Review Transposons As Tools for Functional Genomics

Home , Mutant, Transposon mutagenesis, Transposon tagging

Srinivasan Ramachandran, Venkatesan Sundaresan*

Institute of Molecular Agrobiology, National University of Singapore, 1, Research Link, Singapore 117 604, Singapore

Received 19 December 2000; accepted 25 January 2001

Abstract – Transposons have been used extensively for insertional mutagenesis in several plant species. These include species where highly active endogenous systems are available such as maize and Antirrhinum majus, as well as species where heterologous transposons have been introduced through transformation, such as Arabidopsis thaliana and tomato. Much of the past use of transposons has been in traditional ‘forward genetics’ approaches, to isolate and molecularly characterize genes identiﬁed by mutant phenotypes. With the rapid progress in the genome projects of different plants, large-scale transposon mutagenesis has become an important component of functional genomics, permitting assignment of functions to sequenced genes through reverse genetics. Different strategies can be pursued, depending upon the properties of the transposon such as the mechanism and control of transposition, and those of the host plant such as transformation efficiency. The successful use of these strategies in A. thaliana has made it possible to develop databases for reverse genetics, where screening for the knockout of a gene of interest can be performed by computer searches. The extension of these technologies to other plants, particularly agronomically important crops such as rice, is now feasible. © 2001 Éditions scientiﬁques et médicales Elsevier SAS gene tagging / reverse genetics / transposons

Ac, Activator element / Ds, Dissociation element / dSpm, defective Suppressor-mutator element / En, Enhancer / FST, ﬂanking sequence tag / I, Inhibitor /Mu,Mutator / Spm, Suppressor-mutator

1. INTRODUCTION of Nicotiana tobaccum has been mutated by site- specific base substitution using self-complementary The advent of large-scale sequencing projects in chimeric oligonucleotides [9] but this method is also several plants, together with the anticipated comple- too labour intensive for use in functional genomics. tion of whole genome sequences for Arabidopsis Strategies for sense and anti-sense suppression have thaliana and possibly rice as well, has resulted in an been developed for the inactivation of known genes [8, explosion of gene sequence information in plants. As 37] but as these strategies require generation of several more and more sequences are available in the data- independent transgenic lines for each gene they are bases, it becomes critical to assign functions to the currently limited to the study of single genes. Cur- thousands of new genes identified. There are several rently the most widely used approach for large-scale approaches being attempted to tackle this problem. gene function analysis in plants is random insertional The most direct approach to determine the functions mutagenesis. Either T-DNA (reviewed in [2, 42]) or of the sequenced genes in an organism is to disrupt or transposons (reviewed in [50, 77, 84]) can be used as generate mutations in the genes and analyse the insertional mutagens in plants. consequences. Methods that have been developed in Insertional mutagenesis using Agrobacterium medi- plants for this purpose include gene replacement, sense ated T-DNA integration into plant genomes (primarily and anti-sense suppression, and insertional mutagen- in A. thaliana, and more recently in rice) has proved to esis. Although recently it has been shown that targeted be very successful [2, 34, 42]. This approach has the gene replacement by homologous recombination is advantage of simplicity as each transformant yields a possible in A. thaliana, the frequency is very low [39], stable insertion in the genome and does not need which makes the method laborious for generating large additional steps to stabilize the insert. Several groups numbers of gene knockouts. Recently, a nuclear gene have used this approach in A. thaliana to generate tens of thousands of independent lines that can be used for *Correspondence and reprints: fax +65 872 7007. reverse genetics. Recently, a National A. thaliana E-mail address: [email protected] (V. Sundaresan). Knockout Facility has been established at the Univer- 244 S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243–252 sity of Wisconsin, USA, with access to 60 480 inser- Table I. Endogenous transposons in different plants. tion lines [42]. Modified T-DNA insertions have been used in A. thaliana as gene [3], promoter traps [46] Element Plant Similar to Reference and in activation tagging [85]. Recently, Jeon et al. Activator, Ac Maize – [15, 16, 52] [34] have also been using T-DNA insertions for Tam3 Antirrhinum Ac of maize [14, 28] functional genomics in rice. Despite the extremely majus Tph1 Petunia hybrida Ac of maize [23] successful use of T-DNA in A. thaliana, there are Tag1 Arabidopsis Distantly related [19, 79] however a few disadvantages to this approach. The thaliana to Ac of maize integration of the T-DNA is generally complex, result- Slide Tobacco Ac of maize [25] ing in tandem direct and inverted repeats and deletions Spm/En Maize – [53, 63, 64, 65] in one or more