There are many well described site-specific recombination systems, but none is operationally simpler than systems that rely on a single-polypeptide recombinase to cause the specific reciprocal exchange between two short identical DNA sequences [1] [2]. Three such systems currently being deployed in plant research are known as Cre-lox, FLP-FRT, and R-RS, where Cre, FLP and R are the recombinases, and lox, FRT and RS are the recombination sites. All are of microbial origin: Cre-lox is from bacteriophage P1, FLP-FRT is from the 2µ plasmid of Saccharomyces cerevisiae, and R-RS is from the pSR1 plasmid of Zygosaccharomyces rouxii. To be functional, lox and FRT sites require, at the minimum, an 8 bp core sequence between 13 bp inverted repeats, and RS sites must have a 7 bp core flanked by 12 bp inverted repeats. The asymmetric core sequence determines the site's orientation, and thus the types of recombination product.

Many applications of site-specific recombination have been reviewed recently [3•] [4•]. In this update, I make brief mention of the application of recombinases to transgene engineering; however, greatest emphasis is placed on the current trend for the use of site-specific recombination in plants, the manipulation of chromosomes.

Rearrangement of transgenes

Regardless of whether recombination sites are placed within the same DNA molecule in direct or opposite orientation, or placed on unlinked linear or circular DNA molecules, the corresponding recombinase can catalyze the reciprocal exchange to produce a deletion, inversion, translocation or co-integration event. The reactions are reversible, but the recombination of linked sites is kinetically more favorable than that of sites that are unlinked. Thus, the excision and inversion of transgenes are inherently easier to obtain than the stable co-integration of two molecules. The kinetic argument also predicts that the probability of a recombination reaction would be higher between closely linked sites than between sites separated by great distances. Consequently, site-specific deletion and inversion of transgenes harboring closely linked recombination sites were the first reactions to be demonstrated in higher eukaryotes.

In plants, many variations of site-specific deletion/inversion of transgenes to activate or deactivate gene expression have been described using the Cre-lox [5] [6] [7] [8] [9] [10•] [11], the FLP-FRT [12] [13] [14•] [15] or the R-RS system [16] [17]. More recently, site-specific integration of circular DNA into the tobacco genome through Cre-lox recombination has also been reported [18••]. Pairs of different mutant lox sequences were used such that the recombination reaction produced one non-functional product site. The lack of two fully functional lox sequences impedes subsequent excision of the integrated molecule.

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Rearrangement of chromosomes

Chromosomal rearrangements are most often revealed by aberrant phenotypes resulting from anomalous expression of the displaced genes. These aberrations have important genetic, evolutionary and medical consequences. Induced chromosomal rearrangements can be obtained by radiation or chemical mutagenesis. Identification of aberrant chromosome structures requires cytogenetic analysis, which makes the screening of large numbers difficult. Moreover, this method of inducing rearrangements lacks predictability and often causes additional mutations. A different approach to manipulating genome structure makes use of site-specific recombination systems. Recombination sites within a host genome provide targets for chromosomal recombination and molecular tags for analysis. This approach was first described in S. cerevisiae diploids, where R-RS site-specific recombination produced a translocation, an inversion and a deletion [19]; large chromosomal aberrations were not tolerated in haploid cells. It is possible that plants with high ploidy levels may be more tolerant of chromosomal rearrangements.

Inter-chromosomal recombination

In higher plants, recombinase-mediated chromosomal translocations have been produced in the tobacco Nicotiana tabacum [20••]. With Agrobacterium-mediated transformation, transgenic lines hemizygous for either a lox–hpt or a P–lox–cre construct were generated. The lox–hpt transgene has a 34 bp lox site incorporated into the transcriptional leader of a promoterless hpt, the coding region for hygromycin phosphotransferase. The P–lox–cre construct expresses cre from P, a plant viral promoter. The two types of transgenic line were crossed to produce progeny containing both the lox–hpt chromosome and the P–lox–cre chromosome. Cre causes a reciprocal exchange of chromosome arms at the lox sites, resulting in new transgenes: P–lox–hpt and lox–cre. The fusion of P to hpt confers a hygromycin-resistant (Hyg^R) phenotype, and a promoterless lox–cre minimizes reversal of the reaction by terminating cre expression. Providing that the two lox sites are situated in the same direction with respect to the centromeres, this exchange results in a balanced translocation (Fig. 1). As genetic material is conserved, these plants are likely to be viable. On the other hand, if the two lox sites are oriented in an opposing direction with respect to the centromeres, the translocation produces dicentric and acentric chromosomes and inviability is the likely result.

