| SAR/MAR sequences have been hypothesized as forming the bases of DNA loops in metaphase chromosomes. To test the structural role of SAR/MAR sequences, we created stable cell lines containing chromosomes with regions of high density of integrated SAR/MAR sequences. CHO cells were isolated containing multiple copies of a vector containing a 256 copy lac operator repeat and a DHFR gene flanked by the 5' and 3' MARs from the human binterferon gene. Control experiments in which SAR/MAR sequences were not included in the vector were conducted in parallel. Cell clones were isolated which showed 0.1--0.5-um wide chromosome bands, containing a high density of integrated vector copies with more than a 5-fold increase in the local density of SAR/MARs up to 0.1 kbp-1. Lac repressor staining and FISH revealed normal metaphase chromosome diameter over these chromosome bands, and distribution of vector sequences throughout the entire chromosome width. Quantitative analysis of chromosome "halos" prepared by high salt or diiodosalicylate extraction showed no exclusive targeting of SAR/MAR sequences to the chromosome scaffold. We showed, however, that the amounts of condensins and phosphorylated histone H3 inside vector concatamers were similar to surrounding chromosomal arms in both expressing and not expressing GFP-lacI cells. |
