Analyses of single-copy Arabidopsis T-DNA-transformed lines show that the presence of vector backbone sequences, short inverted repeats and DNA methylation is not sufficient or necessary for the induction of transgene silencing.
In genetically transformed plants, transgene silencing has been correlated with multiple and complex insertions of foreign DNA, e.g. T-DNA and vector backbone sequences. Occasionally, single-copy transgenes also suffer transgene silencing. We have compared integration patterns and T-DNA/plant DNA junctions in a collection of 37 single-copy T-DNA-transformed Arabidopsis lines, of which 13 displayed silencing. Vector sequences were found integrated in five lines, but only one of these displayed silencing. Truncated T-DNA copies, positioned in inverse orientation to an intact T-DNA copy, were discovered in three lines. The whole nptII gene with pnos promoter was present in the truncated copy of one such line in which heavy silencing has been observed. In the two other lines no silencing has been observed over five generations. Thus, vector sequences and short additional T-DNA sequences are not sufficient or necessary to induce transgene silencing. DNA methylation of selected restriction endonuclease sites could not be correlated with silencing. Our collection of T-DNA/plant DNA junctions has also been used to evaluate current models of T-DNA integration. Data for some of our lines are compatible with T-DNA integration in double-strand breaks, while for others initial invasion of plant DNA by the left or by the right T-DNA end seem important.
Meza
Trine J
TJ
Division of Molecular Biology, Department of Biology, University of Oslo, PO Box 1031 Blindern, N-0315 Oslo, Norway. t.j.meza@bio.uio.no
Stangeland
Biljana
B
Mercy
Inderjit S
IS
Skårn
Magne
M
Nymoen
Dag A
DA
Berg
Anita
A
Butenko
Melinka A
MA
Håkelien
Anne-Mari
AM
Haslekås
Camilla
C
Meza-Zepeda
Leonardo A
LA
Aalen
Reidunn B
RB
D017360:Arabidopsis / Q000235:genetics*; D001483:Base Sequence; D002461:Cell Line, Transformed; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000378:metabolism; D018628:Gene Dosage; D018506:Gene Expression Regulation, Plant; D020868:Gene Silencing; D005822:Genetic Vectors; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D017384:Sequence Deletion; D014170:Transformation, Genetic; D019076:Transgenes
D004269:DNA, Bacterial; D018744:DNA, Plant; C036483:T-DNA