Escherichia coli DH10B was designed for the propagation of large insert DNA library clones. It is used extensively, taking advantage of properties such as high DNA transformation efficiency and maintenance of large plasmids. The strain was constructed by serial genetic recombination steps, but the underlying sequence changes remained unverified. We report the complete genomic sequence of DH10B by using reads accumulated from the bovine sequencing project at Baylor College of Medicine and assembled with DNAStar's SeqMan genome assembler. The DH10B genome is largely colinear with that of the wild-type K-12 strain MG1655, although it is substantially more complex than previously appreciated, allowing DH10B biology to be further explored. The 226 mutated genes in DH10B relative to MG1655 are mostly attributable to the extensive genetic manipulations the strain has undergone. However, we demonstrate that DH10B has a 13.5-fold higher mutation rate than MG1655, resulting from a dramatic increase in insertion sequence (IS) transposition, especially IS150. IS elements appear to have remodeled genome architecture, providing homologous recombination sites for a 113,260-bp tandem duplication and an inversion. DH10B requires leucine for growth on minimal medium due to the deletion of leuLABCD and harbors both the relA1 and spoT1 alleles causing both sensitivity to nutritional downshifts and slightly lower growth rates relative to the wild type. Finally, while the sequence confirms most of the reported alleles, the sequence of deoR is wild type, necessitating reexamination of the assumed basis for the high transformability of DH10B.Molecular biology studies rely heavily on Escherichia coli for essential operations, ranging from the simple propagation of plasmid DNA to the creation of large clone libraries for wholegenome sequence determination. Among the strains developed as hosts for these everyday applications, DH10B (17) is commonly used across the research community, taking advantage of particularly useful properties exhibited by the strain. These include high transformation efficiency, the ability to take up and stably maintain large plasmids, the lack of methylation-dependent restriction systems (MDRS), and colony screening via lacZ-based ␣-complementation. However, analysis of sequenced bacterial artificial chromosome (BAC) clones derived from DH10B shows a high incidence of insertion sequence (IS) transposition from the chromosome into the cloned fragment (25).The genome of DH10B was constructed before the modern era of molecular biology, through a series of genetic manipulations (Fig. 1). The progenitors were all K-12 strains, with the exception of D7091F, in which a region surrounding the ⌬(araA-leu)7697 deletion had been derived from E. coli B SB3118 by P1 transduction (John Wertz, personal communication). Ultimately, MC1061 (9) served as a starting point for Hanahan and coworkers to replace alleles by using a series of P1 transductions that resulted in DH10B (17). Among the engineered gene replacements were recA1 ...

The design of vectors for plant transformation has recently progressed to the development of intragenic systems. This involves identifying plant-derived DNA sequences similar to important vector components. The most useful approach involves adjoining two fragments from plant genomes to form sequences that have the functional equivalence of vector elements such as: T-DNA borders for Agrobacterium-mediated transformation, bacterial origins of replication and bacterial selectable elements. Such DNA fragments have been identified from a wide range of plant species, suggesting that intragenic vectors can be constructed from the genome of any plant species. Intragenic vectors provide a mechanism for the well-defined genetic improvement of plants with the entire DNA destined for transfer originating from within the gene pool already available to plant breeders. In this manner, genes can be introgressed into elite cultivars in a single step without linkage drag and, most importantly, without the incorporation of foreign DNA. The resulting genetically modified (GM) plants are non-transgenic, although they are derived using the tools of molecular biology and plant transformation. The genetic makeup of the resulting plants is equivalent to minor rearrangements or micro-translocations that could theoretically arise through natural or induced rearrangements of the endogenous genome. Given the public concerns over the deployment of GM crops in agriculture, especially for food crops, intragenic vector systems offer a more socially acceptable and responsible way forward for genetic engineering. It may especially help us to alleviate some of the ethical issues associated with the transfer of DNA across wide taxonomic boundaries. Plants derived from the use of intragenic vectors raise important issues concerning the definition, regulation and testing of GM plants. This review critically evaluates the progress toward the development and use of intragenic vectors and the implications of their use for the genetic improvement of crops.

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