Abstract
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.
Highlights
MAR estimated far by in situ mapping experi- on the nuclear scaffold using simple binding assays with ments
The DNA segments involved in the matrix binding, designated MAR’ or SAR, have been identified in many gene regions and their functional significance has been implicated by a number of bind DNA in a sequence-independent fashion, a similar experimental strategy should be applicable to identify the attachment sites for nuclear DNA by using a plasmid DNA containing MAR sequences
We show that the nuclear scaffold selectivelybinds MAR-containing DNA fragments and that ina Southwestern binding assay the binding occurs to a scaffold protein with apparent molecular size of
Summary
Scaffold minimal length ofMAR for efficient binding, bind to MAR loading buffer containing 2% SDS and 5% (v/v) 2-mercaptoethanol in a cooperative manner, and localize in the internanl etwork portion of the scaffold Despite these similarities, SP120 and ARBP are probably different proteins, since the molecular size of ARBP issignificantly smaller (75-80 kDa in mammals) by incubation at 37 "C for 10 min, before applying to a 7.5% polyacrylamide mini gel (without sample wells made). Purified and renatured scaffold proteins were incubated at 30 'C for Preparation of the Nuclear Scaffoldand DNA Binding Studies with 15 min with end-labeled MAR and unlabeled competitor DNAs, either. DNA Binding Studies with Protein Blots (Southwestern Blotting)- immunization, the antibody titers in seraof the four mice were tested. The stained samples were viewed undera fluorescence microscope and photographed
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