A. Kohli, X. Fu, R. M. Twyman and P. Christou
Molecular Biotechnology Unit, John Innes Center, Colney Lane,
Norwich NR4 7UH, UK
Transgene expression in plants is
highly variable, even among plants independently transformed with the same
construct There is also no guarantee that primary transformants showing
strong transgene expression will give rise to progeny with the same characteristics.
The production of transgenic plants with stable, high-level transgene expression
is important for the success of crop improvement programs based on genetic
engineering. Many factors may be responsible for variable transgene expression,
including the tendency for exogenous DNA to undergo rearrangement prior
to integration, position effects, the effects of transgene copy number,
and the effects of DNA methylatjon (Meyer 1998). It is therefore important
to learn as much as possible about the mechanisms of transgene integration,
and how transgene structure and organization affects expression and stability.
In this communication, we discuss recent experiments investigating mechanisms
of transgene integration and rearrangement, and the effect of such rearrangements
on transgene expression.
Perhaps one of the most surprising
aspects of particle bombardment..mediated transformation is that genes
introduced on separate plasmids are just as likely to cointegrate (integrate
at the same locus) as genes actually linked on the same vector. We have
begun to unravel the molecular basis of this phenomenon through a detailed
examination of transgene structure at multiple copy transgenic loci. Such
studies revealed an unexpected multiple-tier organization (Fig. 1), in
which clusters of transgene copies apparently joined end-to-end were interspersed
with regions of genomic DNA (Kohli eta!. 1998). We propose that such structures
result from a two-phase integration mechanism. In the first phase, occurring
before integration, exogenous DNA becomes ligated together to form transgene
arrays lacking genomic DNA. These arrays are the substrates for integration,
presumably by interacting whh randomly-occurrjng breaks in the endogenous
chromosomes. While this process could be repeated throughout the genome,
resulting in many unlinked transgenic loci, further transgene arrays instead
tend to integrate at the same locus. The reason for such clustering may
reflect the recruitment of DNA repair complexes to the original site of
integration, resulting in the introduction of many local double strand
DNA breaks. Strand exchange and recombinatjon at such loci could also result
in the elimination of variable-length regions of host DNA at the integration
site, another common observation in lransgenic plants. FISH analysis has
revealed a further level of organization where such clustered transgene
arrays are interspersed with larger regions of genomic DNA. This may reflect
localized damage, caused by the metal particles, to DNA in a specific region
of the nucleus, where tertiary structure brings looped DNA strands into
close physical proximity. Notwithstanding this hierarchical organization,
the individual transgene copies are sufficiently close together so that
rice transformation by particle bombardment generally produces a single
transgenic locus.
We have also carried out detailed
investigations of the molecular mechanisms underpinning transgene rearrangements
(Kohli et a!. 1999). Within each of the transgene arrays that characterize
the typical transgenic locus, individual transgenes are joined together
at sites that can be termed plasmid-plasnud junctions. The nature of such
junctions is poorly understood, especially in monocots, and their investigation
could show how plasmids undergo rearrangement prior to integration and
how this affects transgene expression. Ultimately, information derived
from such studies could lead to the design of better transformation vectors.
We analyzed 12 independent transgenic
rice lines, each containing several plasmidplasmid junctions. One of the
most striking revelations was the involvement of the same region of the
plasmid in more than one third of the rearrangements characterized. Specifically,
a 19-bp palindromic sequence surrounding the TATA box of the CaMV 35S promoter
was often involved in recombination events. Notably, this represented the
region that, in the wild type cauliflower mosaic virus RNA, is responsible
for viral recombination events in planta. Almost all of the junctions appeared
to have arisen by microhomology mediated illegitimate recombination (Table
1), a ubiquitous process in which short complementary tails from two non-homologous
DNA duplexes first overlap, followed by repair synthesis to join the parental
molecules together. In a few rare cases, the junctions appeared to have
been generated by the direct end-to-end ligation of the two recombination
partners, i.e. there was no sequence overlap between the strands. The presence
of direct repeats flanking some junctions suggested the involvement of
a transposition-like process, perhaps the utilization of exogenous plasrnid
DNA as an illegitimate substrate by endogenous transposases. Other characteristics
of the junctions included the presence of filler DNA (several nucleotides
inserted at the junction, with no homology to either of the recombining
partners), the presence of topoisomerase binding sites (suggesting topoisomerases,
which introduce nicks and breaks in plasmids to relieve supercoiling, and
remove knots and catenated links, may have been responsible for some of
the strand breaks that preceded junction formation) and purine-rich tracts
(suggesting the involvement of DNA tertiary structure in junction formation).
Overall, the junctions generated during particle bombardment were similar
to those described for other transformation procedures and indicated common,
underlying mechanisms of transgene rearrangement regardless of transformation
method. Through further such studies, transformation vectors could be optimized
on the principle of avoiding recombinogenic sequences within transgene
expression cassettes and deliberately including them in external regions.
This would promote favorable recombination events during the preintegrative
phase and avoid destructive transgene rearrangements.
References
Kohli A., S. Griffiths, N. Palacios, R.M. Twyman, P. Vain,
D.A. Laurie and P. Christou, 1999. Molecular
characterisation of transforming plasmid rearrangements in transgenic rice
reveals a recombination hotspot in the CaMV 35S promoter and confirms the
predominance of microhomology-mediated recombination. Plant J. 17: 591-601.
Meyer, P., 1998. Stabilities and instabilities in tranagene expression. in Trausgenic Plant Research, Lindsey, K. (ed.). Harwood Academic Publishers, Switzerland, p 263-275. |