Fig. 6. (A-G) Nondegradable Scc1 induces the cut phenotype in early passage human fibroblasts with telomeric DNA present on the DNA bridge. Early passage fibroblasts (PD 15-18) were infected serially with ND-SCC1, and PNA-FISH with centromeric and telomeric probes was performed 2-3 days following the last infection. (A-F) Telomere signals are detected with Cy3 (red) and centromeric signals with FITC (green). Nuclei are stained with DAPI (blue). (A) An example of a nucleus with an abnormal morphology induced by the ND-Scc1mutation. In addition to the irregular nuclear shape, additional micronuclei are present in the cell, hybridizing to centromeric and telomeric probes. One of these micronuclei is shown more clearly in the inset. These nuclei are probably the result of lagging chromosomes. (B) A typical cut phenotype. Note that a thin strand of DNA connects the two nuclei most distantly separated in this field. (C) A cut phenotype with a thick DNA bridge containing multiple telomeric signals. The region of the DNA bridge, connecting the two sister nuclei, is enlarged at the bottom left. (D-E) The cut phenotype with a thin DNA bridge. Several telomeric signals are present on the bridge, whereas no hybridization with the centromeric probe is evident. The region of the DNA bridge is enlarged and presented in the bottom frame of each panel. (F) A nucleus containing a torn DNA bridge. An enlargement of the torn DNA strand is present at the bottom frame of the panel. (G) Staining of a GFP-positive cell with the cut configuration with CREST (anti-centromeric) and anti-GFP antibodies. The centromeric regions are detected with Cy3 (red) and the GFP with FITC (green). DNA is stained with DAPI (blue).