element primers. These products
were cloned and sequenced. Sequence analysis
showed that in one clone the I element insertion
was present in an exon of the pectinesterase gene
identified by ESSA I and had generated a 3 bp
target site duplication, characteristic for the En/
Spm transposon family (Fig. 2A and B) (Aarts et
al., 1993).
Other clones identified five different sequences
of 400 bp between two I elements that were in the
same orientation, since itir2 and itir3 sequences
were found at either end of the fragment. This data
suggests that transposition had resulted in clustering of I-elements. Analysis of the sequences
showed that these insertions were present on chro-
mosome II (BAC T3F17) in a putative Ap2 domain containing protein, on chromosome II (BAC
T11A7) in an unknown protein, on chromosome V
(P1 clone MTH16) in a gene which has similarity
to the transcriptional activator Ra, and two insertions were identified in gene sequences which have
not yet been identified by the Arabidopsis sequencing programmes but which showed homologies to
a phenylalanine ammonia lyase and a protein
serine/threonine kinase like gene, respectively.
3.3. Local mutagenesis on chromosome IV
The other genes that were tested for insertions
on chromosome IV are APETALA2 (AP2 ),
PRHA, DD1, Mek1, GA5, Cer2, CH42, AGAMOUS (AG), GA1 and genes identified by ESSA
I (Fig. 3). The genes described by ESSA I were
annotated as having a ‘strong similarity’or ‘similarity’ to known genes. The three-dimensional insertion patterns obtained for all these genes were
similar to what was observed for pectinesterase.
All insertions identified were also somatic and
heritable and patterns were complex. The number
of insertions identified varied between the genes,
but a correlation between the relative distance of
I-201 and the tested genes and the number of
insertions identified was not observed. The total
number of putative three-dimensional insertions
was low for GA1 and AP2, positioned at 45 cM
and 31 cM from I-201, respectively. However,
equally low numbers were also observed for GA5,
very near (3cM) I-201 and CH42 at the same
genetic position as I-201. This might be due to
PCR primer discrepancies or insertion-site preferences of I-elements.
Fig. 2. Analysis of a pectinesterase::I tagged site. A. Drawing of the pectinesterase gene showing the position and orientation of an
I insertion and the relative positions of both gene-specific primers. The bar represents 125 bp. B. Nucleotide sequences surrounding
the insertion site in the somatic pectinesterase::I allele. The target site duplication (TSD) is underlined.
E. Speulman et al. / Journal of Biotechnology 78 (2000) 301–312
Fig. 3. Genes tested for I insertions. Schematic representation
of chromosome IV. Genes tested for I insertions are shown in
bold. Sequences representing genes found by ESSA I are
shown on the left. Numbers represent the position (cM) of the
gene or marker on chromosome IV (Lister and Dean, 1997).
To determine if similar patterns of somatic
insertions were obtained with a population which
also contained multiple I insertions per line but
with 75 000 I insertions distributed randomly over
the genome (Speulman et al., 1999), the genes
HAT-1 and pectinesterase were tested for insertions using identical PCR conditions as described
here. Similar multiple patterns of somatic insertions were obtained, suggesting that the obtained
patterns are gene-specific and or a consequence of
the multiple I element lines, c.q. the genetic background used.
The most likely explanation for heritability of
somatic insertions is that a donor I element, possibly originating from I-201 has transposed close to
the target gene and is selected for in the process of
the population screen. In the next generation,
transposition events from this linked site will
again produce somatic insertion events at random
in the gene tested when the transposase source is
present, although certain positions might be preferred. Overall, this data indicates that I-201
transposed locally as well as over longer distances
and that the En–I system can be used in Arabidopsis for local as well as long-distance
mutagenesis.
309
A high degree of somatic insertions does not
guarantee detection of germinal insertions at a
high frequency in the next generation. However,
use of DNA from germinal tissue for testing
would greatly increase the likelihood of finding
progeny with germinal insertions. Although we
used inflorescence material for the analysis of the
progeny, it might be necessary to isolate DNA
from pollen of plants showing strong hybridisation signals as described for the isolation of Dsinsertions in the gene polygalacturonase, using
similar multiple insertion lines of tomato (Cooley
and Yoder, 1998). Alternatively, it might be sufficient to analyse very small flower buds for insertions in the gene of interest, followed by selective
crosses of several flowers, originating from the
same inflorescence meristem and located in the
near vicinity of the analysed flower bud, with
wild-type pollen to segregate out the transposase.
When using very small flowerbuds, the amount of
germline specific tissue and the probability of
finding a germinal insertion is considerably higher
as opposed to older flower buds.
Systematic selection for germinal events as a
system was demonstrated by Cooley and Yoder
(1998), who isolated germinal insertions in the
polygalacturonase (PG) gene from plants that displayed high somatic transposition. Four thousand
progeny were screened in this experiment and five
were found containing germinal transmitted Ds
insertions in PG. This procedure can also be
applied to the population we produced for chromosome IV transpositions.
The population we generated was established to
identify germinal insertions in genes located on
chromosome IV using active transposition from
the I-201 launching pad. In addition to the I-201
element, four other I elements, including one
mapped on chromosome V and another on chromosome III are present in the background. Assuming uniform transposition in the target region
on chromosome IV, from I-201 with 50% transposition frequency (data not shown) and the other
elements, we estimate about 500 independent germinal insertions in this region of approximately
21 000 kb (http://genome-www3.stanford.edu/
Arabidopsis/chromosome). If insertions are distributed randomly over this chromosome, one
310
E. Speulman et al. / Journal of Biotechnology 78 (2000) 301–312
insertion should be present every 40 kb. For a
target of an average gene of about 2 kb (using
PCR from one side) this gives a chance of about
1/20 or 5% to hit a specific gene. The targeted
genes in the ESSA I region are located in a 430 kb
interval, with a total of 121 genes. The average
length of a gene in this region is 2.3 kb and an
average of one insertion every 2 kb is necessary to
be able to identify an insertion in any random
gene chosen on chromosome IV. As the chance of
identifying a germinal insertion within the coding
sequences of any of the genes in this region is only
about 5%, our failure to identify germinal insertions directly is therefore due to a low genome
coverage of the transposons. In a larger population for randomly distributed genome wide coverage of transposons with multiple transposons per
plant (Speulman et al., 1999), about 50% insertions in genes were found that were heritable. The
evidence of these insertion mutants was also provided with phenotypes of tagged mutants, showing that this transposon system can be useful for
reverse genetics methods.
Despite an absence of germinal insertions in
any of the genes tested, we show that this population contains a genetic background with a very
high insertion activity that can be used for insertional mutagenesis of any gene of interest on
chromosome IV. However, more refined and thorough screening procedures and larger progeny
populations will be necessary to subsequently
identify germinal insertions in the gene of interest.
Acknowledgements
The authors would like to thank Bert Schipper
and Bas te Lintel Hekkert for technical assistance.
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