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Home , Mutagenesis, Mutant, Transposon mutagenesis

PB3207.QXD 02/24/2000 10:12 Page 103

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Saturation using transposons Virginia Walbot

Transposon mutagenesis facilitates discovery by tagging Table 1 for cloning. New genomics projects are now cataloging Population size estimates for to transposon sites to define all maize genes. Once achieve at least one mutant per gene at a specific coverage. identified, transposon insertions are ‘hot spots’ for generating new that are useful in functional studies. Gene estimate Probability (%) Population size (n)

Addresses 30,000 95 ~91,000 Department of Biological Sciences, 385 Serra Mall, Stanford 50,000 95 ~150,000 University, Stanford, California 94305-5020, USA; 50,000 99 ~230,000 e-mail: [email protected] 70,000 95 ~214,000 Current Opinion in 2000, 3:103–107 Using the formula (1 – probability) = n × log (not desired class), the 1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. required population size (n) can be calculated for a specific probability All rights reserved. of finding at least one in each unit. Because the number of functional units in maize is currently unknown, the estimate of ~50,000 Abbreviations genes is often used; this figure is about twice the gene estimate for bp base pairs Arabidopsis, to accommodate the tetraploidy of maize [38]. MTM Maize Targeted Mutagenesis NSF National Science Foundation monitored with a reporter that has a that TIR terminal TUSC Trait Utility System for Corn beomes visible after the excision of a transposon (Figure 1a). Saturation mutagenesis of all genes requires a robust muta- genized population. An estimate of the distinct units (genes, Introduction for example) and the confidence level of the coverage are The analysis of mutant yields an understand- used to calculate the population size required to provide one ing of gene function in a whole- context. insertion per unit, assuming random mutagenesis (Table 1). Consequently, the generation, evaluation, maintenance and distribution of seed containing verified or Ac/Ds are ‘cut and paste’ transposons (Figure 1b). New seed with populations of mutations suitable for high- insertions can occur throughout the life : somatic throughput screening are essential in the modern genetic insertions that occur in the apical meristem can be appar- analysis of maize and other . ent in the shoot and can be transmitted to the next generation [3]. Most newly identified mutations, however, Saturation mutagenesis of maize genes involves two ambi- are recovered in single progeny, indicating that, in practice, tious goals: to define all of the genes and to find the insertions in gametes are the main source of new muta- phenotype of every individual gene. This review will out- tions. Germinal revertants are readily recovered (at a line how can be used to achieve frequency of 10–1 to 10–2), indicative of late somatic or both goals. Transposons are already the primary tool for gametophytic excision events. These germinal revertants tagging and cloning maize genes. Studies so far demon- often differ in sequence [4], and are a rich source of allelic strate that specific transposons are well suited for either diversity for functional analysis [5•,6•], as they have been global mutagenesis and gene discovery or multiple rounds over evolutionary time [7]. Because only a few mobile of mutagenesis at a defined target gene [1]. New genomics Ac/Ds exist in a mutagenic line and these elements insert approaches, employing strategies for screening by PCR preferentially into sites that are closely linked to the origi- and for rescue, are now providing indexed collec- nal element locations [3], Ac/Ds is an inefficient global tions of mutations and the sequences flanking transposon (with a forward mutation frequency of 10–6 [1]) insertion sites. Many researchers will soon identify trans- but can be highly efficient regionally. There are also maize poson-generated mutants in specific genes after querying a transposons with a high insertion preference for particular database rather than searching a corn field. genes [8]: these are a restricted tool but one that is highly useful for those studying the target(s). Insertional mutagenesis across the : primer on key transposon properties Ac/Ds elements spaced at 10–20 cM intervals across the The Ac/Ds and MuDR/Mu maize transposons have been recombination map would facilitate intensive mutagenesis used in most recent mutagenesis experiments [1–3]. In of the entire maize genome [9•]. Because mobile Ac/Ds copy stocks with the transcriptionally active regulatory elements number is low, confirmation that a particular transposon Ac or MuDR, family members with essential - insert is the cause of a mutant phenotype is easily obtained binding sites in the terminal inverted repeats (TIRs) are [3]. There are several strategies for exploiting Ac/Ds in maize mobilized to create new insertion mutations. During a gene- that depend on enriching for transposed copies, which may tagging experiment, transposon activity is conveniently be linked or unlinked to the original location [3] (Figure 2). PB3207.QXD 02/24/2000 10:12 Page 104

