Biomedical subjects

Inderjit S Mercy

Publications and source records attributed to Inderjit S Mercy.

2 recordsLinked to original sources

PubMed2006

An inverted repeat transgene with a structure that cannot generate double-stranded RNA, suffers silencing independent of DNA methylation.

Transgene silencing in plants is most often dependent on homologous sequences, e.g. tandemly repeated T-DNAs. We have identified an Arabidopsis line (ex2-4 line 4) displaying silencing of the T-DNA-born nptII gene. This line contains a truncated copy of the T-DNA encompassing the nptII gene with its nos promoter adjacent to an intact T-DNA copy. The orientation of the intact and the truncated copies preclude the generation of a double-stranded nptII transcript. Therefore, we have investigated the genomic landscape surrounding T-DNA insertion in the silenced ex2-4 line 4 and five single-copy ex2-4 lines without silencing in search of features that might explain the silencing phenomenon. GC content, putative matrix-attachment regions and transcriptional interference from neighbouring genes could all be ruled out as major causes of silencing. Bisulphite sequencing revealed de novo methylation of the nos promoter both in non-silenced and silenced plants of this line, thus silencing was not correlated to DNA methylation level. Also, the methylation pattern deviated from that characteristic for RNA-mediated DNA methylation and silencing. Our data therefore suggest that ex2-4 line 4 represents a case where silencing is due to DNA-DNA pairing, i.e. pairing between the intact T-DNA and the adjacent truncated, inverted T-DNA copy.

Arabidopsis↗

PubMed2002

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.

Arabidopsis↗