Plant genetic engineering has, until now, relied on the incorporation of foreign DNA into plant genomes. Public concern about the extent to which transgenic crops differ from their traditionally bred counterparts has resulted in molecular strategies and gene choices that limit, but not eliminate, the introduction of foreign DNA. Here, we demonstrate that a plant-derived (P-) DNA fragment can be used to replace the universally employed Agrobacterium transfer (T-) DNA. Marker-free P-DNAs are transferred to plant cell nuclei together with conventional T-DNAs carrying a selectable marker gene. By subsequently linking a positive selection for temporary marker gene expression to a negative selection against marker gene integration, 29% of derived regeneration events contain P-DNA insertions but lack any copies of the T-DNA. Further refinements are accomplished by employing Ω-mutated virD2 and isopentenyl transferase cytokinin genes to impair T-DNA integration and select against backbone integration, respectively. The presented methods are used to produce hundreds of marker-free and backbone-free potato (Solanum tuberosum) plants displaying reduced expression of a tuber-specific polyphenol oxidase gene in potato. The modified plants represent the first example of genetically engineered plants that only contain native DNA

SummaryAcrylamide is produced in starchy foods that are baked, roasted or fried at high temperatures. Concerns about the potential health issues associated with the dietary intake of this reactive compound led us to reduce the accumulation of asparagine, one of its main precursors, in the tubers of potato ( Solanum tuberosum ). This metabolic change was accomplished by silencing two asparagine synthetase genes through 'all-native DNA' transformation. Glasshouse-grown tubers of the transformed intragenic plants contained up to 20-fold reduced levels of free asparagine. This metabolic change coincided with a small increase in the formation of glutamine and did not affect tuber shape or yield.Heat-processed products derived from the low-asparagine tubers were also indistinguishable from their untransformed counterparts in terms of sensory characteristics.However, both French fries and potato chips accumulated as little as 5% of the acrylamide present in wild-type controls. Given the important role of processed potato products in the modern Western diet, a replacement of current varieties with intragenic potatoes could reduce the average daily intake of acrylamide by almost one-third.

(23,25). Only in the cases of the tetracenomycin (40) and actinorhodin (5,19,34,55) PKSs, however, have biochemical characterizations also been carried out. Thus, most of the information on type II PKSs is derived from the strong conservation of gene structure among the PKS genes (23,25) and cross-functionality of the components (5,26,34,41). We describe here the structure of a gene region from Streptomyces sp. strain C5 that putatively encodes daunomycin biosynthesis and show that it has a significantly different overall structure from other type II PKS gene regions. MATERUILS AND METHODSBacterial strains and media used. Streptomyces sp. strain C5 and mutants derived from it have been described previously (3,4). Streptomyces lividans TK24 (22), used as a recombinant host strain, was obtained from D. A. Hopwood. Streptomyces coelicolor mutants, described by Rudd and Hopwood (37), were obtained from H. G. Floss. Streptomyces galilaeus ATCC 31671, which lacks a functional polyketide reductase (PKR), has been described previously (5, 50).

(4,5,8,9,33), a pathway in which two methyltransferase reactions have been proposed (8,9,33). Aklanonic acid methyltransferase (AAMT) catalyzes the methyl esterification of aklanonic acid (Fig. 1A) (8). Aklanonic acid methyl ester (AAME) cyclase catalyzes the formation of aklaviketone from AAME, and in wild-type anthracycline producers, aklaviketone is reduced to form aklavinone (8). We have generated mutants specifically blocked in AAMT activity (dauC mutants) and AAME cyclase activity (dauD) (4, 5).Carminomycin 4-O-methyltransferase (CMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 4-O-position of carminomycin and 13-dihydrocarminomycin (Fig. 1B) to form daunomycin and 13-dihydrodaunomycin, respectively (9, 10). CMT has been purified to near homogeneity and is an apparent homotetramer with an M r of ca. 161,000 (10). A gene from S. peucetius ATCC 29050 encoding CMT has been isolated and sequenced (25). In this article, we show the isolation and sequence analyses of AAMT, AAME cyclase, and CMT of Streptomyces sp. strain C5. On the basis of a comparison of the sequence similarities and differences, the two methyltransferases apparently belong to two different subgroups. The AAME cyclase appears to be unusual with respect to both its activity and proteins in the databases. We also show the sequence of dauP, encoding an esterase-like function which we hypothesize also to be involved in daunomycin biosynthesis. MATERIALS AND METHODSBacterial strains, plasmids, and media. Streptomyces sp. strain C5 and mutants derived from it have been described previously (4, 5). Streptomyces lividans TK24 (18) was obtained from D. A. Hopwood. Streptomyces strains normally were grown in YEME (18) supplemented with 20% sucrose. R2YE medium, also used for growth as well as for preparation of streptomycete protoplasts, was prepared as described by Hopwood et al. (18). Nitrate-defined-plus-yeast-extract (NDYE) medium, used for growth of Streptomyces sp. strain C5 and its mutants, has been described previously (9). Strains carrying streptomycete plasmids pIJ486 (38), pIJ702 (18), and pWHM3 (36) or derivatives of them were grown and stored on plates containing 40 g of thiostrepton per ml.Escherichia coli JM83 was used to propagate plasmids for sequencing and restriction analyses. E. coli was grown in Luria-Bertani medium, and plasmids were introduced into E. coli by transformation done by standard procedures (26). For E. coli strains harboring plasmids, ampicillin was added at a final concentration of 100 g/ml.General genetic manipulations. The procedures for protoplast formation, transformation, and regeneration of protoplasts for Streptomyces sp. strain C5 have been described elsewhere (24). The procedures used for the preparation of Streptomyces plasmid and chromosomal DNA have been described by Hopwood et al. (18). The digestion of DNA with restriction endonucleases was carried out according to the manufacturers' directions. Restriction mapping and other routine molecular methods used in th...

Summary