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Figure 1

(a) (b) (c) synthesis at the insertion site during repair synthesis. If timed to conicide with replication, Ac/Ds copy number can increase. It is often observed that plant Original site Empty site Old sectors or kernels on an ear differ in Ac copy Target number, in twin sectors. If the transposon is excised from an old site after its DNA DNA repair replication and is inserted into a new site Target for new insertion Old before DNA replication, one daughter cell New receives two copies of Ac (old site and new site) and one daughter cell receives one copy Current Opinion in Plant Biology (new site). (c) Replicative transposition. Transposase makes two single-strand nicks Transposon properties. (a) Somatic excision transposase liberates a double-stranded next to the 3′ ends of the transposon in the of a transposon from a gene of the transposon from the original site by executing original site. In the model, transposase makes anthocyanin pathway can result in spots of four strand breaks (arrowed at top left). two staggered nicks in the target (9 bp apart pigmentation in cell lineages with a functional Transposase creates staggered nicks at the for Mu elements). A single strand (dotted line) allele. This visible phenotype demonstrates target site (8 bp apart) and inserts the double- is, in effect, transferred to a new site. Massive somatic mutability and transposon activity. A stranded transposon into a new site. The host DNA replication is required to make both the range of phenotypes is observed, because chromosome is repaired (with modest old and new site double-stranded. A host revertant alleles differ in sequence and changes) at the old site, and an 8 bp host sequence duplication is also created at the function. (b) Cut and paste transposition. The sequence duplication is created by DNA new insertion site.

These strategies have also been implemented in transgenic [19]; consequently, insertions would be expected to affect dicots expressing maize transposons [10–15]; with genetical- only one gene. With short introns, maize genes are as com- ly engineered Ac/Ds, gene and trap transposons pact as those of Arabidopsis [20]. Thus, transcription, have also been developed [10–12]. recombination [21] and DNA transposon insertion are all highly biased for the genes, and for these important process- MuDR/Mu transposons are active late in tissue develop- es absolute genome size is of relatively little importance. ment, resulting in tiny somatic sectors from ‘cut and paste’ events. MuDR/Mu undergo duplicative transposition in DNA sequencing strategies for genomic DNA pre-meiotic and gametophytic cells (Figure 1c); conse- next to transposon insertions: methods used quently, few germinal revertants exist (excision frequency to analyze transposons in a single plant <10–5, which is less than a new germinal insertion) [1]. Initial applications of transposon tagging in maize relied on Mutator stocks typically contain several copies of MuDR correlating the inheritance of a plant phenotype with a and many Mu elements [16], resulting in a high forward band on a DNA hybridization blot. Particularly for the mutation frequency (~10–4, range 10–3–10–5 in any gene multi-copy Mu elements, demonstrating tight linkage [1,16]). When Mu element copy number is low, the muta- required some luck in restriction choice and in tion frequency is similar to that of Ac/Ds [17]. Because hybridization probes to resolve specific Mu elements (Mu1, epigenetic silencing is stochastic and frequent, somatic Mu2, Mu3, Mu8 and MuDR); the Mu TIR probe detects too instability (Figure 1a) and unmethylated Mu TIRs should many bands to be useful. Proof that the correct gene was be verified before mutagenesis [2]. cloned after recovery of the transposon-tagged genomic fragment requires the analysis of independent mutants, or Mu insertions occur preferentially into low-copy DNA; there a complementation test. Given the difficulty of maize is no apparent local bias [16]. Recent analyses of hundreds of transformation, complementation has so far been useful somatic (M Raizada, V Walbot, unpublished data) and only for traits that can be assayed in transient expression germinal Mu element insertions (K Edwards, personal com- assays with protoplasts or tissues amenable to particle gun munication) confirm insertion into or near exon-like bombardment [22]. sequences. Although high copy numbers make MuDR/Mu efficient , the resulting multiple mutant genes com- Several techniques for amplifying and sequencing genomic plicate the assignment of phenotypes to specific genes [2]. DNA next to transposon ends are amenable to genomics approaches. PCR primers anchored on transposons are ‘read Transposon tagging in maize is much more efficient than out’ into genomic DNA [23,24,25••] with a wide choice of would be predicted from the large genome size, ~2.3 × strategies for priming from the flanking genomic DNA of 109 base pairs (bp) [18]. More than half of the maize unknown or specified sequence (Figure 2) [26••]. Initial genome, however, consists of inactive retrotransposons methods relied on the separation of the PCR products by found in complex arrays; these create the spacers containing size, with manual recovery and analysis of bands that segre- 20–200 thousand base pairs (kbp) between maize genes gated with a phenotype. Nowadays, simplifications include PB3207.QXD 02/24/2000 10:12 Page 105

