Chromosome fusion points as revealed by FISH. (A-H) tert/nbs1 double homozygotes, (I-P) tert. (A-D) tert/nbs1 mutant, generation G6, line K12. Sequential FISH on mitotic anaphase bridge revealed involvement of the right ends from chromosome 1. (A) DAPI stained anaphase with the bridge consisting of two chromatids. (B) The signals from BAC probe F5I6 (Cy3 labelled, red) are presented on both bridges. In the second FISH (C) no signals on the bridges are presented whereas in (D) the green probe (SpectrumGreen labelled, BAC F5I6) highlights the same positions as in (B). According to the FISH setup, alternation of colours unambiguously disclosed the fusion of the 1R chromosome end. (E-H) FISH with telomere-specific probe. Using telomere-specific Cy3-labelled PNA-C3TA3-probe, fusion points were detected on two chromatid anaphase bridges from line K12, generation G3 plants (F), whereas in higher generations (G4) the signals from PNA-probe were not detectable on the anaphase bridges (H). To demonstrate the morphology of bridges DAPI stained images are presented (E, G). (I) DAPI stained anaphase displaying twin anaphase bridges of tert mutant, line 69–2, generation G6. (J-L) Sequential FISH. (J, K) Left ends of chromosomes 3 were sequentially labelled by 3 L-specific T4P13 BAC, green in first FISH (J), and red in second FISH (K). In the third FISH, green signals from the probe for 45S rDNA illuminated the 2 L chromosome end (L). Thus, direct reciprocal fusions of 3 L and 2 L chromosome ends were proven. (M-P) Anaphase bridges from tert mutants probed by telomere PNA probe. (N) tert line 69–1, G5 and (P) line 69–2, generation G6. In both lines fusion points were decorated by telomere-specific probes from the generation G5 on. (M, O) DAPI stained anaphases with bridges. Bar represents 10 μm for all images.