borders. Such rearrangements can make Tam1 A. majus Spm/En [58] Tnr3 Rice Spm/En [56] the subsequent molecular analysis difficult in many Psl P. hybrida Spm/En [72] cases, and adversely affect the success of large-scale Mutator Maize [67] strategies such as flanking sequence databases (described later). Secondly, complex and multiple insertions are more likely to lead to artefactual patterns of reporter gene expression when using entrapment 2. ENDOGENOUS TRANSPOSABLE vectors such as gene and enhancer traps. Finally, while ELEMENTS IN DIFFERENT PLANT SPECIES the T-DNA approach is extremely useful for plant species where quick and efficient transformation meth- Transposable elements like Activator (Ac), ods are available, it may not be feasible in those plant Suppressor-mutator/Enhancer (Spm/En) and Mutator species where the transformation methods are slow or (Mu) were originally discovered and molecularly char- labour intensive. acterized in maize (table I [15, 16, 52, 53, 63, 64, 67]). For these reasons, insertional mutagenesis using Subsequently, a number of endogenous transposable transposable elements offers some advantages over elements in other plant species that are similar or T-DNA mutagenesis. The insertions generated by trans- distantly related to the maize Ac element have been posons are generally single intact elements, which lend identified including Tam3 in Antirrhinum majus [14, themselves easily to molecular analysis. Such inser- 28], Tag1 in A. thaliana ecotype Landsberg [79] and tions are also less likely to result in artefactual patterns slide1 in tobacco [25]. Also Spm/En-like transposable of expression if the transposon is being used as a gene elements have been reported in A. majus (Tam1 [58]), trap or enhancer trap. An additional advantage is that rice (Tnr3 [56]) and in Petunia (Psl [72]). many transposons can be excised from the disrupted Endogenous transposable elements have been used gene in the presence of transposase. Such excisions to clone genes in maize, A. majus and Petunia hybrida. can result in phenotypic reversion to the wild type or In maize, all three families (Ac, Spm and Mu)of give rise to alleles with weaker phenotypes. This transposable elements have been widely used as ‘tags’ property of many transposons provides ready confir- to isolate genes (reviewed in [24, 83]). The Tam mation that the mutation was really tagged by the elements from A. majus (reviewed in [24]) and the transposon, as well as the possibility of generating an dTph elements from P. hybrida [73] have proved allelic series. In addition, another property of several similarly valuable in their host species. Some families transposons to preferentially insert into genetically of endogenous elements are present in high-copy linked sites [7, 33, 36], can be used to perform local numbers in their hosts. For example elements of the mutagenesis in a particular region of interest by Mu family in maize and dTph family in P. hybrida re-mobilizing the transposon [31, 35, 69]. Finally, exist in more than 100 copies per genome. This is very transposons can be used for insertional mutagenesis in advantageous for large-scale mutagenesis, as a few plant species where transformation is inefficient, since thousand lines will be sufficient to cover the whole the generation of new insertions occurs through cross- genome. On the other hand, there are a few disadvan- ing or propagation rather than through transformation. tages in using high-copy number endogenous trans- In this review, we discuss the most widely used posons. First, since there is a number of insertions per transposons and how these elements have been exploited line, there will be a continuous transposition of ele- successfully in heterologous plant species as an inser- ments. If there was a significant frequency of germinal tional mutagen and as a tool for ‘reverse genetics’ for excisions, this would result in mutations that are due to functional genomics. the excision footprints, and therefore not tagged. S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243–252 245

Secondly, confirmation that a mutation is indeed caused Table II. Transposons activity in heterologous plants. by a particular copy of the transposon may require Plant of origin Transposon Activity in Reference several out-crosses to remove other elements, which is type heterologous a time consuming process. Finally, the use of gene/ plants enhancer trap elements is not feasible, as the patterns Maize Ac/Ds A. thaliana [7, 81] of expression due to several insertions of the elements Rice [32, 57, 70] per plant will be additive and therefore inaccurate. Tomato [35, 54, 90] One of the most successfully used high-copy num- Petunia [13, 26, 66] ber transposon families is the Mu transposable element Flax [17, 44] family consisting of the autonomous element MuDR Tobacco [5, 18, 86] and the non-autonomous elements Mu1toMu8 Carrot [81] Lettuce [89] (reviewed in [83]). These elements have been used to Potato [40] clone several genes in maize [10]. The Mu elements Maize Spm/En A. thaliana [1, 12] have been particularly effective tool for gene tagging Potato [21] for the following reasons: (a) higher forward mutation Tobacco [51, 62] frequency when compared to Ac or Spm element (20 to Arabidopsis Tag1 Rice [48] 50 % higher) (reviewed