Rice Genetics IV - IRRI books - International Rice Research Institute

sertion of an adventitious DNA fragment. Such events are thought to be responsiblefor the generation of processed pseudogenes, which make up a significant componentof many higher eukaryotic genomes (Berg and Howe 1989). The direct repeats wouldin this case represent repair synthesis over the staggered break, generating the “targetsite duplication” characteristic of transposition events in all species, including theintegration of T-DNA into the plant genome (Tinland 1996). Gorbunova and Levy(1997) found insertions ranging from 2 bp to 1.2 kbp in nearly 30% of the plasmidjunctions they studied. This so-called “filler DNA” was sometimes genomic in origin,sometimes it appeared to derive from the transforming plasmid, and in othercases the origin was uncertain. The similarities between recombination junctions generatedby particle bombardment in our study and those generated by alternative transformationmethods in both monocots and dicots in other studies strongly suggest thatthe underlying mechanisms controlling plasmid rearrangement and transgene integrationin plants are likely to be the same. The evidence we have provided suggeststhat several categories of illegitimate recombination are likely to be involved in thisprocess, although microhomology-mediated recombination predominates (Kohli etal 1999; Fig. 3).Anos terminator (gusA) Backbone Ubi-1 promoter (bar)65.....GTTACTAGATCGGGAATTCAAGCTTGCATGCCTGCAGT.....306...GGAATTCACAAAGCTTGCAG............CTCCGATTCACTGCAGTGCAG..Maize α-zein geneBCDnos terminator 37-493.....GTTACTA..........ATGGTTCTCCACTTCGATAAACG......Ubi-1 promoter 834-568nos terminator 37-286.....GTTACTAGATCGCGAAAACTGTGGAATT..........gusA coding sequencenos terminator 37–286.....GTTACTAGATCCGATGATAAGCTGTCAAA..........Plasmid backboneFig. 3. Microhomology-mediated recombination in transgenic maize. The figure showstransgene junction sequences from transgenic loci in four transgenic maize plants. In eachcase, short regions of homology (3–10 nt) are involved in the recombination process andthese regions of common sequence between the two recombining partners are shown asunderlined. This can result in (A) the insertion of genomic DNA into transgene (B) “head-tohead”junctions or (C, D) “head-to-tail” junctions.456 Christou et al