Integration of Agrobacterium tumefaciens T-DNA in the Saccharomyces cerevisiae genome by illegitimate recombination

Abstract

Agrobacterium tumefaciens can transfer part of its Ti plasmid, the T-DNA, to plant cells where it integrates into the nuclear genome via illegitimate recombination. Integration of the T-DNA results in small deletions of the plant target DNA, and may lead to truncation of the T-DNA borders and the production of filler DNA. We showed previously that T-DNA can also be transferred from A. tumefaciens to Sac-charomyces cerevisiae and integrates into the yeast genome via homologous recombination. We show here that when the T-DNA lacks homology with the S. cerevisiae genome, it integrates at random positions via illegitimate recombination. From 11 lines the integrated T-DNA was cloned back to Escherichia coli along with yeast flanking sequences. The T-DNA borders and yeast DNA flanking the T-DNA were sequenced and characterized. It was found that T-DNA integration had resulted in target DNA deletions and sometimes T-DNA truncations or filler DNA formation. Therefore, the molecular mechanism of illegitimate recombination by which T-DNA integrates in higher and lower eukaryotes seems conserved.

Figure 1

Integration of nonhomologous T-DNA into the genome of S. cerevisiae is random at the chromosome level. (A) A CHEF gel showing the separated chromosomes of an untransformed colony of S. cerevisiae strain RSY12. Each chromosome is indicated. (B) Chromosomes from Ura⁺ strains obtained after cocultivation of LBA1126(pRAL7102) with RSY12 were separated on a CHEF gel and blotted to a nylon membrane. The blot was probed with a labeled 1.1-kb HindIII URA3 fragment. Lane 1, RSY12 (not cocultivated); lane 2, Ura⁺ S. cerevisiae M5-1a strain obtained after cocultivation of M5-1a with LBA1100(pRAL7100). The T-DNA has integrated via a double crossover on chromosome V (11). Lane 3, as in lane 2, but the whole binary vector pRAL7100 has integrated via a single crossover on chromosome V, causing a shift in chromosome V mobility. Lanes 4–11, RSY12 Ura⁺ strains obtained after cocultivation of RSY12 with LBA1126(pRAL7102).

Figure 2

Nucleotide sequences of yeast target sites and the insertion points of the T-DNA 3′ end [left border (LB)] and 5′ end [right border (RB)]. The diagram at the top of the figure presents the rationale used to present the sequence data. All the sequences are shown in the 3′ to 5′ orientation. The line marked “T-DNA” shows the termini of the processed bottom strand of the T-region that constitutes the T-DNA. This DNA is transferred to the yeast cell during cocultivations. The extent of T-DNA border truncation found in each strain after T-DNA integration can therefore be calculated by comparison of the integrated borders with the intact T-DNA. Yeast target DNA is shown in lowercase letters, and deleted yeast DNA is shown in boldface type. Filler DNA is shown in lowercase, underlined, italic type. The rescued and sequenced T-DNA borders are shown on the line above (the LB) and below (the RB) the yeast target sequence. The T-DNA borders are positioned to correspond with the left and right ends of the deleted yeast DNA. Numbers on the same line as the T-DNA border sequences indicate the number of bases lost from that T-DNA border. The T-DNA in strain 10 was fused to mitochondrial sequences, and we were therefore unable to determine the extent of the target DNA deletion.