in [83, 84]); (b) equal transposition to linked and unlinked sites when compared to other transposable elements [47]; (c) characteristi- heterologous species including A. thaliana, rice, tomato, cally late excisions which stabilizes new alleles in P. hybrida, flax, carrot, lettuce, potato, etc. (table II). sibling progeny and reduces the probability of foot- Similarly maize Spm/dSpm (En/I) has been utilized print mutations. However, transfer of the Mu system successfully in tobacco, potato and A. thaliana (table into heterologous plant species has not yet been II). Apart from maize transposable elements, the A. successfully accomplished. thaliana transposon Tag1 which is distantly related to Retrotransposons, which transpose through a RNA the maize Ac, has been shown to be active in tobacco intermediate, have been shown to generate spontane- [19] and in rice [48]. ous mutations in maize [82] and are the major class of In order to better regulate the transpositional events transposable elements in most plants (reviewed in and obtain stable insertions, two-component systems [11]). Retrotransposons are potentially very useful for are preferred. In a typical two-component system, a gene tagging as these elements generate stable inser- transgenic line will be generated which harbours an tions, and unlike many DNA transposons they inte- immobilized (‘wings clipped’) autonomous element grate into unlinked sites. However, the relatively low (Ac or Spm/En) that will provide the transposase frequencies of transpositions of most plant retrotrans- source. Second line transgenic for the non-autonomous posons have restricted their uses for large-scale gene element (Ds or dSpm/I), which cannot transpose unless tagging. Recently however, the Tos17 retrotransposon the transposase source is available, will be generated. has been developed as a promising system for rice Selectable markers such as antibiotic resistance or [29]. The endogenous rice Tos17 retrotransposons herbicide resistance markers can be engineered in the appear to be inactive during normal growth conditions, non-autonomous elements to select for the presence of but they are reactivated by tissue culture, resulting in transposed elements. In order to monitor the transpo- high transposition frequencies suitable for insertional sition events, the non-autonomous transposon can be mutagenesis [29]. inserted between a promoter and the marker gene so that excision results in expression of the selectable marker gene (reviewed in [76]). Plants homozygous 3. TRANSPOSONS for transposase and for the dependent element are used IN HETEROLOGOUS SYSTEMS as starter lines and crossed to facilitate transposition. Lines carrying stable single insertions can be obtained The first report that maize transposable element subsequently by segregation of the transposase source, Ac/Ds can be active in a heterologous system came which can be simplified by the use of negative from studies with transgenic tobacco (Nicotiana tobac- selection markers [76] (figure 1B). cum) by Baker et al. [4, 5]. Since then, the maize The ability of Spm/En elements to amplify through Ac/Ds elements have been shown to transpose actively continued propagation in A. thaliana has been exploited and been exploited in tagging studies in a number of to increase the number of insertions per plant [74, 88]. 246 S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243–252

permits near-saturation mutagenesis of a plant genome with a relatively small number of plants. However, there are also some disadvantages in using this approach, arising from the continuous nature of the transposition events. First, the PCR strategies to identify a gene knockout are complicated by somatic insertion events that will result in the detection of insertions which are not transmitted through the germ line. Second, the presence of footprints in genes due to imprecise excisions will lead to mutations that are not tagged. Most transposable elements, including Ac/Ds and En/Spm, have a tendency to preferentially transpose to genetically linked sites [7, 33, 36]. This feature can be advantageous for directed tagging of a specific target gene or for performing local (regional) insertional mutagenesis in a selected region of a chromosome when a transposable element is inserted close to the target gene or within the derived chromosomal region (figure 1A) [31, 35, 69]. In another instance, Ac/Ds transposons and cDNA scanning methods were used together to perform regional insertional mutagenesis on genes from CIC7E11/8B11 and 5CIC5F11/CIC2B9 loci on A. thaliana chromosome V [31, 69]. This allows cloning of cDNAs from a small region in the genome. The flanking sequences of insertions showed that 14–20 % of the transpositions were located in about 1 Mb of genomic DNA surrounding Ds donor sites. Figure 1. Schematic diagram of different approaches to transposon When random saturation mutagenesis is desired, the tagging. In both A and B, the Ds element is mobilized in the presence high fraction of closely linked site transpositions poses of Ac transposase. A, Directed tagging; B, random tagging. 