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Figure 2

Identifying transposon insertions. (a) (b) (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 Current Opinion in Plant Biology somatic excision, kernels with fewer spots probably contain both the original donor site and a new transposed Ac [3]. (b) The PCR [26••], TIRs form intramolecular artificial joining of the genomic DNA flanking amplification of sequences next to duplexes in single-stranded DNA; the flanking the left and right ends of the transposon. transposons typically uses a ‘read out’ primer gene is in the pan and the transposon is the Primers within the TIR sequence are used to (arrows) complementary to the TIRs and a handle. Several restriction digestion and amplify around the circle of genomic DNA; the strategy to allow priming from the contiguous ligation steps are required to achieve this second primer is in the transposon body to genomic DNA (star) such as a restriction site structure [9•,26••]. One restriction enzyme amplify back in the other direction when first- overhang ligated to an adapter. In ‘panhandle’ site within the pan structure represents the strand extension is completed.

shotgun cloning combined with high-throughput sequenc- was developed by Pioneer Hi-Bred International; it is ing. Selective PCR primer strategies can examine a subset available to academic researchers who sign material trans- of transposon family members or a subset of insertion sites fer agreements to receive the seeds containing putative (with primers spanning the joint with genomic DNA). ‘hits’ to user-supplied sequence motifs. TUSC contains Because transposon insertions result in characteristic host DNA samples and progeny seed from ~44,000 Mutator sequence duplications (9 bp for Mu [16], 8 bp for Ac/Ds [3]), plants that probably contain >106 independent Mu inser- compiling the continuous DNA sequence at any given tions. DNA samples, from single plants or post-preparation insertion site uses the duplication for matching up the left pools, are screened for Mu insertions into a target gene or and right genomic sequences. motif by using a PCR primer that reads out of Mu TIRs and a target-specific primer. All mobile Mu elements share ‘Panhandle’ PCR, in which transposon TIRs form a very highly conserved sequence in the terminal 25 bp of intramolecular duplexes in single-stranded DNA [26••] the TIRs [16]; consequently, a single ‘read out’ primer suf- (Figure 2), is the foundation for a new National Science fices. TUSC has been used successfully by both Pioneer Foundation (NSF)-funded genomics project that aims to Hi-Bred International [29] and by academic researchers recover and sequence maize genomic DNA adjacent to [30,31]. One drawback of a PCR strategy with a high cycle transposed Ac [9•]. Plasmid rescue is an alternative strate- number is that both germinal and somatic insertions are gy that is much