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Fig. 2. FISH detection of inserted S/MARs (from the human IFN-ß locus) following DNase I digestion of the halo, demonstrating different nuclear matrix association patterns. The yellow FISH signals of the S/MAR anchor sequences show that they are tightly bound to the nuclear matrix and are therefore protected from nuclease digestion. (A) A 100-copy S/MAR-containing insert following DNase I digestion that displays multiple FISH signals in a pattern of many yellow dots. Because the halo or loop portion has been digested away, this pattern indicates that many but not all the S/MAR copies were tightly bound and were positioned to serve as loop anchors (indicated by arrows). (B) The FISH signal of a single-copy insert following DNase I digestion displays a single yellow spot localized to the nuclear matrix (indicated by the arrow). The great majority of single-copy S/MAR-containing inserts were detected on the nuclear matrix, whereas multiple-copy S/MAR-containing inserts all had a portion of the S/MARs associated with the nuclear matrix as well as detected on the loop. This illustrates that, regardless of the number of copies, those sequences that are anchored to the nuclear matrix are indeed strongly anchored and resistant to DNase I digestion.
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