We have used the Escherichia coli beta‐glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta‐glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta‐glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.

Point mutations result from errors made during DNA replication or repair, so they are usually expected to be homogeneous across all regions of a genome. However, we have found a region of chloroplast DNA in plants related to sweetpea (Lathyrus) whose local point mutation rate is at least 20 times higher than elsewhere in the same molecule. There are very few precedents for such heterogeneity in any genome, and we suspect that the hypermutable region may be subject to an unusual process such as repeated DNA breakage and repair. The region is 1.5 kb long and coincides with a gene, ycf4, whose rate of evolution has increased dramatically. The product of ycf4, a photosystem I assembly protein, is more divergent within the single genus Lathyrus than between cyanobacteria and other angiosperms. Moreover, ycf4 has been lost from the chloroplast genome in Lathyrus odoratus and separately in three other groups of legumes. Each of the four consecutive genes ycf4-psaI-accD-rps16 has been lost in at least one member of the legume ''inverted repeat loss'' clade, despite the rarity of chloroplast gene losses in angiosperms. We established that accD has relocated to the nucleus in Trifolium species, but were unable to find nuclear copies of ycf4 or psaI in Lathyrus. Our results suggest that, as well as accelerating sequence evolution, localized hypermutation has contributed to the phenomenon of gene loss or relocation to the nucleus.

The suitability of potato virus X (PVX) as a gene vector in plants was tested by analysis of two viral constructs. In the first, the GUS gene of Escherichia coli was substituted for the viral coat protein gene. In the second, GUS was added into the viral genome coupled to a duplicated copy of the viral promoter for the coat protein mRNA. The viral construct with the substituted coat protein gene accumulated poorly in inoculated protoplasts and failed to spread from the site of infection in plants. These results suggest a role for the viral coat protein in key stages of the viral infection cycle and show that gene replacement constructs are not suitable for the production of PVX-based gene vector. The construct with GUS coupled to the duplicated promoter for coat protein mRNA also accumulated less well in protoplasts than the unmodified PVX, but did infect systemically and directed high level synthesis of GUS in inoculated and systemically infected tissue. Although there was some genome instability in the PVX construct, much of the viral RNA in the systemically infected tissue had retained the foreign gene insertion, especially in infected Nicotiana clevelandii plants. These data point to a general utility of PVX as a vector for unregulated gene expression in plants.

We used DNA sequencing and gel blot surveys to assess the integrity of the chloroplast gene infA , which codes for translation initiation factor 1, in Ͼ 300 diverse angiosperms. Whereas most angiosperms appear to contain an intact chloroplast infA gene, the gene has repeatedly become defunct in ‫ف‬ 24 separate lineages of angiosperms, including almost all rosid species. In four species in which chloroplast infA is defunct, transferred and expressed copies of the gene were found in the nucleus, complete with putative chloroplast transit peptide sequences. The transit peptide sequences of the nuclear infA genes from soybean and Arabidopsis were shown to be functional by their ability to target green fluorescent protein to chloroplasts in vivo. Phylogenetic analysis of infA sequences and assessment of transit peptide homology indicate that the four nuclear infA genes are probably derived from four independent gene transfers from chloroplast to nuclear DNA during angiosperm evolution. Considering this and the many separate losses of infA from chloroplast DNA, the gene has probably been transferred many more times, making infA by far the most mobile chloroplast gene known in plants. INTRODUCTIONMany genes have been lost from the chloroplast genome during plant and algal evolution. Most of these losses occurred in the murky interval between the original endosymbiosis of a cyanobacterium (with perhaps 2000 proteincoding genes) and the last common ancestor of all existing chloroplast genomes (with ‫ف‬ 210 protein-coding genes; . Many other genes were lost during the early evolution of photosynthetic eukaryotes, often in parallel in different algal lineages, and some of these losses were the result of gene transfers to the nuclear genome . During the evolution of land plants, relatively few changes occurred to the set of genes found in chloroplast DNA (cpDNA) Palmer and Delwiche, 1998). Nonetheless, the most recent changes are likely to provide the most information about the evolutionary mechanisms involved.Among the six completely sequenced chloroplast genomes from angiosperms (excluding the nonphotosynthetic plant Epifagus virginiana ; Wolfe et al., 1992a), 74 proteincoding genes are held in common and an additional five are present in only some species. These five genes are accD , ycf1 , and ycf2 (pseudogenes in rice and maize; Hiratsuka et al., 1989;Maier et al., 1995), rpl23 (pseudogene in spinach; Thomas et al., 1988), and infA (pseudogene in tobacco, Arabidopsis, and Oenothera elata ; Shinozaki et al., 1986;Wolfe et al., 1992b;Sato et al., 1999; Hupfer et al., 2000). Other chloroplast gene losses in angiosperms that have been confirmed by sequencing include rpl22 , rps16 , and ycf4 (open reading frame 184), all of which have been lost in 1 To whom correspondence should be addressed. E-mail (in Dublin) khwolfe@tcd.ie; fax 353-1-6798558. 646The Plant Cell some or all legumes (Gantt et al., 1991;Nagano et al., 1991; Doyle et al., 1995; K.H. Wolfe, unpublished data), and ycf2 and ndhF , both of which have been lost ...

SummaryTransgene expression from the plant's plastid genome represents a promising strategy in molecular farming because of the plastid's potential to accumulate foreign proteins to high levels and the increased biosafety provided by the maternal mode of organelle inheritance.In this article, we explore the potential of transplastomic plants to produce human