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Intrachromosomal Homologous Recombination in Arabidopsis Induced by a Maize Transposon

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Intrachromosomal Homologous Recombination in Arabidopsis Induced by a Maize Transposon Mol Gen Genet (2000) 263: 22±29 Ó Springer-Verlag 2000 ORIGINAL PAPER Y.-L. Xiao á T. Peterson Intrachromosomal homologous recombination in Arabidopsis induced by a maize transposon Received: 28 September 1999 / Accepted: 19 November 1999 Abstract In plants, the frequency of spontaneous in- Introduction trachromosomal homologous recombination is low. Here, we show that a maize transposable element greatly Transposable element sequences make up a large por- stimulates intrachromosomal homologous recombina- tion of many eukaryotic genomes, and hence may be able tion between direct repeat sequences in Arabidopsis. to in¯uence genetic recombination in a number of ways. Plants were transformed with a construct (GU-Ds-US) For example, transposon insertions may have a negative containing a Ds (Dissociation) transposable element in- impact on recombination; in heterozygous condition serted between two partially deleted GUS reporter gene they constitute sequence heterologies that may interrupt segments. Homologous recombination between the pairing or branch migration of Holliday junctions overlapping GUS fragments generates clonal sectors (Dooner and Martinez-Ferez 1997; Biswas et al. 1998). visible upon staining for GUS activity. Plants containing Alternatively, transposable elements may provide dis- the GU-Ds-US construct and a source of Ac (Activator) persed sequence homologies that can participate in ec- transposase showed an over 1000-fold increase in the topic recombination (Montgomery et al. 1991; Clegg incidence of recombination relative to plants containing et al. 1997; Caceres et al. 1999). Moreover, transposable the same construct but lacking transposase. Transposon- elements may aect recombination more directly induced recombination was observed in vegetative and through the action of transposon-encoded proteins that ¯oral organs, and several germinally transmitted events cleave and rejoin DNA during the transposition process. were recovered. Transposon-induced recombination The possible eects of transposable elements on re- appears to be a general phenomenon in plants, and thus combination have been investigated since their discovery may have contributed to genome evolution by inducing by McClintock (1949). In E. coli,Tn3 was reported to deletions between repeated sequences. promote general recombination in neighboring regions (Kondo et al. 1989). Also, Tn7 transposition can create Key words Direct repeat á Recombination á a hotspot for homologous recombination at the trans- Transposon á Arabidopsis á b-Glucuronidase position donor site (Hagemann and Craig 1993). Inter- molecular transposition of IS10 greatly stimulates, and is coupled to, homologous recombination between the donor and acceptor molecules at the transposition site Communicated by H. Saedler (Eichenbaum and Livneh 1995). In Drosophila, the P element can signi®cantly increase recombination fre- Y.-L. Xiao quencies in the male germline (Hiraizumi 1971; Kidwell Interdepartmental Genetics Program, and Kidwell 1976) and somatic cells (Sved et al. 1990). Department of Zoology and Genetics, 2288 Molecular Biology Building, The P element also promotes ecient gene conversion, Iowa State University, Ames, IA 50010, USA which enables site-speci®c gene replacement and can T. Peterson (&) induce a variety of associated chromosomal rearrange- Interdepartmental Genetics Program, ments (Engels et al. 1990; Gloor et al. 1991; Preston and Department of Zoology and Genetics, Engels 1996; Preston et al. 1996). In humans, it was and Department of Agronomy, found that two inherited peripheral neuropathies are 2206 Molecular Biology Building, Iowa State University, Ames, IA 50010, USA caused by a recombination hot spot located near a E-mail: [email protected] Mariner transposon-like element (Kiyosawa and Chance Tel.: +1-515-294-6345; Fax: +515-294-6755 1996; Reiter et al. 1996). In maize, early reports indi- 23 cated that the transposable element Activator (Ac)orDs Recovery of the germinally transmitted recombination events either reduced (McClintock 1953), or had no eect on Seeds were harvested from the siblings of plants of the genotype (Fradkin and Brink 1956), crossing over in the region (GU-Ds-US/-, sAc/-) that gave a high frequency of blue spots upon ¯anking the element. However, it has been observed that staining for GUS activity. A portion of the seeds from each of 18 Ac insertions can induce homologous recombination plants were grown on MS plates until two true leaves had devel- between directly repeated sequences in the maize P locus oped, at which stage the plants were stained for GUS activity. If (Athma and Peterson 1991). Ac may also