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Crosses between lox–hpt and P–lox–cre lines yielded progeny that gave rise to Hyg^R plants [20••]. Molecular analyses indicated that the Hyg^R plants have the newly acquired P–lox–hpt and lox–cre junctions and lack the parental P–lox–cre and lox–hpt junctions. DNA hybridization analysis showed the exchange of flanking DNA. Transmission of the chromosomes containing the translocations was determined in the progeny of an outcross to wild-type plants. As translocation results in heterozygotes, the first meiotic division produces gametes that harbor both normal chromosomes, both translocated chromosomes, or a normal chromosome with a translocated chromosome in a 1:1:2 ratio (see Fig. 1). The last class, with its associated duplications and deficiencies, would less likely be viable. Transmission of the two transgenes, lox–cre and P–lox–hpt, and by inference their respective chromosomes, was determined by DNA hybridization. Consistent with balanced genomes having higher viability rates, co-transmission rates were 67%, 80%, 90% and 100% in the translocation lines. Cases where co-transmission frequencies were below 100% may result from the allotetraploid nature of N. tabacum, which can tolerate the loss of certain chromosomes.

Recombinase-mediated chromosomal translocations have also been generated in animal cell culture. Smith et al. [21••] and Van Deursen et al. [22••] have reported Cre-lox-mediated reciprocal chromosomal translocations in mouse embryonic stem cells. A major advantage of animal over plant model systems is the availability of homologous recombination, which can direct the placement of recombination sites to cause the pre-determined genome aberration.

Intra-chromosomal recombination

To generate internal chromosomal inversions and deletions, the intervening DNA must be flanked by a pair of recombination sites. This is difficult to achieve through the random placement of transgenes. Unlike some fungal and animal systems, the insertion of transgenes through homologous recombination in plants is currently not a practical option. Therefore, a rather elaborate scheme has been devised to place two recombination sites in cis [23]. The generic scheme is illustrated in Fig. 2. A dissociation element (Ds), a non-autonomous derivative of the plant activator element transposon (Ac) [24], is used to carry a recombination site. A second recombination site is located outside the Ds element. To facilitate detection of Ds transposition, the Ds derivative is inserted between the promoter and the coding region of a selectable marker such that excision allows marker expression. Ac transposase is introduced through a sexual cross to transactivate excision of Ds. Because Ac and Ds transpose preferentially to linked sites, transposition of the Ds element to a nearby location will flank a DNA segment with recombination sites. Depending on the relative orientation of the two recombination sites, deletion or inversion of the intervening DNA segment will occur when the recombinase is introduced through sexual hybridization.

Variations of this generic scheme have been tested by several laboratories using the Cre-lox system ([25••] [26]; M van Haaren, personal communication) or the R-RS system [27••]. In N. tabacum, our group [25••] has detected a deletion and three independent inversion events. Yet, only an inversion between two lox sites ~ 10 cM apart passed to progeny without the co-transmission of a cre gene. A second inversion was passed to progeny, but only when co-transmitted with an unlinked cre gene. This suggests that the inversion was not viable in the gamete, but could be somatically generated again in the next generation by Cre-mediated recombination. The remaining inversion and deletion lines failed to transmit the rearrangement to the next generation. The strategy used by Osborne et al.[26] detects only rearrangements that transmit to progeny. In their work on Arabidopsis, two inversions, flanked by lox sites separated by 5.6 cM and 16.5 cM, were obtained. Machida et al. [27••] have employed a strategy in Arabidopsis where two RS sites of opposite orientation are placed at the origin and in the Ds element. Consequently, only deletions (not inversions) can be generated stably. In 18 out of 38 lines, the Ds element was found transposed within 800 kb of the origin. So far, one deletion spanning 12 kb of DNA has been obtained, but this failed to transmit to progeny.

