R. Abranches et al., Widely separated multiple transgene integration sites in wheat chromosomesare brought together at interphase, PLANT J, 24(6), 2000, pp. 713-723
We have investigated the organization of transgenes delivered by particle b
ombardment into the wheat genome, combining conventional molecular analysis
with fluorescence in situ hybridization (FISH) and three-dimensional confo
cal microscopy. We selected a representative population of transformed whea
t lines and carried out molecular and expression analysis. FISH on metaphas
e chromosomes showed that transgene integration sites were often separated
by considerable lengths of genomic DNA (>1 Mbp), or could even be on. oppos
ite chromosome arms. Plants showing multiple integration sites on a single
chromosome were selected for three-dimensional confocal analysis of interph
ase nuclei in root and embryo tissue sections. Confocal microscopy revealed
that these sites lay in close physical proximity in the interphase nuclei.
Our results clearly show that multiple transgenes physically separated by
large intervening regions of endogenous DNA at metaphase can be brought tog
ether at interphase. This may reflect the original physical organization of
the endogenous DNA at the moment of transformation, with DNA strand breaks
introduced into several co-localized DNA loops by the intruding gold parti
cles. Alternatively, the transgenes may be brought together after transform
ation, either by an ectopic homologous pairing mechanism, or by recruitment
to a common transcription site.