The type of DNA attachment sites recovered from nuclear matrix depends on isolation procedure used.
A large variety of DNA sequences have been described in nuclear matrix attachment regions. It could be most likely a result of the different methods used for their isolation. The idea about how different types of known DNA sequences (strongly attached to the nuclear matrix, weakly attached, or not attached) directly participate in anchoring DNA loops to the nuclear matrices isolated by different experimental procedures was tested in this study. Matrix-attached (M) and matrix-independent or loop (L) fractions as well as nuclear matrices were isolated using extractions of nuclei with 25 mM lithium 3,5-diiodosalicylate (LIS), 2 M NaCl, 0.65 M ammonium sulphate containing buffers followed by DNase I/RNase A digestion, or according to so designated conventional method. Using PCR-based and in vitro binding assays it was established that LIS and ammonium sulphate extractions gave similar results for the type of attachment of sequences investigated. The harsh extraction with 2 M NaCl or the conventional procedure led to some rearrangements in the attachment of DNA loops. As a result a big part of matrix attached sequences were found detached in the loop fractions. However, the in vitro binding abilities of the MARs to the nuclear matrices isolated by different methods did not change.
Donev
R M
RM
Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia.
D000645:Ammonium Sulfate; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002458:Cell Fractionation / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D007463:Iodobenzoates; D015530:Nuclear Matrix / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012459:Salicylates
D017931:DNA Primers; D007463:Iodobenzoates; D012459:Salicylates; C020891:3,5-diiodosalicylic acid; D004247:DNA; D000645:Ammonium Sulfate