Identification and characterization of a nuclear scaffold protein that binds the matrix attachment region DNA.
The association of nuclear DNA with the nuclear matrix (scaffold) is mediated by defined segments of DNA called matrix association region (MAR). By using a plasmid harboring a portion of the Ig kappa gene within which MAR had been located, we searched for proteins recognizing MAR in the nuclear scaffold components electrophoretically separated and blotted onto a membrane. In the presence of nonspecific competitor DNA, the labeled plasmid selectively bound to a protein with apparent molecular weight of 120,000 (designated SP120). The protein was purified directly from SDS-polyacrylamide gels and renatured by a guanidine hydrochloride procedure. The DNA region in the plasmid responsible for the binding to the solubilized SP120 coincided with the 365-base pair HindIII-HinfI fragment that had been identified as MAR. In solution, SP120 exhibited a cooperative mode of interaction with the end-labeled MAR fragment. Measurement of relative affinities of MAR subfragments to SP120 showed that the whole region is required for efficient binding. This is consistent with the minimal length for MAR estimated thus far by in situ mapping experiments. The MAR derived from another gene, fushitarazu, also bound specifically to SP120. Immunostaining of whole cells and isolated nuclei with a monoclonal antibody raised against SP120 indicated that the protein is localized in a nuclear skeletal structure. These results suggest the involvement of SP120 in the MAR-mediated anchorage of nuclear DNA to the nuclear scaffold.
Tsutsui
K
K
Department of Molecular Biology, Okayama University Medical School Japan.
Tsutsui
K
K
Okada
S
S
Watarai
S
S
Seki
S
S
Yasuda
T
T
Shohmori
T
T
D000818:Animals; D034961:Antigens, Nuclear; D001483:Base Sequence; D001667:Binding, Competitive; D001710:Biotin; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000302:isolation & purification / Q000378:metabolism*; D004330:Drosophila / Q000235:genetics; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D015183:Restriction Mapping
D034961:Antigens, Nuclear; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D001710:Biotin; D004247:DNA