Gene 218 (1998) 27–35 Structures of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: Junction regions can bind to nuclear matrices Tatsuya Sawasaki 1, Misa Takahashi, Naoki Goshima, Hiromichi Morikawa * Graduate Department of Gene Science, Faculty of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 724, Japan Received 19 January 1998; received in revised form 24 June 1998; accepted 15 July 1998; Received by H. Uchimiya Abstract To clarify the molecular structure of the integration sites of transgenes, we used particle bombardment to examine the DNA sequences of transgene loci. Three transgenic Arabidopsis lines gave a single Southern hybridization band with a selectable gene as the probe. Junction regions flanked by the transgenes were cloned by the inverse polymerase chain reaction method, and the characteristics of the DNA sequences of the 10 junction regions were investigated. All but two of these were AT-rich sequences bearing motifs characteristic of a scaffold/matrix-attachment region (S/ MAR). Calculations showed that seven of them should have a propensity for curvature. An assay of in-vitro binding to tobacco nuclear matrices showed that all the junction regions bound to nuclear matrices and that the two input DNAs did not bind. The 12 chromosome/transgene (CT ) junctions in these three transgene loci were investigated. Cleavage sites for topoisomerase I were found at 10 of the 12, near the junction point. The other two junctions had sites within 6 bp of the junction point. The sequence near one terminal of the transgene in the transgene loci was compared with that near the other terminal. Short, direct repeats consisting of 4–6 bp were present within 10 bp of the junction points in the sequence. We speculate that the transgene introduced by particle bombardment is delivered on AT-rich S/MAR that has a propensity for curvature, and then a nucleotide near the short, direct repeat on the transgene is joined near the cleavage sites on the genome for topoisomerase I. © 1998 Elsevier Science B.V. All rights reserved. Keywords: AT-rich region; Hot spot; Integration; S/MAR; Topoisomerase I; Transgene 1. Introduction for the analysis of genes and for practical uses. Bombardment-mediated transformation has been shown Gene transfer techniques, including particle bombard- to be the most versatile and effective way for creating a ment, are important tools in genetic engineering both variety transgenic organisms that include microorga- nisms, mammalian cells, and a large number of plant * Corresponding author. Tel: +81 824 24 7449; Fax: +81 824 24 0749; species. We previously reported transformants of e-mail: hmorikaw@sci.hiroshima-u.ac.jp Arabidopsis thaliana (Seki et al., 1991; Sawasaki et al., 1 Present address: Department of Applied Chemistry, Faculty of 1994; Takahashi and Morikawa, 1996). There is, how- Engineering, Ehime University, Matsuyama 790, Japan. ever, no information on the integration mechanism or Abbreviations: A, adenosine; ARS, autonomous replication the site of integration of transgenes introduced by sequence(s); ATP, adenosine triphosphate; bar, gene coding phosphi- particle bombardment. nothricin acetyltransferase; bp, base pair(s); C, cytidine; CEN4, The eukaryotic genome in the nucleus is thought to the minimal centromere domain of chromosome 4; CT, chromosome/transgene; CTAB, cetyltrimethyl ammonium bromide; form a chromatin structure that has important roles dNTP, deoxyribonucleoside triphosphate; EDTA, ethylenediaminetet- in replication, transcription, and repair processes. raacetic acid; EST, expressed sequencing tag; G, guanosine; hpt, gene According to the current model, chromatin is organized coding hygromycin phosphotransferase; IPCR, inverse polymerase in chromosomal loop domains, the basal parts of which chain reaction; kb, kilobase(s) or 1000 bp; NADH, nicotinamide-ade- nine dinucleotide reduced form; nt, nucleotide(s); PCR, polymerase are attached to a proteinaceous scaffold, also referred chain reaction; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium to as the nuclear matrix. Accordingly, the responsible dodecyl sulfate; S/MAR, scaffold/matrix-attachment regions; T, thym- DNA sequences have been called ‘scaffold-attachment idine; TAE, 4.84 g Tris base/1.142 ml glacial acetic acid/2 ml 0.5 M regions’ (SARs) or ‘matrix-attachment regions’ EDTA (pH 8.0) per liter; T-DNA, transfer DNA; tRNA, transfer RNA; W, adenosine, or thymidine. ( MARs). These S/MAR elements insulate the chromatin 0378-1119/98/$19.00 © 1998 Elsevier Science B.V. All rights reserved. PII: S0378-1119(98)00388-6