1, 2 and 3 represent different chromosomes, and X, Y, Z different genes. C, a serious limitation. In principle, this limitation can be Insertion of gene trap or enhancer trap element. SA, splice acceptor; overcome by using many starter lines, with Ds/dSpm TA, minimal promoter; E, enhancer sequence in the genome; X, insertions distributed evenly over different chromo- unknown gene and GUS, glucuronidase. D, Insertion of activation somes. Alternately, in the Ac-Ds system of Sundaresan tagging element. X denotes an unknown gene. TE, transposable et al. [77], the propensity of Ds elements to transpose element; LB, left border of T-DNA; RB, right border of T-DNA; PSM, positive selection marker; NSM, negative selection marker. to linked sites is overcome by selection against the donor site using a negative selection marker. By simultaneous selection against the transposase source In one strategy, the autonomous Spm/En element was and the donor site, a population of stable, unlinked, transformed into A. thaliana and propagated for five transposon insertion lines can be generated (figure 1B) generations by a single seed descent. This yielded [61, 77]. In the modified Spm/En system designed by 15 000 insertions in 3 000 lines [88]. Alternatively, a Tissier et al. [78], the Spm transposase and the mobile two component (in-cis) En-I (Spm/dSpm) system has dSpm elements are contained within the same T-DNA been introduced into A. thaliana, where a mobile I transformed into A. thaliana. This system eliminates (dSpm) element and an immobilized trans-active En the need for crossing, and also reduces the number of (Spm) transposase are contained in the same T-DNA. progeny required for the selection by a factor of four, This in-cis element system was used to generate as the negative selection is applied against only a 2 592 A. thaliana lines containing multiple insertions single locus as opposed to two loci when the trans- (approx. twenty inserts/line) of mobile I element [74]. posase is introduced separately. However, the mainte- This approach of amplifying the number of insertions nance of starter lines becomes problematic, as the per plant has the very significant advantage that it dSpm elements will continually transpose in the pres- S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243–252 247 ence of the Spm transposase, so that this system expressing a gene can provide dominant gain-of- requires a relatively large number of independent function mutations that produce informative mutant starter lines obtained through transformation. phenotypes. A strategy to generate mutants as a con- sequence of increased or expanded expression of a tagged gene is known as ‘activation tagging’. The 4. SPECIALIZED TAGGING SYSTEMS principle involves using strong enhancers in T-DNA or a transposon resulting in ectopic expression or over- 4.1. Gene and enhancer trap elements expression of nearby genes through transcriptional activation. The first successful activation tagging in Insertional mutagenesis by transposon tagging is plants was reported by Hayashi et al. [27], who useful when disruption of a gene leads to an obvious introduced four copies of enhancer elements from the phenotype. But in eukaryotic systems, disruptions of cauliflower mosaic virus 35S promoter (CaMV 35S) genes frequently do not result in visible phenotypes into T-DNA, generating gain-of-function mutations due to functional redundancy, or they may result in and novel phenotypes. This approach has been used to early lethality that obscures late-acting functions when generate a large-scale tagging population by Wiegel et the same gene has multiple functions in development. al. [85]. The principle of activation tagging has also To overcome these difficulties, modified transposons been adapted for transposable elements by Wilson et called gene trap or enhancer trap transposable ele- al. [87]. They used the Ds element with a complete ments have been developed (reviewed in [43, 75, 76]). CaMV 35S promoter pointing outwards from the Such modified elements were first used in Drosophila element (figure 1D), and generated a population of melanogaster and mouse [71, 87], and were subse- insertions through crosses with Ac transposase. This quently extended to plant transposon systems [75–77]. approach was used to identify dominant mutations at Gene/enhancer trap elements carry a reporter gene various loci in A. thaliana, including TINY, Late whose expression pattern reflects the activity and Elongated Hypocotyl (LHY), and Short Internodes regulation of the disrupted genes (figure 1C). The (SHI) [22, 68, 87]. A limitation of this approach is that enhancer traps contain a reporter gene driven by a the observed frequency of dominant mutations through minimal or weak promoter in the dependent element. activation tagging has been significantly lower than The reporter gene expression is achieved (by utilizing that of recessive mutations arising from insertional the endogenous enhancer sequences) only when the inactivation [85], suggesting that many genes may be transposition occurs within or close to a gene (figure over-expressed without resulting in observable pheno- 1C). A variation of this vector called a promoter trap types. Nevertheless, activation tagging has proven to contains a promoterless reporter gene, which will be a valuable complementary approach for the identi- express only when the transposon is inserted down- fication of gene functions. stream of an active endogenous plant promoter. In