the DNA ends of two molecules are ligated together in a process also known as DNA
end-joining or illegitimate recombination. In some lower eukaryotes, such as the
budding yeast, the homologous recombination between introduced DNA and host DNA
occurs at a high frequency. In most other higher eukaryotes, however, nonhomologous
recombination is the predominant route by which new DNA is incorporated into the genome.
Homologous recombination has great potential for genome engineering. The homolo-
gous replacement of a host gene by an altered version of that gene can help elucidate
gene function, as well as create new genetic varieties through the precise alteration of a
host sequence. The use of homologous gene replacement is becoming more common in
animal research, particularly in mice, although these are still rare events occurring in a
small fraction of transgenic cells. Among plants, the engineering of host DNA by
homologous recombination has been practical with a moss (Physcomitrella patens) that
is becoming a model system for basic genetic studies (Schaefer 2001). With other higher
plants, however, there have been only a few reports of successful gene replacement via
homologous recombination that were detected at a percent or less of transformation events.
Some of the potentially most useful biotechnologies for increased precision aresite-
specific recombination systems, most of which are described from the viruses and plasmids
of bacteria or yeast. Bacterial viruses, known asphages, often integrate their DNA into the
genome of the bacterial host at a designated site. This site-specific integration process is
highly efficient and depends on recombinase protein(s) encoded by the phage genome.
Integration into the host genome enables a phage to hide within the bacterial genome for
many generations until it elects to leave by a reversal of the integration process, or site-
specific excision, which is also mediated by phage-encoded excisionase protein(s). Some
plasmids also encode site-specific recombination systems. When plasmids replicate,
mother and daughter plasmid chromosomes sometimes recombine as a result of host-
mediated homologous recombination. The plasmid-encoded site-specific recombination
system resolves these dimers back into monomers, thus ensuring the partitioning of
plasmid molecules to dividing cells. Although these recombination systems originate
from prokaryote or lower eukaryotes, many of them also function in higher eukaryotic
cells. Since the mid-1980s, scientists have been using these site-specific recombination
systems to cause a variety of site-specific deletion, inversion, or integration events in
animal or plant cells.
The types of site-specific recombination systems that function in higher eukaryotes can
be divided into several groups. In the first group, the genetic crossover occurs between two
recombination substrates that are identical or nearly identical in sequence. The two product
sites generated by the recombination event are therefore identical or similar to the substrate
sites, and the recombination reaction is fully reversible. Notable examples from this group
are Cre-lox, FLP-FRT, and R-RS, where Cre, FLP, and R are therecombinasesandlox,
FRT, andRSare the respectiverecombination sites. These recombination systems can
perform the full range of recombination both within and between DNA molecules
(Fig. 16.1). In a second group, the substrates and products of the recombination reaction
are also identical or very similar to each other. However, only one of the two types of intra-
molecular recombination is possible, either deletion or inversion, and the reaction is not
reversible. Examples of deletion systems from this group areb-six, ParA-MRS, and
CinH-RS2, whereb, ̇ParA, and CinH are the recombinases (also known asresolvases)
that catalyze DNA excision between pairs of sites known respectively assix, MRS, and
res.In a third group, the recombinase (also known asintegrase) recombines two substrates
that do not share extensive similarity, typically known as bacterial and phage attachment
360 THE FUTURE OF PLANT BIOTECHNOLOGY