Journal: Nucleic Acids Research
Article Title: DNA of a circular minichromosome linearized by restriction enzymes or other reagents is resistant to further cleavage: an influence of chromatin topology on the accessibility of DNA
Figure Lengend Snippet: Minichromosome DNA is cut at only one site by SpeI or SwaI, which have seven and two cutting sites, respectively. ( A ) Circular minichromosome DNA showing SpeI, SwaI and PacI sites; SpeI fragment lengths are not shown for clarity. ( B ) Minichromosome DNA from permeabilized cells incubated with SpeI or ( C ) with SwaI, and deproteinized. Lane DNA, SwaI fragments produced in deproteinized cells. ( D ) The SwaI cleavage site mapped by gel hybridization. DNA linearized by SwaI (200 U/ml, 2 h) was isolated from a PFGE gel, cut by PacI (100 U/ml, 18 h), and the products were separated by PFGE. Lanes M, oligomers with HindIII fragments of λ DNA. The four fragments produced (∼140, 100, 72 and 29 kb) represent a mixture of the pairs produced after SwaI had cut at only one of its two sites in different minichromosomes. ( E ) The SwaI cleavage site mapped by fibre-FISH. The positions of the SwaI sites and hybridization probes (green) are shown above; the biotin-labelled probes were detected with anti-biotin antibodies (green) and BrdU-labelled DNA with anti-BrdU antibodies (red). Images show representative linear molecules from the two classes observed, which had been cut by SwaI (shown by the extremities of the molecule) at either the left (upper panel) or the right (lower panel) site on the map. The probe positions were aligned approximately considering the variable stretching during combing ( 22 , 23 ). Below, linear molecules cleaved at the single PacI site for comparison.
Article Snippet: Restriction fragments of this DNA cut with SpeI or SwaI (100 U/ml) were separated by PFGE in 1% LMP agarose at 190 V/cm for 7 or 20 h and switch time ramped linearly from 0.4 to 6 or 0.3 to 3 s, respectively, excised from gels and purified on Ultrafree columns (Millipore).
Techniques: Incubation, Produced, Hybridization, Isolation, Fluorescence In Situ Hybridization