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First published online 2 August 2005
doi: 10.1242/jcs.02498

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Tandem repetitive transgenes and fluorescent chromatin tags alter local interphase chromosome arrangement in Arabidopsis thaliana

Ales Pecinka^1,, Naohiro Kato^2,*,, Armin Meister¹, Aline V. Probst³, Ingo Schubert¹ and Eric Lam^2,

¹ Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany
² Biotech Center for Agriculture and the Environment, Rutgers, The State University of New Jersey, 59 Dudley Road, Foran Hall, New Brunswick, NJ 08901, USA
³ Laboratory of Plant Genetics, University of Geneva, 30 Quai Ernset Ansermet, CH-1211 Geneva 4, Switzerland

View larger version (37K): [in a new window]   Fig. 1. GFP spot numbers in living guard cell nuclei (2C) from cotyledons of dexamethasone-treated transgenic Arabidopsis seedlings. (A) Percentage of nuclei with 0, 1, 2, 3 and 4 spots in hemizygous EL702C plants (n=92), homozygous EL702C plants (n=197) and homozygous EL700S plants (n=56). (B) Representative images of nuclei with 0, 1, 2, 3 and 4 spots from each of the lines and schematic view of the lac operator array loci on chromosome 3 in each line.

View larger version (35K): [in a new window]   Fig. 2. Colocalization of GFP spots and lac operator FISH signals in Dex-treated 2C leaf nuclei of homozygous EL702L plants. (A) Percentage of nuclei with one to four GFP spots compared to FISH signals. Fewer GFP spots than FISH signals may occur in one nucleus. (B) Examples of nuclei with 1, 2, 3 or 4 GFP spots coinciding with lac operator FISH signals. Bars, 3 µm.

View larger version (42K): [in a new window]   Fig. 3. The double transgene insertion in EL702C is accompanied by a paracentric inversion between the integration points. (A) Painted regions between BACs F2O10 and F28J15 (yellow) and MBK21 and MSL1 (red) schematically positioned on the top arm of chromosome 3 of the wild-type (left) and together with the transgene (green) of the EL702C genotype (right). (B) FISH of the complex probe to pachytene chromosomes of the wild type (left) and homozygous EL702C (right). Arrows indicate the top arm end of chromosome 3 bivalent. Bars, 5 µm.

View larger version (34K): [in a new window]   Fig. 4. Ectopic and allelic pairing of the lac operator array loci in EL702C plants. (A) Percentage of loci showing ectopic pairing (untreated hemizygous nuclei) or ectopic/allelic pairing (untreated and Dex-treated homozygous nuclei). (B) Scheme of chromosome 3 (EL702C) with the transgene insertion positions and the flanking regions used as markers for FISH. (C) Hemizygous nuclei showing ectopic pairing (top) or separation (bottom) of transgenic loci. (D) Homozygous nuclei with ectopic pairing of both transgenic loci (top, could be intrachromosomally or between homologues), allelic pairing of only the distal locus (middle) or separation of both loci (bottom). Bars, 3 µm.

View larger version (34K): [in a new window]   Fig. 5. Homologous pairing and ectopic association of regions flanking the lac operator transgene. (A) Percentage of homologously paired MGL6 loci, homologously paired F18C1 loci and association between both regions in the wild type, hemizygous and homozygous EL702C nuclei without or after Dex treatment. Significant differences *P<0.05 and ***P<0.001 were found compared to levels in the wild type determined using 2 test. (B) Schemes of chromosome 3 (wild type and EL702C) showing the position of BACs MGL6 and F18C1 used for FISH. (C) Homozygous EL702C nuclei showing homologous pairing of MGL6 (top) or ectopic association intrachromosomally or between two homologues (bottom). Bars, 3 µm.

View larger version (21K): [in a new window]   Fig. 6. Association frequency of the lac operator arrays with heterochromatic domains compared to that of flanking MGL6/F18C1 sequences. The percentage of FISH signals associated with heterochromatin in nuclei of wild type (MGL6, F18C1) and of homozygous EL702C plants without and after Dex treatment (MGL6, F18C1, lac operator) is shown. ***P<0.001 compared to the wild-type situation determined using 2 test.

View larger version (45K): [in a new window]   Fig. 7. The association frequency of the transgenic hygromycin phosphotransferase (HPT) locus with endogenous heterochromatin is similar in line A and in the mom1-1 background. Top row, Arabidopsis nuclei with intense DAPI-stained chromocenters. Middle row, the same nuclei after FISH with the 178 bp centromeric repeat (red) and pGL2 sequence labeling the HPT locus (green). From left to right: two, one or none of the separated allelic HPT signals associated with centromeric heterochromatin, and paired HPT signals associated or not associated with centromeric heterochromatin. Note the appearance of the HPT locus as small DAPI-positive chromocenter(s) in the second, third and fifth nucleus of the upper panel (arrows). Bottom row, the percentage of nuclei showing the corresponding type of association of HPT signals with centromeric heterochromatin (line A, n=269 nuclei; mom1-1, n=355 nuclei). Of the total HPT signals 30% were paired (see fourth and fifth nucleus) in line A and 21% in mom1-1. In nuclei of line A, 61% of the 271 HPT sites colocalizing with centromeric heterochromatin did so as single sites and 39% as paired sites. In mom1-1 nuclei, 65% of the 346 HTP sites colocalized with centromeric heterochromatin as single sites and 35% as paired sites.

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