Integration of foreign genes by homologous recombination

The era of plastid transformation enables the detailed study of recombination events in plastids. In algae and plants foreign DNA integrates by homologous recombination between common DNA sequences in the transforming vector and resident plastid genome. Large regions of donor plastid DNA integrate into the resident plastid genome well beyond the markers used to select transformants resulting in incorporation of all or almost all of a 6.2 kb recombinant plastid sequence in N. tabacum plastid transformants (Staub and Maliga 1992). Reciprocal recombination or gene conversion events between transforming plasmid and resident plastid genome will result in transgene integration (Fig. 10a). A variety of plastid DNA sequences have been used to target integration of foreign genes to different sites in the plastid genome (Chapter 14). This indicates the plastid recombination machinery is not limited to specific substrates but can act on a wide selection of DNA sequences. When a vector containing a gene-of-interest flanked by targeting DNA is introduced into angiosperm plastids, double recombination events in both arms will insert the transgene into the plastid genome. Alternatively, the entire plasmid can integrate as a result of homologous recombination in

Fig. 9. Double rolling circle model of DNA replication gives rise to large circular multimers from a single round of replication initiation (Futcher 1986). Not to scale: the small single copy region is enlarged to illustrate the model. O= replication origin.

one flanking region of plastid DNA. The resulting co-integrate contains duplications of left and right targeting regions. The co-integrate can be selected by placing the marker gene in the vector sequences (Klaus et al. 2004). When selection is removed further homologous recombination events between these duplicated sequences excise vector and marker sequences to leave either the gene-of-interest or

Plastid Genome

Fig. 10. Integration of foreign DNA into the plastid genome by homologous recombination. a) Integration requires homology and involves reciprocal recombination and possibly gene conversion events. b) Integration of homeologous DNA reveals multiple crossover events between target and donor DNA (Kavanagh et al. 1999).

Plastid Genome

Fig. 10. Integration of foreign DNA into the plastid genome by homologous recombination. a) Integration requires homology and involves reciprocal recombination and possibly gene conversion events. b) Integration of homeologous DNA reveals multiple crossover events between target and donor DNA (Kavanagh et al. 1999).

WT plastid sequences (see Chapter 14; Klaus et al. 2004). These co-integrate experiments show that the crossover events in both arms required for integration are not tightly linked and this integration pathway must reflect properties of the DNA-RRR pathways in angiosperm plastids.

In E. coli, homologous recombination is stimulated by 8 base 5'GCTGGTGG chi sequences which are recognised by the recBCD complex (Kowalczykowski 2000). The eight base chi motif is absent in N. tabacum plastids (Shinozaki et al. 1986) but is present in the 16S ribosomal RNA genes in grass plastid genomes (Hiratsuka et al. 1989). Any role for chi sequences in plastids would appear to be ruled out by the finding that cyanobacterial and angiosperm genomes do not appear to contain homologues of genes encoding the recBCD complex. In C. reinhardtii, sequences that appear to stimulate recombination have been identified within the large inverted repeat and have been localised to a 400 bp region of plastid DNA containing the 3' end of the psbA gene (Newman et al. 1992).

Was this article helpful?

0 0

Post a comment