Single-site integration of foreign DNA into Arabidopsis showed
rearrangements and deletions of both plant DNA and foreign DNA.
Arabidopsis thaliana (Thale Cress; mustard family).
Various gene constructs containing in each case a neomycin
phosphotransferase marker gene and one or more different reporter genes.
Agrobacterium was used as a vector.
Goal of This Study:
Investigate how a transgenic gene construct is integrated into the plant's
Results of This Study:
Initial screening indicated that in 80% of the
transgenic lines the gene construct had been inserted at a single site
(locus). About 22% of these contained a single intact copy of the
gene construct. From this group, 122 lines were selected for further
investigation, since the gene construct was of "sufficient length and
quality to determine unambiguously the position of integration in the
Arabidopsis genome" (p. 165). In connection with these 122 lines,
the researchers found:
The researchers summarize: "In the majority of cases, single-copy T-DNA
[that is, the transgenic construct] were associated with small or large
rearrangements such as deletions and/or duplications of target site
sequences, deletions and/or duplications of T-DNA sequences and gross
chromosomal rearrangements such as translocations" (p. 161).
The gene construct was identified in all five of the plant's chromosomes;
there was no apparent bias toward any particular chromosome.
Over half of the insertions were directly upstream or downstream from
plant genes (where regulatory DNA sequences usually lie) or within plant
genes. The rest were in non-coding regions.
Only three of 71 lines investigated contained a gene construct that
corresponded precisely to the full-length strand of DNA in the originally
inserted construct; in all the others there were small deletions at one
or both ends of the sequence.
In 88.7% of the lines, the integration of the gene construct resulted in
deletions of plant DNA at the site of integration. Only 2.7% "inserted
exactly into the target site" (p. 168).
In over half the lines, short "filler sequences" of DNA were found
between the borders of the construct and plant DNA that flanked those
borders. In those cases in which the origin of this filler DNA could
be identified, it was found to consist of DNA fragments from either the
plant or the transgenic construct.
In two of the lines "extensive chromosomal rearrangements associated with
the integration" (p. 169) of the gene construct were found. Chromosomal
fragments were swapped between different chromosomes (reciprocal
The authors point out that the frequency of DNA
rearrangements "is likely to be even higher if transgenic lines
containing multiple T-DNA inserts are analyzed" (p. 173), and in this
regard refer to other research.
Forsbach, A., D. Schubert, B. Lechtenberg, M. Gils et al. (2003).
"A Comprehensive Characterization of Single-Copy T-DNA Insertions
in the Arabidopsis thaliana Genome," Plant Molecular Biology
vol. 52, pp. 161-76.
Max-Planck Institut für Molekulare Pflanzenphysiologie,
Golm, Germany; Coley Pharmaceutical, Langenfeld, Germany; Icon Genetics,
Halle (Saale), Germany.
German Ministry for Education and Research (BMBF) and fellowships
from the Max Planck Society.
basic research; not related to the development of a
potential commercialized product.
Copyright 2008 The Nature