effects at high copy number. Unlike chromosomal DNA, plasmid DNA is dispensable to
the host, so why does the host keep it? To be maintained, plasmid DNA molecules must
provide their host cells with a selective advantage over their competitors. Plasmid selection
is a natural phenomenon that has allowed the evolution of plasmid DNA and its mainten-
ance in bacterial host cells. They encode genes, such as bacteriocins or antibiotics, enabling
the host to kill other organisms competing for nutrients. The first bacterial plasmid ident-
ified was the fertility factor (F factor) inE. coli, discovered in 1946 by Joshua Lederberg
and Edward Tatum. This F factor enables bacteria to donate genes to recipients by conju-
gation [for a review, see Clark and Warren (1979)], providing a mechanism for adaptive
evolution, permitting, for example, plasmid-mediated transfer of antibiotic resistance
genes or pathogenicity genes.
7.2.1 DNA Vectors for Plant Transformation
Many bacterial plant pathogens benefit from plasmid borne, pathogenicity genes, which
provide them with the ability to infect or parasitize plants. One such organism,
Agrobacterium tumefaciens, benefits from a tumor-inducing (Ti) plasmid (Fig. 7.6),
which plays a central role in crown gall disease in a wide variety of plants.
The ability ofA. tumefacienscontaining a Ti plasmid to hijack a plant’s protein synthesis
machinery and genetically engineer the host genome, prompted the development of plasmid
vectors forAgrobacterium-mediated plant transformation. In plant transformation vectors,
the T-DNA contains only the genes intended for transfer to the nuclear genome of the
Figure 7.6.The Ti plasmid ofAgrobacterium tumefaciensshowing the origin of replication, the
region encoding the virulence (vir) genes, and the transfer-DNA (T-DNA). The T-DNA is flanked
by 25-bp direct repeats, known as theleftandright bordersequences (LB and RB, respectively).
Thevirgenes are required for T-DNA processing and transfer to the plant cell. The T-DNA is
stably integrated into the nuclear genome of the plant cell, and genes encoded within it, necessary
for the biosynthesis of the plant growth hormones, cytokinin and auxin result in the formation of
the characteristic tumorous growth associated with crown gall disease. The T-DNA also encodes
opines (nopaline and octapine) that provide theAgrobacteriumwith an exclusive nitrogen source.
This provides Agrobacterium carrying the Ti plasmid with a competitive advantage over
Agrobacteriumthat do not.
166 RECOMBINANT DNA, VECTOR DESIGN, AND CONSTRUCTION