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Plant Molecular Biology 23: 525-533, 1993. © 1993 Kluwer Academic Publishers. Printed in Belgium.

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Prospects of applying a combination of DNA transposition and site-specific recombination in plants: a strategy for gene identification and cloning Mark J.J. van Haaren 1 and David W. O w 2 1Department of Genetics, Institute for Molecular Biological Sciences, Biocentrum Amsterdam, De Boelelaan 1087, 1081 H V Amsterdam, Netherlands; 2Plant Gene Expression Center, U.S. Department of Agriculture, 800 Buchanan Street, Albany, CA 94710, USA Received 22 March 1993; accepted in revised form 15 July 1993

Key words: transposition, AciDs, site-specific recombination, Cre/lox, chromosomal rearrangements, deletion mutagenesis Abstract

The concept of gene identification and cloning using insertional mutagenesis is well established. Many genes have been isolated using T-DNA transformation or transposable elements. Maize transposable elements have been introduced into heterologous plant species for tagging experiments. The behaviour of these elements in heterologous hosts shows many similarities with transposon behaviour in Zea mays. Site-specific recombination systems from lower organisms have also been shown to function efficiently in plant cells. Combining transposon and site-specific recombination systems in plants would create the possibility to induce chromosomal deletions. This 'transposition-deletion' system could allow the screening of large segments of the genome for interesting genes and may also permit the cloning of the DNA corresponding to the deleted material by the same site-specific recombination reaction in vitro. This methodology may provide a unique means to construct libraries of large DNA clones derived from defined parts of the genome, the phenotypic contribution of which is displayed by the mutant carrying the deletion.

Introduction

A desirable goal in plant molecular biology is the isolation of genes that are only known for the mutant phenotype they cause upon mutation. Currently, map-based cloning, insertional mutagenesis and subtraction cloning are the three best developed strategies for such gene cloning experiments. In this article, we introduce a new theoretical approach for the identification and cloning of genes that is based on the combined use of DNA transposition and site-specific recombination. Although this approach is still in an

infancy stage of development, there is recent and considerable interest for its use in the generation of physically tagged deletions in the plant genome. A mutant phenotype arising from the deletion event will have delimited the location of the gene(s) in question and therefore allow for its isolation.

Map based gene cloning

One conventional approach has been based on prior knowledge of the genome location of the

526 gene. Successful cloning of a gene using this technique depends on the availability of molecular markers in the vicinity of the gene of interest. These markers, such as RAPD and RFLP markers that are obtained in many different ways, should eventually produce a high-density linkage map of the region of interest. By identifying the genetic linkage in segregation analysis between a mutant phenotype and a known marker, the region of interest corresponding to the mutant phenotype can be delimited to specific yeast artificial chromosome (YAC) or cosmid clones within a gene fragment library. Final verification entails the successful isolation of a gene by the subsequent complementation of a recessive trait by an introduced subfragment of the YAC or cosmid clone. Although this technique has proved to be successful in Arabidopsis thaliana [3, 7, 22] for which both a molecular and genetic map is available [42], high-density molecular maps are a prerequisite for successful map-based cloning experiments. Until such high-density maps are available, map-based cloning could be very labour-intensive and will be increasingly difficult as larger genomes are being studied.

set up transposon tagging experiments in hosts such as tobacco, tomato, Arabidopsis and rice [26]. Recently, using random tagging strategies, a petunia Ph gene was tagged by Ac [8], and A. thaliana MS2 and DRLI genes were tagged by En and Ds, respectively [1, 5]. The observed frequency of insertional inactivation in maize is low, ranging from 1 0 - 4 - 1 0 - 6 [17], which indicates the necessity to screen large numbers of independent transposition events. The most successful method of gene identification using a heterologous element thus far has been T-DNA-mediated gene tagging in A. thaliana. The transferred D N A (T-DNA) from the Agrobacterium tumefaciens tumour-inducing (Ti) plasmid integrates with little site specificity into the plant genome. At least in A. thaliana numerous instances have been reported for the isolation of a gene from a mutant whose aberrant phenotype is attributed to the insertion of a T-DNA [19, 32]. Commonly described phenotypes are recessive to the wild-type allele, consistent with a disruption of gene function. More recently, a strategy for activating silent genes by insertion of an enhancer near a gene has yielded a dominant trait [32, 61].

