Structures of transgene loci in transgenic Arabidopsis plants obtained by particle bombardment: junction regions can bind to nuclear matrices.
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 6bp 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 4-6bp were present within 10bp 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.
Sawasaki
T
T
Graduate Department of Gene Science, Faculty of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima 724, Japan.
Takahashi
M
M
Goshima
N
N
Morikawa
H
H
D017360:Arabidopsis / Q000235:genetics*; D001665:Binding Sites; D003001:Cloning, Molecular; D018744:DNA, Plant; D005821:Genetic Techniques; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D012689:Sequence Homology, Nucleic Acid; D014026:Nicotiana / Q000235:genetics; D019076:Transgenes
D018744:DNA, Plant