simpler to use in practice but requires found; ~10–20% genuine germinal insertions are verified more effort to set up. Transgenic plants with transposons among candidate insertions from TUSC screening [28•]. engineered to contain a bacterial plasmid allow the selec- tive cloning of transposon insertion sites after A convenient field organization into rows and columns for transformation of Escherichia coli with total genomic DNA; collecting pooled plant (and hence DNA) samples was only plant DNA with a bacterial origin of replication and implemented at about the same time as TUSC [32]; this an resistance marker is maintained in the trans- method overcomes the identification of ‘false positive’ formed . This approach was pioneered by using somatic insertions. DNA samples representing pooled leaf Ac/Ds-based elements introduced into dicots via punches from a row of plants and separate sets of leaf Agrobacterium-mediated transformation [27]; now RescueMu punches from each column of plants are screened by PCR; based on Mu1 are being used to clone maize two successful reactions define the row and the column and genes [28•]. hence a specific plant that has the germinal insertion muta- tion (Figure 3). The Maize Targeted Mutagenesis (MTM) Indexed mutant collections and parallel project, funded in the NSF plant genomics program, is now searches for transposon insertions offering screening of a Mutator collection of ~50,000 plants Traditional genetics starts with the mutant individual and and providing seed for the phenotypic evaluation of ‘hits’ seeks the gene, whereas genomics methods start with the [33•]. Project personnel are evaluating plant phenotypes at DNA sequence and search for the phenotypes. The first the kernel, seedling and adult plant stages to build a data- genomics approach, TUSC (Trait Utility System for Corn), base of maize mutant phenotypes. To use MTM services, PB3207.QXD 02/24/2000 10:12 Page 106

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Figure 3 few base changes in addition to the host sequence dupli- cation and, more rarely, larger deletions, ‘filler DNA Individual plants numbered within a row additions’ or rearrangements. Of particular utility is the Row 1 1 2 3 4 5 6 7 8 9 10 11 12 ...... n preference of Ac/Ds for local transposition; an Ac/Ds that

Row 2 1 2 3 4 5 6 7 8 9 10 11 12 ...... n transposes nearby can readily transpose back to the target gene, providing many new types of mutant allele [34,35]. Row 3 1 2 3 4 5 6 7 8 9 10 11 12 ...... n For example, the can be saturated with inser- Row 4 1 2 3 4 5 6 7 8 9 10 11 12 ...... n tions at different sites followed by selection for minor Row M 1 2 3 4 5 6 7 8 9 10 11 12 ...... n alterations that affect regulation, or the requirements for splicing can be explored from insertion sites in or near the Columns in the vertical dimension 1 to n Current Opinion in Plant Biology conserved intron motifs.

