Global Architecture of Genetic Interactions on the Protein Network Protein-protein Genetic

490 plants retain both of the adjacent cpDNA NcoI sites that are outside the experimental construct. Most digested DNA samples from the kanamycin-resistant plants show two hybridization fragments that mirror those in the transplastome (Fig. 3a,b in ref. 1). The generally large size of the integrants is confirmed by sequence data from two of the kanamycin-resistant plants (Fig. 3e,f in ref. 1). For example, there are 1,775 bp of vector chloroplast DNA and 5,917 bp of nonvector chloroplast DNA (see Supplementary Information to ref. 1) between the junction with nuclear DNA and the aadA gene in kr1. Similarly, there are 1,165 bp of vector chloroplast DNA and 934 bp of nonvector chloroplast DNA adjacent to the junction site downstream of neo in the nuclear integrant of kr17. Consequently, there can be no doubt that most of these integrants contain more DNA of chloroplast origin than was present in our experimental cassette (see Fig. 1). Integrants that are shorter than the chloroplast transformation vector may also be present. Regarding the concern over multiple integrants, we do not yet understand the complexity of the transfer process, but we do know that single Mendelian loci are involved in all but four of the kanamycinresistant plants from the screen. Multiple integrations do not require multiple transposition events. The lysis of a single plastid would release tens to hundreds of plastid genomes into the cytoplasm, some of which could integrate into a common genomic location. This process could be analogous to the high-copy number of transgenes delivered into the cell via biolistic transformation, so it is not surprising to find a proportion of multiple integrants among the kanamycin-resistant plants. We did not conclude that chloroplast-specific genes, such as aadA in our experiment, will not function when transposed to the nucleus. What we did show, in all cases where we selected for nuclear kanamycin resistance, was that the relocated neo gene was accompanied by the adjacent aadA gene (and other flanking native chloroplast DNA). We noted that the latter gene was not expressed to confer spectinomycin resistance. A News & Views commentary4 that accompanied our article in Nature suggested that we undertake a much larger screen to search for spectinomycin resistance to determine whether a chloroplast specific gene rarely could be expressed after integration into an appropriate nuclear environment. This is an evolutionary experiment in the true sense, but the likely scale of such a screen is daunting. However, such an approach may greatly increase our understanding of the evolution of nuclearencoded plastid genes. Finally, we stated in our paper that there was likely to be an equilibrium between the ingress of chloroplast DNA sequences and their elimination. The fact that Daniell and Parkinson know of no such mechanism merely demonstrates that there is still much to learn about the processes of genome evolution. Chun Y. Huang, Department of Molecular Biosciences, The University of Adelaide, South Australia, 5005, Australia, Michael A. Ayliffe, CSIRO Plant Industry, GPO Box 1600, ACT 2601, Australia, and Jeremy N. Timmis The University of Adelaide, South Australia, 5005, Australia