Download Saturation mutagenesis using maize transposons Virginia Walbot

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Saturation mutagenesis using maize transposons
Virginia Walbot
Transposon mutagenesis facilitates gene discovery by tagging
genes for cloning. New genomics projects are now cataloging
transposon insertion sites to define all maize genes. Once
identified, transposon insertions are ‘hot spots’ for generating
new alleles that are useful in functional studies.
Table 1
Addresses
Department of Biological Sciences, 385 Serra Mall, Stanford
University, Stanford, California 94305-5020, USA;
e-mail: [email protected]
30,000
95
~91,000
50,000
95
~150,000
50,000
99
~230,000
70,000
95
~214,000
Current Opinion in Plant Biology 2000, 3:103–107
Using the formula (1 – probability) = n × log (not desired class), the
required population size (n) can be calculated for a specific probability
of finding at least one mutation in each unit. Because the number of
functional units in maize is currently unknown, the estimate of ~50,000
genes is often used; this figure is about twice the gene estimate for
Arabidopsis, to accommodate the tetraploidy of maize [38].
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
bp
base pairs
MTM
Maize Targeted Mutagenesis
NSF
National Science Foundation
TIR
terminal inverted repeat
TUSC Trait Utility System for Corn
Introduction
The analysis of mutant phenotypes yields an understanding of gene function in a whole-organism context.
Consequently, the generation, evaluation, maintenance
and distribution of seed containing verified mutations or
seed with populations of mutations suitable for highthroughput screening are essential in the modern genetic
analysis of maize and other plants.
Saturation mutagenesis of maize genes involves two ambitious goals: to define all of the genes and to find the
phenotype of every individual gene. This review will outline how transposon mutagenesis can be used to achieve
both goals. Transposons are already the primary tool for
tagging and cloning maize genes. Studies so far demonstrate that specific transposons are well suited for either
global mutagenesis and gene discovery or multiple rounds
of mutagenesis at a defined target gene [1]. New genomics
approaches, employing strategies for screening by PCR
and for plasmid rescue, are now providing indexed collections of mutations and the sequences flanking transposon
insertion sites. Many researchers will soon identify transposon-generated mutants in specific genes after querying a
database rather than searching a corn field.
Insertional mutagenesis across the genome:
primer on key transposon properties
The Ac/Ds and MuDR/Mu maize transposons have been
used in most recent mutagenesis experiments [1–3]. In
stocks with the transcriptionally active regulatory elements
Ac or MuDR, family members with essential transposasebinding sites in the terminal inverted repeats (TIRs) are
mobilized to create new insertion mutations. During a genetagging experiment, transposon activity is conveniently
Population size estimates for insertional mutagenesis to
achieve at least one mutant per gene at a specific coverage.
Gene estimate
Probability (%)
Population size (n)
monitored with a reporter allele that has a phenotype that
beomes visible after the excision of a transposon (Figure 1a).
Saturation mutagenesis of all genes requires a robust mutagenized population. An estimate of the distinct units (genes,
for example) and the confidence level of the coverage are
used to calculate the population size required to provide one
insertion per unit, assuming random mutagenesis (Table 1).
Ac/Ds are ‘cut and paste’ transposons (Figure 1b). New
insertions can occur throughout the life cycle: somatic
insertions that occur in the apical meristem can be apparent in the shoot and can be transmitted to the next
generation [3]. Most newly identified mutations, however,
are recovered in single progeny, indicating that, in practice,
insertions in gametes are the main source of new mutations. Germinal revertants are readily recovered (at a
frequency of 10–1 to 10–2), indicative of late somatic or
gametophytic excision events. These germinal revertants
often differ in sequence [4], and are a rich source of allelic
diversity for functional analysis [5•,6•], as they have been
over evolutionary time [7]. Because only a few mobile
Ac/Ds exist in a mutagenic line and these elements insert
preferentially into sites that are closely linked to the original element locations [3], Ac/Ds is an inefficient global
mutagen (with a forward mutation frequency of 10–6 [1])
but can be highly efficient regionally. There are also maize
transposons with a high insertion preference for particular
genes [8]: these are a restricted tool but one that is highly
useful for those studying the target(s).
Ac/Ds elements spaced at 10–20 cM intervals across the
recombination map would facilitate intensive mutagenesis
of the entire maize genome [9•]. Because mobile Ac/Ds copy
number is low, confirmation that a particular transposon
insert is the cause of a mutant phenotype is easily obtained
[3]. There are several strategies for exploiting Ac/Ds in maize
that depend on enriching for transposed copies, which may
be linked or unlinked to the original location [3] (Figure 2).