Insertional mutagenesis Genomic substraction Another promising strategy for the identification of genes is based on the disruption of gene function by insertion of a characterized DNA element. Classical examples are those whose gene function are disrupted or altered by the presence of a transposon. A number of genes have already been cloned in this manner from the genomes of Zea mays and Antirrhinum majus [8, 18, 29]. In plants where an indigenous transposable element has not been characterized, considerable efforts are being made to adapt heterologous elements for the same purpose. In this respect the maize Activator/Dissociation (Ac/Ds) transposable elements have received considerable attention. These elements have been shown to retain their transposition activity when introduced in a number of heterologous hosts. In many laboratories, the Ac/Ds two-element system is being utilized to

A third procedure that has shown promise is the genomic subtraction cloning strategy. This technique utilizes the minor differences between a homozygous deletion mutant and its wild-type relative. In this manner, Sun et al. [58] were able to clone the GA1 locus ofA. thaliana after conducting multiple rounds of hybridization between the mutant and wild-type DNA. This technique will be especially useful when isogenic deletion mutants are available, the induction of which is the goal of the strategy presented below. In the sections that follow, we present a strategy to induce deletions in the plant genome that is based on the combination of D N A transposition and site-specific recombination. This strategy incorporates many of the key features common to the three current approaches described

527 above plus a new technology being developed for manipulating plant genomes: the ability to recombine D N A at specific sequences introduced into the genome.

Site-specific recombination in plants Recently, several well characterized prokaryotic and lower eukaryotic site-specific recombination systems have been introduced in higher eukaryotes and have been shown to operate successfully in these new hosts. In plant and animal cells, functional site-specific recombination has been shown for the recombination systems from bacteriophages P1 (Cre-lox) [11, 34, 43, 47] and Mu (Gin-gix) [38], and from the inversion plasmids of Saccharomyces cerevisiae (FLP-frt) [23, 35, 37, 41, 45] and Zygosaccharomyces rouxii (R-RS) [46]. In each of these systems, no additional factor aside from the recombinase and target sequences is required for recombination. The four systems listed above are strikingly similar. Since activity of the Cre-lox system in plant cells is well established, we will confine further discussion to this system. It should be understood, however, that the same general principles apply regardless of which recombination system will be used. The Cre-lox site-specific recombination system of bacteriophage P 1 has been studied extensively in vitro and in Escherichia coli [2, 10]. Functionality has been demonstrated in yeast [55], mammalian [34, 47, 56] and plant cells [6, 11, 12, 43, 54]. Expression of the 38.5 kDa Cre protein is sufficient to cause recombination between 34 bp lox sites that consist of 13 bp inverted repeats separated by an 8 bp asymmetric spacer sequence. The result of a recombination reaction is dependent on the relative position of the recombination sites. Recombination of sites within the same chromosome will result in an inversion (recombination sites in inverted repeat) or a deletion (recombination sites in direct repeat) of the intervening DNA. When two recombination sites reside on different chromosomes of the same genome, a recombination event will result in a balanced translocation of chromosomes. Cre-

mediated recombination of two lox sites resulting in a translocation has been shown to occur [50, M. Qin et al., submitted]. These results open the way for the generation of reciprocal translocation libraries that can be useful in genetic analysis. The fact that Cre-mediated translocations are possible suggests that the recombination reaction can be completed even when the lox sites are separated over a considerable distance.

Transposition-deletion system To create the opportunity for deletion mutagenesis in plant genomes, we describe the feasibility of using a combination of site-specific recombination and transposition in plants. This system, based on the mobility of one of the recombination sites introduced into the plant genome, provides the possibility to induce several forms of genome rearrangements. As depicted in Fig. 1, an A. tumefaciens-transferred T-region harbouring a lox site and a Ds element carrying a second lox site is integrated into the plant genome. Transactivation of the Ds element with the Ac-transposase function will result in relocation of the Ds element, and the consequent separation of the two lox sites over different unknown distances on the same chromosome or on different chromosomes. Subsequent Cre-induced recombination between these separated recombination sites will lead to deletion, inversion or translocation of D N A segments, dependent on the relative orientation and chromosome location of the lox sites. The tendency of Ds elements to transpose over relatively short distances on the same chromosome [15, 16, 24, 30, 48, 53] suggests that most of the transposition-recombination events may result in the deletion or inversion of a D N A segment on the chromosome where the T-DNA resides. In contrast to insertional mutagenesis with transposable elements, the deletion mutants induced by the transposition-deletion system should be stable even if the Ae element remains present in the genome. This is because the deletion removes half of the Ds element, and the single remaining Ds end is unable to undergo further

