Specific interaction of mouse major satellite with MAR-binding protein SAF-A.
A DNA-binding activity specific to the major mouse satellite (satMa) has been detected in a nuclear matrix protein extract by electrophoretic mobility shift assays (EMSA) after fractionation by ion exchange chromatography. An antibody raised against the satMa-protein complexes recovered from preparative EMSA recognizes on Western blots one major polypeptide with an apparent molecular mass of 120 kDa. The protein also has a similar affinity for a matrix-associated region (MAR) fragment. We demonstrate that the protein is a murine homologue of SAF-A which has been shown to bind selectively to MARs and is responsible for the satMa-binding activity in the chromatographic fractions. SatMa has significant homology to the mouse minor satellite fragments, but its binding of SAF-A shows much less affinity. No protected regions of significant length were found by footprinting, but multiple T residues scattered within the satMa sequence are protected, indicating that the whole fragment is involved in the binding to SAF-A. Combined immunofluorescence (SAF-A) and FISH (satMa) with in situ nuclear matrix procedures reveal that SAF-A and satMa colocalize. SAF-A appears as bright dots in interphase nuclei, presumably associated with MARs, predominantly surrounding and covering heterochromatic areas. A scheme based on morphological observations and biochemical data of SAF-A double satMa/MAR specificity is discussed.
Lobov
I B
IB
Institute of Cytology RAS, St Petersburg, Russia.
Tsutsui
K
K
Mitchell
A R
AR
Podgornaya
O I
OI
D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004276:DNA, Satellite / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D017404:In Situ Hybridization, Fluorescence; D007399:Interphase; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D015530:Nuclear Matrix / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism*; D017830:Octoxynol / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry / Q000378:metabolism*
D034961:Antigens, Nuclear; D004276:DNA, Satellite; D004268:DNA-Binding Proteins; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D012261:Ribonucleoproteins; D017830:Octoxynol; D004247:DNA