contrast, gene trap vectors are designed to contain one or more splice acceptor sequences preceding the reporter 5. FORWARD VS. REVERSE GENETICS gene. This allows expression of the reporter only when it inserts in a transcribed region. The splice donor sites Cloning of genes that produce mutant phenotype or from the intron of the endogenous gene and the splice function forms the basis of ‘forward genetics’ (i.e. acceptor sequences, which precede the reporter gene, phenotype or function to gene). On the other hand, if will be spliced to generate a fusion transcript (reviewed the gene sequence is known and the biological func- in [50, 75, 76]). Thus far only the Ac/Ds transposon tion of that gene is not known, a knockout mutant can system has been modified for enhancer and gene trap be generated and analysed to determine its function. approaches. This forms the basis for ‘reverse genetics’, i.e. from gene to phenotype or function (reviewed in [42, 50, 60]). Since genome sequencing projects for various 4.2. Activation tagging elements plant species are progressing rapidly, more and more Gene disruptions through insertional mutagenesis sequences encoding predicted genes are available in nearly always generate recessive loss-of-function muta- the databases. Reverse genetics strategies will be of tions. Such mutations do not always produce an great importance for the purpose of assigning func- obvious phenotype due to various factors such as tions to predicted genes. As discussed earlier, gene functional redundancy (see next section). In such disruption by transposons constitutes a powerful tool cases, increasing the expression level or ectopically for reverse genetics. 248 S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243–252

6. STRATEGIES FOR REVERSE GENETICS SCREENING In order to perform reverse genetic screens effi- ciently, it is necessary to generate a large population of transposon tagged mutants. The number of lines to be screened is dependent on the genome size and the number of genes of a given plant species, the type of transposon used (single or multi-copy) etc. A population containing insertions with a reasonable probability of finding at least one insertion in any given gene is important for the success of this approach. In order to screen for an insertion in a particular gene, a PCR-based strategy has been applied in D. melanogaster for site-specific selection of insertions using P elements [6, 38]. In this approach, a gene- specific primer and an insertion-specific primer are used for PCR amplification. Several insertion lines are pooled together and the DNA extracted is then used as a template for PCR reactions. Samples of 20 to 100 insertion lines are pooled to extract genomic DNA, and a gene-specific primer and an insertion- specific primer were used for PCR [55, 78]. Any pool showing a positive signal is re-screened using DNA from individual lines, to identify the line carrying the insertion of interest. Several pools can be combined to form a super pool if the number of insertion lines in the population is very high [78]. Alternatively a three-dimensional matrix pooling strategy has been tested for P. hybrida lines carrying the multi-copy Figure 2. Flow chart for the use of insertional mutants in a reverse dTph1 transposon [41]. The leaf material from a genetics approach to functional genomics. The steps involved from the population of 1 000 plants were pooled according to a identification of an insertion in a gene of interest to the phenotypic three-dimensional matrix (columns, rows and blocks). characterization of the mutant are detailed. DNA samples were extracted from ten blocks of 100 lines. Similarly DNA is extracted from ten columns and ten rows each of them containing 100 lines. display and AIMS techniques utilize adapters that are Altogether thirty DNA samples were used to perform ligated to the genomic DNA, which has been digested PCR, which can identify a single plant that contains with restriction enzyme(s). The appropriate restriction the insertion in the specific gene. This method is enzyme(s) are selected which recognizes one site in advantageous as it requires fewer amplifications, and the insertion element and the other in the flanking directly identifies the single plant with insertion (figure region. A chimeric adapter and transposon primer is 2 [41]). used from one end and an adapter primer from the An alternate approach to identify the genes that other end to amplify the flanking region in the trans- have been tagged in a population of insertion lines poson display technique. On the other hand, adapter involves random amplification of the DNA flanking primers are used as forward and reverse primers for the insertions. Several different methods can be fol- amplification in AIMS approach. Flanking regions lowed within this approach. Transposon display [80] obtained by amplifications are displayed which can be and amplification of insertion mutagenized sites (AIMS) cloned and sequenced subsequently ([20, 80] and [20] are techniques used to identify tagged genes in reviewed in [50]). families with multi-copy transposons like dTph1 in A method called inverse display of insertions (IDI) Petunia and Mu in maize. For example, the maize Bx1 has been developed by Tissier et al. [78] to isolate the gene