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T-DNA

~ Transposition

Fig. 1. Schematic representation of the transposition-deletion system. The Ds transposable element (shaded oval) excises from the T-DNA (between brackets) and reintegrates into a new locus. After transposition of the Ds-element, the lox recombination sites (triangles) are separated by a certain distance on the same chromosome. Cre-induced recombination of the lox sites will then result in a chromosomal deletion and the excision of a circular D N A molecule.

transposition. In addition, Cre-induced recombination can no longer occur due to a single lox site remaining in the genome. Another advantage of chromosomal deletions over insertional gene inactivation is that the former is no longer dependent on precise integration of the transposon into the gene of interest. Insertions in the vicinity of the gene may also result in its deletion after Cremediated recombination, providing that Ds transposition leads to the flanking of the gene of interest with lox sites. This would mean that on average a smaller number of individual plants would have to be screened before a certain desirable phenotype would be obtained. In addition, deletions may allow for the identification of a gene that would otherwise not be susceptible to transposon or T - D N A insertional inactivation.

Selection for genomic recombination events Selectable markers can be used to monitor the occurrence of D N A rearrangement events. A deletion marker in combination with a transposon excision and integration marker, should permit the monitoring of the steps of the transpositiondeletion event. Different schemes for the selection of deletions utilizing negative and positive selectable marker genes can be envisioned. Negative selectable markers, such as the tms2 gene of A. tumefaciens [14] or the nitrate reductase gene [60], can be inserted into the T-region adjacent to the lox site, so that upon recombination this gene

Transposon deletion.

~¢ ~xdsion' ~ ~



~excision-I ~ Transposition ~

~

Recombination

Fig. 2. Schematic representation of a construct allowing selection for the occurrence of Ds transposition and Cre-loxinduced deletions. After excision of the transposon (between brackets), the promoter (P) will drive the expression of the excision marker. After subsequent recombination of the lox sites (triangles), the excision marker will be deleted and the promoter will drive the expression of the deletion marker.

becomes part of the deleted D N A segment. A positive selectable deletion marker can be used in a similar fashion as the excision marker for transposable elements [4, 51, 57]. When this approach is followed, the same promoter can be used to drive the expression of both the excision and deletion marker (see Fig. 2).

Chromosomal deletions The transposition-deletion system induces chromosomal deletions starting at the original site of T - D N A integration. From this site, deletions of

529 different length can be obtained depending on the integration site of the transposable element after its relocation (Fig. 1). A mutant phenotype arising from a deletion event delimits the location of the gene(s) responsible for this phenotype to the region between the T-DNA and the Ds reintegration site. After comparison of the phenotype of different deletion mutants derived from the same parent plant, the location of these gene(s) can be mapped more precisely. If, for instance, deletions larger than 100 kb induce the mutant phenotype and a deletion of 50 kb does not, then the genes of interest are deduced to be in the region between 50 and 100 kb away from the T-DNA. Mutant phenotypes caused by the deletion of large D N A segments can be the result of a combination of mutations that are added up in the phenotype. Complex phenotypes can be resolved in its constituent components by comparing the phenotypes of different overlapping deletion mutants. In theory, the availability of a deletion library could increase our ability to hunt for interesting mutants by at least an order of magnitude compared to T-DNA or transposon tagging strategies. The frequency of insertional inactivation of a particular gene (average length 5 kb) if random transposon integration occurs in a genome size of 10 6 kb would be ca. 0.5 x 10 5. Considering the possibility to induce deletions of an average size of 100 kb, the chance that a certain gene of interest becomes deleted would be 10 - 4. In tomato, with a haploid genome size of 0.7 x 10 6 kb, the number of functional genes has been estimated to be 60000 [31 ]. This could mean that a deletion of 100 kb in the tomato genome will on average result in the removal of nine genes.

of Cre to recombine sites on different chromosomes in tobacco [50, Qin etal., submitted]. Therefore, it is expected that deletions of 200 kb and larger are within the capability of site specific recombinases. Early cytogenetic studies on the tomato genome show that at least some large chromosomal deletions, up to complete chromosome arms (over 25 Mb), are tolerated [33]. In order to delete any specific part of the plant genome, it may be necessary to saturate the genome with lox containing T-DNAs. Calculations made for the tomato genome with an approximate genome size of 0.7 × 10 6 kb and an average deletion size of 100 kb in both directions, predicts that ca. 10000 insertions are needed to be able to delete a specific part of the genome with a probability of over 95~o. However, since large genomes contain a lot of repetitive DNA, it is likely that on average much larger deletions may be tolerated. If the average deletion extends to 250 kb, then the number of tomato transformants needed would drop to ca. 4000 (P