destabilize a any uniform blue seedlings were detected, the remaining sibling seeds were planted in soil. At the 4-leaf stage, one leaf from each tandem duplication in the maize Bz locus (Dooner and plant was stained for GUS activity. In the case of those plants Martinez-Ferez 1997), albeit at a much lower frequency which gave a uniformly staining leaf, a second leaf was picked and than that observed for the maize P locus. In addition, Ac stained for GUS activity. In this way, we identi®ed uniformly is reported to induce low levels of somatic recombina- GUS-positive plants which were used as a source of DNA for Southern analysis of the recombination event. tion between ectopic sites in transgenic tobacco (Shalev and Levy 1997). The aim of this study was to test directly the ability of PCR and Southern hybridization a transposable element to induce recombination between Genomic DNA samples (Dellaporta et al. 1983) were ampli®ed by homologous repeat sequences in plants. For this pur- PCR for 35 cycles of denaturation at 94 °C for 30 s, annealing at pose, we examined transgenic Arabidopsis plants con- 57 °C for 30 s, and elongation for 1 min at 72 °C. The primer taining a recombination substrate composed of a maize sequences used were: P1, 5¢-GAAGACTCAGACTCAGACT-3¢; Ds transposable element inserted between overlapping G1, 5¢-GGTGGGAAAGCGCGTTACAAG-3¢; H3, 5¢-CGTCT- GGACCGATGGCTGTG-3¢; H2, 5¢-TTCGGGGCAGTCCTCG- segments of a bacterial gene (uidA) encoding b-glucu- G-3¢; H1, 5¢-GATGTAGGAGGGCGTGG-3¢; D1, 5¢-GATCCG- ronidase (GUS). The results indicate that activation of GTTCTCTCCAAATG-3¢; S1, 5¢-CTGTCTGGCTTTTGGCTG- the Ds insertion by Ac transposase supplied in trans TG-3¢;X,5¢-GGATATTCTGCAACCCTTCCCCTCC-3¢; and Y, causes a high level of recombination between the 5¢-CTCGCAGGTATGTTTGTCTC-3¢. ¯anking repeat sequences. Recombination may be PCR products were cloned into the pT7Blue T-vector (Nov- agen) and sequenced at the ISU DNA Sequencing and Synthesis stimulated in part by a double-strand break induced by Facility. Probe preparations and Southern hybridizations were transposon excision, and possibly by additional performed as described. unknown properties of the Ac transposase. Results Materials and methods Detection of somatic recombination events Construction of the binary vector GU-Ds-US in transgenic plants Vector pWS31 (Sundaresan et al. 1995), obtained from Dr. We inserted a Ds element between two partially over- V. Sundaresan, was digested with SalI to remove the GUS gene lapping, non-functional segments of the b-glucuronidase fragment, then religated to form pWS31Y, which contains a Ds element with NPTII as selection marker. Plasmid pWS31Y was gene (gus; Jeerson et al. 1987) to generate a binary cleaved with SacI to release the 4.7-kb band containing Ds,to plant transformation vector termed GU-Ds-US (Fig. 1). which SacI-PstI linker fragments (synthesized by the Sequencing The homologous direct repeat sequences are 618 bp in and Synthesis Facility at ISU) were ligated. Plasmid pGU.US length, and the distance between them is 6.3 kb, in- (Tinland et al. 1994) was cleaved with PstI and ligated to the Ds element with attached SacI-PstI linkers to generate GU-Ds-US. cluding the 4.7-kb Ds element. Recombination between Restriction enzyme digestion, ligation and plasmid preparation the direct repeats in the GU and US segments would were performed according to standard protocols and enzyme generate a functional GUS gene driven by a strong manufacturers' instructions (Sambrook et al. 1989). constitutive (CaMV 35S) promoter. A. thaliana ecotype Columbia was transformed with the GU-Ds-US con- Plant transformation and histochemical assays struct by vacuum in®ltration (Bechtold et al. 1993). The original transformed plants were allowed to self-polli- Arabidopsis thaliana ecotype Columbia was transformed by vac- nate, and progeny were analyzed by Southern hybrid- uum in®ltration (Bechtold et al. 1993) with Agrobacterium strain ization (data not shown). Three independent transgenic ASE (obtained from E. Meyerowitz) containing the binary vector GU-Ds-US, and transformed seeds were selected by growth on starter lines with single-copy GU-Ds-US transgene in- agar media containing 30 lg/ml kanamycin. Kanamycin-resistant sertions were selected for analysis. Plants homozygous lines were further screened by progeny testing and Southern for the GU-Ds-US transgene were crossed with lines analysis to identify three independent lines (DsI5, DsI6, DsII7) expressing the Ac transposase in the No-O (CS 8037 and that carried a single integrated GU-Ds-US transgene. Ac trans- posase lines (CS8037, CS8038, CS8045) were obtained from the CS 8038) and Landsberg (CS 8045) backgrounds. As a Arabidopsis Biological Resource Center (ABRC, Columbus, control, the transgenic GU-Ds-US plants were also Ohio). Plants were grown as described by Koncz et al. (1992). crossed with
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