Row and column pooling strategies for high-throughput transposon insertion site screening. Plant samples such as leaf punches are An important feature of all maize transposons is that they collected from every plant in a row (individuals 1 to n in the horizontal are somatically unstable in the presence of the transposase- direction). A single DNA sample is prepared from each row for PCR encoding element. Consequently, somatic tissue is a screening or plasmid rescue. Similarly, plant samples are collected of mutant and revertant cells of many different 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 phenotypes. To produce somatic tissue of a single pheno- column define a specific plant with the germinal insertion of interest, type, revertants can be selected, but not all of these are for example, row 3 and column 8 (circled). Somatic insertions are ‘knockout’ alleles. Alternatively for Ac/Ds, individuals lack- amplified only in a row or only in a column in Mutator grids because ing Ac can be recovered to stop somatic excisions. This somatic excisions and insertions occur mainly late in development. 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 investigators supply a gene sequence with intron–exon case of Mu1 insertions in promoters, methylation after annotation; after receiving seed, investigators are obligated silencing can activate a ‘read out’ promoter in the TIRs, in to contribute phenotypic analysis to the database. effect restoring [36]. Stabilization of a fully mutant phenotype is possible by selecting for dele- In both TUSC and the row/column pooling method, DNA tions, which occur with about 10–2 frequency from the samples are finite. The NSF-funded Maize Gene ends of Mu1 elements [37]. Discovery Project uses RescueMu plasmid rescue to create immortalized collections of insertion sites in E. coli [28•]. Conclusions Tagging fields with 2304 transgenic individuals are orga- Transposon-induced phenotypes have long provided nized into 48 rows and 48 columns, generating 96 separate geneticists with beautiful materials and insights into libraries of RescueMu plasmids for each field. Users receive gene expression, development and chromosome a 96-well plate containing 48 row and 48 column libraries mechanics. Cloned transposons have facilitated gene dis- and design their own PCR strategy to identify insertions; covery and cloning. In the genomics era, maize germinal events are present in one row and one column transposons have emerged as the premier method for (Figure 3). To obtain seed, users submit 1 kb of DNA gene discovery and sequencing, as well as the phenotyp- sequence next to the Mu insertion site of the RescueMu ic analysis of gene expression in a whole-organism plasmid of interest. Concomitantly, the Maize Gene context. Transposons allow simultaneous effort in both Discovery Project will sequence row libraries to 95% cov- phases of genomics, gene discovery and functional stud- erage. Within a few years, users could first search the web ies. Transposons are more efficient than a sequential site database [28•] for insertions into genes of interest and approach to gene sequencing followed by the design of then perform PCR or hybridization on column libraries to tools to study gene expression. ascertain which plant has the mutation. Acknowledgements Saturation mutagenesis within a gene I thank Michelle Facette for her comments on the manuscript. Research on One mutation, no matter how instructive, is rarely suffi- Mutator and preparation of this article were supported by grants from the NSF (Maize Gene Discovery, DNA Sequencing and Phenotypic Analysis cient to lead to a comprehensive understanding of a gene’s plant genomics grant 9872657) and the National Institutes of Health function. Most transposon insertions into exons or introns (GM49681). are ‘knockouts’ of gene function; alternative splicing events that use sequences in the ends of the transposon References and recommended reading can result in modest expression in a few cases [1]. Papers of particular interest, published within the annual period of review, Transposon insertions, unlike most mutations induced by have been highlighted as: physical agents or Agrobacterium insertions, are ‘hot spots’ • of special interest •• of outstanding interest for secondary mutations. Of greatest current use in maize 1. Walbot V: Strategies for mutagenesis and gene cloning using are Ac/Ds and Spm/En mutants, from which germinal rever- transposon tagging and T-DNA insertional mutagenesis. tants are readily recovered; these typically contain one or a Annu Rev Plant Physiol 1992, 43:49-82. PB3207.QXD 02/24/2000 10:12 Page 107

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2. Chomet PS: Transposon tagging with Mutator. In The Maize 21. Dooner HK, Martinez-Ferez IM: Recombination occurs uniformly Handbook. Edited by Freeling M, Walbot V. New York: Springer, within the bronze gene, a meiotic recombination hotspot in the 1996:243-248. maize genome. Plant Cell 1997, 9:1633-1646. 3. Dellaporta SL, Moreno MA: Gene tagging with Ac/Ds elements in 22. Nash J, Luehrsen KR, Walbot V: Bronze-2 gene of maize: maize. In The Maize Handbook. Edited by Freeling M, Walbot V. New reconstruction of a wild-type allele and analysis of transcription York: Springer, 1996:219-239. and splicing. Plant Cell 1990, 2:1039-1049. 4. Nordborg M, Walbot V: Estimating allelic diversity generated by 23. Koes R, Sour E, van Houwelinger A, Mur L, Spelt C, Quattrochio F, excision of different transposon types. Theor Appl Genet 1995, Wing J, Oppendijk B, Ahmed SA, Maes T et al.: Targeted gene 90:771-775. inactivation in petunia by PCR based selection of transposon insertion mutants. 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