Identification of a novel 9-kDa polypeptide from nuclear extracts. DNA binding properties, primary structure, and in vitro expression.
Using a modified DNA mobility shift assay, we have identified and purified a novel 9-kDa polypeptide (designated p9) from plasmacytoma nuclear extracts which forms salt-stable DNA-protein complexes without apparent sequence specificity. In competition studies, p9 bound exclusively with naturally occurring DNA relative to RNA and preferentially with polydeoxypyrimidines among 10 homopolynucleotides tested. Nuclear extracts prepared from various murine and human cell lines contained a common factor which, when cross-linked with photoreactive [32P]DNA, co-migrated with covalent [32P]DNA-p9 complexes on polyacrylamide gels. An oligonucleotide, constructed on the basis of the N-terminal 29 amino acid residues of p9, was employed to isolate a 700-base pair cDNA clone encoding the complete polypeptide. On Northern blots, p9 cDNA hybridized with a mRNA species of comparable size from both mouse and human cell lines, suggesting a significant degree of interspecies sequence conservation. Amino acid and cDNA sequence analyses demonstrated that p9 derives from the 77-residue C-terminal domain of a 14-kDa polypeptide comprised of 127 amino acids. DNA binding activity was exhibited by peptides synthesized in vitro from run-off RNA transcripts corresponding to the truncated 9-kDa C-terminal domain, but not to the 14-kDa precursor, implicating proteolysis as a post-translational mechanism required for functional activation of p9.
Ballard
D W
DW
Howard Hughes Medical Institute, New Haven, Connecticut.
Philbrick
W M
WM
Bothwell
A L
AL
D000818:Animals; D001483:Base Sequence; D002467:Cell Nucleus / Q000032:analysis; D004273:DNA, Neoplasm / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000032:analysis*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000032:analysis*; D010954:Plasmacytoma / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D014157:Transcription Factors
D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; C056169:Rpo2tc1 protein, mouse; D014157:Transcription Factors