Chromosomal recombination in vitro

Cre-catalyzed and FLP-catalyzed reactions in vitro on small DNA substrates are well documented. The concept of manipulating megabase-sized DNA segments through site-specific recombination is thus tempting. Parental lines leading to deletions and inversions are marked by flanking recombination targets. Theoretically, it should be possible to excise the intervening DNA through recombination between chromosomally situated and exogenously provided recombination sites.

This concept has been tested on the 5.7 Mb chromosome I of the fission yeast Schizosaccharomyces pombe [28••]. Homologous recombination was used to place lox sites at ade2 and swi4 (Fig. 3). Spheroplasts embedded in agarose beads were lysed to release the chromosomes, and Cre protein and lox-DNA were infused into the beads. The products of recombination between the lox-DNA and the genomic lox sites were resolved by pulsed-field gel electrophoresis. When the reaction was performed on a strain that harbored lox sites in both loci, the 0.92 Mb ade2-to-telomere segment, the 3.0 Mb swi4-to-telomere segment, and the 1.8 Mb ade2-to-swi4 intervening segment were detected.

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Conclusions and prospects

The proof-of-concept studies described above represent a modest beginning for manipulating plant chromosomes through site-specific recombination. Unlike some fungal and animal systems, where targeted insertions are practical for the site-specific introduction of recombination sites, plant biologists do not currently have the option of selecting the exact rearrangement to generate. Until DNA insertion via homology becomes practical — and progress has recently been made in this area [29] [30] [31] [32•] — exploitation of the powers of recombinase-directed genome restructuring will be restricted. Recombination sites in the genome would be most useful if a large collection of plant lines harboring mapped sites existed, such that suitable sites for a particular rearrangement could be chosen. As transgenic plants can be generated in large numbers, it is possible to imagine libraries of random rearrangements in a plant genome.

For the past two years, our group has been assessing whether the construction of a translocation library in a model plant is a realistic goal. A. thaliana was chosen as the model because of its rapid generation time, small genome size, and small number of chromosomes. A large number of lox–hpt and P–lox–cre transgenic lines were generated. As recombination between unlinked sites is infrequent, the F₁ progeny bearing both the P–lox–cre and the lox–hpt chromosomes were selfed to provide an additional generation for germline recombination. Thus far, from nine P–lox–cre and 20 lox–hpt transgenic lines, 39 crosses have produced Hyg^R F₂ progeny that harbor the P–lox–hpt junction. Mapping of the breakpoints of these putative translocation lines is under way. On the basis of progress thus far, it appears that a modest translocation library in a model plant will be achieved within the year. All indications suggest that the generic strategy can be readily adopted for crop plants.

The frequency of generating inversions or deletions is very low. Partly, this is the result of the time and labor associated with using a combination of transposon and recombination systems. Construct designs could be improved, but an inherent problem is that certain rearrangements, particularly deletions, may be difficult to transmit through gametes. This may be less of a problem with species having high ploidy levels.

Site-specific cleavage of chromosomes in vitrocan be readily achieved through the use of rare-cutting endonucleases. However, site-specific recombination may allow the insertion of a retrieved DNA segment directly into a carrier molecule, such as a bacterial artificial chromosome vector. A speculative, but most exciting, possibility is chromosomal cleavage in vivo at selected times during development. For example, plants harboring the P–lox–cre transgene contain Cre. If lox oligonucleotides were introduced into these plants, endogenously supplied Cre could recombine the genomic and exogenously provided lox sequences to split the chromosome. The phenotype associated with the cell lineage dependent loss of a particular chromosome arm could then be observed.

The random shuffling of large blocks of genomic DNA has evolutionary consequences, much the same as those described for the pioneering work on Drosophila ananassae, where inversions and translocations are associated with speciation [33]. Similar to findings in animals, plant genome research is revealing an amazing amount of structural conservation. Plant genomes also appear to be organized into a number of discrete blocks of genetic information, where the order of markers and genes within these linkage blocks are conserved in distantly related species [34] [35] [36]. The major cereals, for instance, appear to be put together with the same set of basic linkage blocks, but in different arrangements [37••]. It is as though primary differences among them lie more so in the order of these genomic blocks than in the novelty of particular genes. This begs the following question: does the shuffling of genomic blocks through site-specific recombination, within and maybe even between plant species, facilitate the evolution of plant diversity?

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