and the Fbp1 gene fragment from Petunia were flanking sequences spanning the dSpm/I element. The isolated by this method [20, 80]. The transposon inverse PCR (iPCR) method [59] has been adapted for S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243–252 249 this strategy. Since the selection of restriction sites (not phenotype caused by the single stable insertion, then too close or too far from the insertion site) is a critical the gene functions may be deduced through detailed factor for a successful iPCR, three different restriction analysis of the phenotype (figure 2). However, there enzymes were used to greatly increase the probability are many instances, where a gene knockout does not of amplifying the flanking region. DNA pools from show an observable phenotype. This could be due to insertion lines were used as template and iPCR reac- the fact that several genes may be required and tions were performed. These products were mixed and expressed only under specific conditions, such as spotted in grid arrays on a nylon membrane, and pathogen infection or environmental stresses. For muta- hybridized with labelled probes from the genes of tions in such genes, in order to detect a phenotype it interest [78]. will be necessary to subject the plants to conditions in Alternatively, the DNA flanking the insertions can which the gene is required. These conditions may be amplified and sequenced individually to catalogue include challenges with pathogens and the use of the insertions by chromosomal location [61, 78]. This specific growth media or growth conditions. For strategy is especially useful when substantial genome example, a T-DNA insertion in the AKT-1 potassium sequence information is available, as in the case of A. channel gene of A. thaliana was identified by a reverse thaliana. A protocol such as iPCR, ligation-mediated genetics approach using pooled PCR and hybridiza- PCR or thermal asymmetric interlaced PCR (TAIL- tion. On most nutritional media, the akt-1 mutant PCR; [49]) can be used to amplify and sequence the plants were indistinguishable from wild type plants, flanking region of single- or low-copy insertion lines. but on media containing a low potassium concentra- With an appropriately constructed database of such tion of 100 µM or less, the growth of the mutant plants flanking sequences, it will be feasible to identify was defective [30]. insertions in a particular gene by simple computer Another reason for not observing a clear phenotype searches, removing the necessity for the more tedious when a gene is knocked out is functional redundancy pooling and hybridization protocols. In addition, even with other genes. In such cases, creation of double or if no ‘hit’ is found, the availability of a transposon triple mutants of the functionally redundant genes will insertion close to a gene of interest can be quickly uncover the phenotype and permit characterization of ascertained by a computer search. As described previ- their functions (figure 2). For example, A. thaliana ously, transposons from the Ac/Ds and Spm/En fami- genes ‘SHATTERPROOF1’ (SHP1) and ‘SHATTER- lies are very useful for mutagenesis of closely linked PROOF2’ (SHP2) regulate fruit dehiscence or pod genes, and a collection of sequenced insertions pro- shatter. Both genes are functionally redundant, as vides launch pads for tagging most of the genes within neither single mutant produces a novel phenotype. the genome. However, shp1 and shp2 double mutants produce fruits or pods that do not shatter [45]. Since the A. thaliana genome-sequencing project is almost complete and the 7. GENE SEQUENCE sequences are available, it is feasible to identify all the TO FUNCTIONAL ANALYSIS closely related members of a gene family in this Once a knockout in a desired gene is identified species. Together with computer searches of flanking through reverse genetics strategies among the popula- sequence databases, it will soon be possible to con- tion of insertional mutants, it becomes necessary to struct combinations of mutants to reveal novel func- functionally characterize the mutant (figure 2). The tions that have gone undetected using the current first step in the characterization process is to obtain a genetics methodologies. mutant that has a single insertion in the gene of interest. If a two-component transposable element system (Ac/Ds for example) has been used in generat- 8. CONCLUSION ing a population of insertion mutants, then obtaining single insertion line is rather straight forward. On the In the post-genome era, sequences of many plant other hand, if multi-copy transposable elements are genomes will be available and functional assessment used to perform the reverse genetics screen, then it is of the genes identified will depend on many approaches necessary to do several out-crosses to make sure that including insertional mutagenesis, polymorphism only a single insertion is in the gene. The next step for analysis, expression microarrays, and bioinformatics. characterization is the identification of phenotypes A large population of insertional mutants generated by caused by gene knockout. If there is an observable transposon mutagenesis can be used to dissect out 250 S. Ramachandran, V. Sundaresan / Plant Physiol. 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