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Figure 1
(a)
(b)
(c)
Original site
Empty site
Old
Target
Target for new insertion
DNA repair
Old
New
Current Opinion in Plant Biology
Transposon properties. (a) Somatic excision
of a transposon from a gene of the
anthocyanin pathway can result in spots of
pigmentation in cell lineages with a functional
allele. This visible phenotype demonstrates
somatic mutability and transposon activity. A
range of phenotypes is observed, because
revertant alleles differ in sequence and
function. (b) Cut and paste transposition. The
transposase liberates a double-stranded
transposon from the original site by executing
four strand breaks (arrowed at top left).
Transposase creates staggered nicks at the
target site (8 bp apart) and inserts the doublestranded transposon into a new site. The host
chromosome is repaired (with modest
changes) at the old site, and an 8 bp host
sequence duplication is created by DNA
These strategies have also been implemented in transgenic
dicots expressing maize transposons [10–15]; with genetically engineered Ac/Ds, gene and enhancer trap transposons
have also been developed [10–12].
MuDR/Mu transposons are active late in tissue development, resulting in tiny somatic sectors from ‘cut and paste’
events. MuDR/Mu undergo duplicative transposition in
pre-meiotic and gametophytic cells (Figure 1c); consequently, few germinal revertants exist (excision frequency
<10–5, which is less than a new germinal insertion) [1].
Mutator stocks typically contain several copies of MuDR
and many Mu elements [16], resulting in a high forward
mutation frequency (~10–4, range 10–3–10–5 in any gene
[1,16]). When Mu element copy number is low, the mutation frequency is similar to that of Ac/Ds [17]. Because
epigenetic silencing is stochastic and frequent, somatic
instability (Figure 1a) and unmethylated Mu TIRs should
be verified before mutagenesis [2].
Mu insertions occur preferentially into low-copy DNA; there
is no apparent local bias [16]. Recent analyses of hundreds of
somatic (M Raizada, V Walbot, unpublished data) and
germinal Mu element insertions (K Edwards, personal communication) confirm insertion into or near exon-like
sequences. Although high copy numbers make MuDR/Mu
efficient mutagens, the resulting multiple mutant genes complicate the assignment of phenotypes to specific genes [2].
Transposon tagging in maize is much more efficient than
would be predicted from the large genome size, ~2.3 ×
109 base pairs (bp) [18]. More than half of the maize
genome, however, consists of inactive retrotransposons
found in complex arrays; these create the spacers containing
20–200 thousand base pairs (kbp) between maize genes
synthesis at the insertion site during repair
synthesis. If timed to conicide with
chromosome replication, Ac/Ds copy number
can increase. It is often observed that plant
sectors or kernels on an ear differ in Ac copy
number, in twin sectors. If the transposon is
excised from an old site after its DNA
replication and is inserted into a new site
before DNA replication, one daughter cell
receives two copies of Ac (old site and new
site) and one daughter cell receives one copy
(new site). (c) Replicative transposition.
Transposase makes two single-strand nicks
next to the 3′ ends of the transposon in the
original site. In the model, transposase makes
two staggered nicks in the target (9 bp apart
for Mu elements). A single strand (dotted line)
is, in effect, transferred to a new site. Massive
DNA replication is required to make both the
old and new site double-stranded. A host
sequence duplication is also created at the
new insertion site.
[19]; consequently, insertions would be expected to affect
only one gene. With short introns, maize genes are as compact as those of Arabidopsis [20]. Thus, transcription,
recombination [21] and DNA transposon insertion are all
highly biased for the genes, and for these important processes absolute genome size is of relatively little importance.
DNA sequencing strategies for genomic DNA
next to transposon insertions: methods used
to analyze transposons in a single plant
Initial applications of transposon tagging in maize relied on
correlating the inheritance of a plant phenotype with a
band on a DNA hybridization blot. Particularly for the
multi-copy Mu elements, demonstrating tight linkage
required some luck in restriction enzyme choice and in
hybridization probes to resolve specific Mu elements (Mu1,
Mu2, Mu3, Mu8 and MuDR); the Mu TIR probe detects too
many bands to be useful. Proof that the correct gene was
cloned after recovery of the transposon-tagged genomic
fragment requires the analysis of independent mutants, or
a complementation test. Given the difficulty of maize
transformation, complementation has so far been useful
only for traits that can be assayed in transient expression
assays with protoplasts or tissues amenable to particle gun
bombardment [22].
Several techniques for amplifying and sequencing genomic
DNA next to transposon ends are amenable to genomics
approaches. PCR primers anchored on transposons are ‘read
out’ into genomic DNA [23,24,25••] with a wide choice of
strategies for priming from the flanking genomic DNA of
unknown or specified sequence (Figure 2) [26••]. Initial
methods relied on the separation of the PCR products by
size, with manual recovery and analysis of bands that segregated with a phenotype. Nowadays, simplifications include
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Figure 2
Identifying transposon insertions.
