Integration of Agrobacterium T-DNA into a tobacco chromosome: Possible involvement of DNA homology between T-DNA and plant DNA

Abstract

We established tobacco tumour cell lines from crown galls induced by Agrobacterium. Restriction fragments containing T-DNA/plant DNA junctions were cloned from one of the cell lines, which has a single copy of the T-DNA in a unique region of its genome. We also isolated a DNA fragment that contained the integration target site from nontransformed tobacco cells. Nucleotide sequence analyses showed that the right and left breakpoints of the T-DNA mapped ca. 7.3 kb internal to the right 25 bp border and ca. 350 bp internal to the left border respectively. When the nucleotide sequences around these breakpoints were compared with the sequence of the target, significant homology was seen between the region adjacent to the integration target site and both external regions of the T-DNA breakpoints. In addition, a short stretch of plant DNA in the vicinity of the integration site was deleted. This deletion seems to have been promoted by homologous recombination between short repeated sequences that were present on both sides of the deleted stretch. Minor rearrangements, which included base substitutions, insertions and deletions, also took place around the integration site in the plant DNA. These results, together with previously reported results showing that in some cases sequences homologous to those in T-DNA are present in plant DNA regions adjacent to left recombinational junctions, indicate that sequence homology between the incoming T-DNA and the plant chromosomal DNA has an important function in T-DNA integration.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular and cytogenetic analyses of stably and unstably expressed transgene loci in tobacco.

“…The right T-DNA borders were better conserved (compared with the theoretical expectation based on the VirD2 nicking site) than were the left ones (Tinland and Hohn,1995). In line H9, the 6 bp of homologous sequence between plant DNA directly at the site of insertion and a short region outside of the left T-DNA border (consisting, in this case, of right T-DNA border sequences that were fused to the left border) is consistent with the short patches of plant DNA and T-DNA homology (7 to 14 bp) found for a number of other T-DNA integration events (Matsumoto et al, 1990;Ohba et al, 1995;Tinland, 1996).…”

An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration

“…T-DNA integration does not take place by homologous recombination, the most common method of foreign DNA integration in prokaryotes and lower eukaryotes, because no extensive homology between the T-DNA and target sequences has been found. T-DNA therefore integrates by illegitimate recombination (16)(17)(18)(19), the predominant mechanism of DNA integration into the genomes of higher plants (20)(21)(22). However, plant factors involved in illegitimate recombination of T-DNA into the plant genome have not yet been identified.…”

Presence of an SAR‐like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency

“…In the transgene integration in eukaryotic cells, a random, non-homologous recombination between the exogenous DNA and genome DNA has been postulated (Lewin, 1997;Matsumoto et al, 1990). Therefore, no consensus sequences in transgene loci in the genome are thought to be present.…”