(a) Enriching for transposed Ac/Ds. In this
diagram, kernels with excision sectors are
white with spots of gray. Early somatic
excisions that restore gene function result in a
sector of gray kernels. By selecting seeds in a
sector of kernels with a functionally revertant
allele at the reporter gene (gray circles)
investigators identify a somatic excision event
from the original site; these kernels should
contain the same transposed Ac at a new
location in the genome. Because Ac shows a
negative dosage effect on the frequency of
somatic excision, kernels with fewer spots
probably contain both the original donor site
and a new transposed Ac [3]. (b) The
amplification of sequences next to
transposons typically uses a ‘read out’ primer
(arrows) complementary to the TIRs and a
strategy to allow priming from the contiguous
genomic DNA (star) such as a restriction site
overhang ligated to an adapter. In ‘panhandle’
(a)
(b)
Current Opinion in Plant Biology
PCR [26••], TIRs form intramolecular
duplexes in single-stranded DNA; the flanking
gene is in the pan and the transposon is the
handle. Several restriction digestion and
ligation steps are required to achieve this
structure [9•,26••]. One restriction enzyme
site within the pan structure represents the
shotgun cloning combined with high-throughput sequencing. Selective PCR primer strategies can examine a subset
of transposon family members or a subset of insertion sites
(with primers spanning the joint with genomic DNA).
Because transposon insertions result in characteristic host
sequence duplications (9 bp for Mu [16], 8 bp for Ac/Ds [3]),
compiling the continuous DNA sequence at any given
insertion site uses the duplication for matching up the left
and right genomic sequences.
‘Panhandle’ PCR, in which transposon TIRs form
intramolecular duplexes in single-stranded DNA [26••]
(Figure 2), is the foundation for a new National Science
Foundation (NSF)-funded genomics project that aims to
recover and sequence maize genomic DNA adjacent to
transposed Ac [9•]. Plasmid rescue is an alternative strategy that is much simpler to use in practice but requires
more effort to set up. Transgenic plants with transposons
engineered to contain a bacterial plasmid allow the selective cloning of transposon insertion sites after
transformation of Escherichia coli with total genomic DNA;
only plant DNA with a bacterial origin of replication and
an antibiotic resistance marker is maintained in the transformed bacteria. This approach was pioneered by using
Ac/Ds-based elements introduced into dicots via
Agrobacterium-mediated transformation [27]; now RescueMu
plasmids based on Mu1 are being used to clone maize
genes [28•].
Indexed mutant collections and parallel
searches for transposon insertions
Traditional genetics starts with the mutant individual and
seeks the gene, whereas genomics methods start with the
DNA sequence and search for the phenotypes. The first
genomics approach, TUSC (Trait Utility System for Corn),
artificial joining of the genomic DNA flanking
the left and right ends of the transposon.
Primers within the TIR sequence are used to
amplify around the circle of genomic DNA; the
second primer is in the transposon body to
amplify back in the other direction when firststrand extension is completed.
was developed by Pioneer Hi-Bred International; it is
available to academic researchers who sign material transfer agreements to receive the seeds containing putative
‘hits’ to user-supplied sequence motifs. TUSC contains
DNA samples and progeny seed from ~44,000 Mutator
plants that probably contain >106 independent Mu insertions. DNA samples, from single plants or post-preparation
pools, are screened for Mu insertions into a target gene or
motif by using a PCR primer that reads out of Mu TIRs
and a target-specific primer. All mobile Mu elements share
a very highly conserved sequence in the terminal 25 bp of
the TIRs [16]; consequently, a single ‘read out’ primer suffices. TUSC has been used successfully by both Pioneer
Hi-Bred International [29] and by academic researchers
[30,31]. One drawback of a PCR strategy with a high cycle
number is that both germinal and somatic insertions are
found; ~10–20% genuine germinal insertions are verified
among candidate insertions from TUSC screening [28•].
A convenient field organization into rows and columns for
collecting pooled plant (and hence DNA) samples was
implemented at about the same time as TUSC [32]; this
method overcomes the identification of ‘false positive’
somatic insertions. DNA samples representing pooled leaf
punches from a row of plants and separate sets of leaf
punches from each column of plants are screened by PCR;
two successful reactions define the row and the column and
hence a specific plant that has the germinal insertion mutation (Figure 3). The Maize Targeted Mutagenesis (MTM)
project, funded in the NSF plant genomics program, is now
offering screening of a Mutator collection of ~50,000 plants
and providing seed for the phenotypic evaluation of ‘hits’
[33•]. Project personnel are evaluating plant phenotypes at
the kernel, seedling and adult plant stages to build a database of maize mutant phenotypes. To use MTM services,
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Figure 3
Individual plants numbered within a row
Row 1
1
2
3
4 5
6
7
8
9 10 11 12 . . . . . . . n
Row 2
1
2
3
4 5
6
7
8
9 10 11 12 . . . . . . . n
Row 3
1
2
3
4 5
6
7
8
9 10 11 12 . . . . . . . n
Row 4
1
2
3
4 5
6
7
8
9 10 11 12 . . . . . . . n
Row M
1
2
3
4 5
6
7
8
9 10 11 12 . . . . . . . n
Columns in the vertical dimension 1 to n
Current Opinion in Plant Biology
Row and column pooling strategies for high-throughput transposon
insertion site screening. Plant samples such as leaf punches are
collected from every plant in a row (individuals 1 to n in the horizontal
direction). A single DNA sample is prepared from each row for PCR
screening or plasmid rescue. Similarly, plant samples are collected
from every column, the vertical direction of the grid; in this example
column 4 is in bold and boxed. Positive PCR results from a row and a
column define a specific plant with the germinal insertion of interest,
for example, row 3 and column 8 (circled). Somatic insertions are
amplified only in a row or only in a column in Mutator grids because
somatic excisions and insertions occur mainly late in development.
investigators supply a gene sequence with intron–exon
annotation; after receiving seed, investigators are obligated
to contribute phenotypic analysis to the database.
In both TUSC and the row/column pooling method, DNA
samples are finite. The NSF-funded Maize Gene
Discovery Project uses RescueMu plasmid rescue to create
immortalized collections of insertion sites in E. coli [28•].
Tagging fields with 2304 transgenic individuals are organized into 48 rows and 48 columns, generating 96 separate
libraries of RescueMu plasmids for each field. Users receive
a 96-well plate containing 48 row and 48 column libraries
and design their own PCR strategy to identify insertions;
germinal events are present in one row and one column
(Figure 3). To obtain seed, users submit 1 kb of DNA
sequence next to the Mu insertion site of the RescueMu
plasmid of interest. Concomitantly, the Maize Gene
Discovery Project will sequence row libraries to 95% coverage. Within a few years, users could first search the web
site database [28•] for insertions into genes of interest and
then perform PCR or hybridization on column libraries to
ascertain which plant has the mutation.
few base changes in addition to the host sequence duplication and, more rarely, larger deletions, ‘filler DNA
additions’ or rearrangements. Of particular utility is the
preference of Ac/Ds for local transposition; an Ac/Ds that
transposes nearby can readily transpose back to the target
gene, providing many new types of mutant allele [34,35].
For example, the promoter can be saturated with insertions at different sites followed by selection for minor
alterations that affect regulation, or the requirements for
splicing can be explored from insertion sites in or near the
conserved intron motifs.
An important feature of all maize transposons is that they
are somatically unstable in the presence of the transposaseencoding element. Consequently, somatic tissue is a
mosaic of mutant and revertant cells of many different
phenotypes. To produce somatic tissue of a single phenotype, revertants can be selected, but not all of these are
‘knockout’ alleles. Alternatively for Ac/Ds, individuals lacking Ac can be recovered to stop somatic excisions. This
strategy is virtually impossible for multi-copy MuDR lines;
however, epigenetic silencing of transposons can occur
spontaneously and is frequent in Mutator lines [16]. In the
case of Mu1 insertions in promoters, methylation after
silencing can activate a ‘read out’ promoter in the TIRs, in
effect restoring gene expression [36]. Stabilization of a
fully mutant phenotype is possible by selecting for deletions, which occur with about 10–2 frequency from the
ends of Mu1 elements [37].
Conclusions
Transposon-induced phenotypes have long provided
geneticists with beautiful materials and insights into
gene expression, development and chromosome
mechanics. Cloned transposons have facilitated gene discovery and cloning. In the genomics era, maize
transposons have emerged as the premier method for
gene discovery and sequencing, as well as the phenotypic analysis of gene expression in a whole-organism
context. Transposons allow simultaneous effort in both
phases of genomics, gene discovery and functional studies. Transposons are more efficient than a sequential
approach to gene sequencing followed by the design of
tools to study gene expression.
Acknowledgements
Saturation mutagenesis within a gene
One mutation, no matter how instructive, is rarely sufficient to lead to a comprehensive understanding of a gene’s
function. Most transposon insertions into exons or introns
are ‘knockouts’ of gene function; alternative splicing
events that use sequences in the ends of the transposon
can result in modest expression in a few cases [1].
Transposon insertions, unlike most mutations induced by
physical agents or Agrobacterium insertions, are ‘hot spots’
for secondary mutations. Of greatest current use in maize
are Ac/Ds and Spm/En mutants, from which germinal revertants are readily recovered; these typically contain one or a
I thank Michelle Facette for her comments on the manuscript. Research on
Mutator and preparation of this article were supported by grants from the
NSF (Maize Gene Discovery, DNA Sequencing and Phenotypic Analysis
plant genomics grant 9872657) and the National Institutes of Health
(GM49681).
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• of special interest
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