((45C48), a function of obvious utility inside a polypeptide component of a complex that functions in recombination. of these switch transcript (15). Since S region transcription is definitely prerequisite to recombination, it is possible that LR1 binding to sites in the S areas might potentiate S region transcription and therefore activate recombination. DNA binding studies have shown the LR1CDNA binding consensus, GGNCNAG(G/C)CTG(G/A), is definitely loose, and LR1 may bind multiple sites in each of the G-rich S areas. This suggests that another possible function for LR1 could be to organize S region DNA before, during, or after recombination. To understand the function of LR1, we have purified SU14813 double bond Z and characterized the activity. LR1 is definitely a complex of two polypeptides, of 106 kDa and 45 kDa (2, 16). With this communication we demonstrate the 106-kDa polypeptide component of LR1 is definitely nucleolin. Nucleolin is an abundant and highly altered protein found in nucleoli, the centers of ribosome biogenesis (17C22). Its N terminus is definitely homologous to histone H1, and like many RNA-binding proteins, nucleolin consists of both RNA acknowledgement motifs (RRM) and Arg-Gly-Gly (RGG) motifs (observe Fig. ?Fig.1).1). The similarity between nucleolin SU14813 double bond Z and histone H1 offers suggested that one part of nucleolin in the nucleolus may be to determine rDNA architecture (23, 24); and nucleolin may function analogously in switching, organizing S region DNA before, during, or after recombination in response to cell cycle controls. Nucleolin has also been thought to regulate rDNA manifestation; and the results we report provide the SU14813 double bond Z 1st conclusive evidence for participation of nucleolin inside a transcription element. Finally, the observation that SU14813 double bond Z nucleolin is definitely a component of a factor that binds specifically to S areas suggests that S areas and rDNA are evolutionarily related. This probability is in accord with early reports of sequence homology between rDNA and portions of the Ig weighty chain locus (25) and with SU14813 double bond Z practical studies showing that rDNA may have unique properties in recombination (26C28). Open in a separate window Number 1 Nucleolin. (DNA, in buffer L comprising 0.2 M KCl; proteinCDNA complexes were captured on streptavidin agarose (30); and protein was eluted with 0.6 M KCL in buffer L (20 mM Hepes, pH 7.5/2 mM EDTA/10% glycerol/1 mM DTT/1 mM phenylmethylsulfonyl fluoride/10 g/ml leupeptin/5 g/ml benzamidine/5 g/ml pepstatin A/5 g/ml aprotinin). The eluate was dialyzed in buffer L comprising 0.1 M KCl and chromatographed on DEAE-Sepharose using a gradient of 0.1C1.0 M KCl in buffer CD121A L. Fractions with LR1CDNA binding activity eluted between 0.45C0.6 M KCl and contained 106 kDa and 45 kDa polypeptides, as observed previously (2). The 106-kDa varieties was isolated by preparative SDS/PAGE and submitted in the gel slice to the W. M. Keck Basis, Yale Medical School, where proteolysis with endoprotease lys C and peptide sequence analysis were carried out. Aware that nucleolin might copurify like a contaminant, we generated highly purified LR1 from your murine pre-B cell collection, PD31. Attempts to remove nucleolin in the 1st purification step by DEAE chromatography resulted in loss of more than 90% of LR1CDNA binding activity, suggesting that, at least in nuclear components, nucleolin may contribute to the stability of the LR1CDNA binding activity. Instead, nuclear draw out (31) from 40 L of cells was fractioned 1st on Heparin Hi-Trap; then by oligonucleotide capture, as explained above; then on Mono Q (Pharmacia), using a 0.05C1.0 M NaCl gradient. Activity assays and immunoblotting showed that most (90%) of the LR1CDNA binding activity flowed through Mono Q, while most ( 95%) of the nucleolin eluted at 0.4 M NaCl, as reported by others (32). The flow-through from your Mono Q column was then subjected to Mono S chromatography, using a 0.05C1.0 M NaCl gradient, and binding activity eluted at about 0.33 M NaCl. At this point the preparation was estimated to be at least 12,000-collapse purified. The purified preparation, which specifically bound to duplex DNA transporting an LR1 site DNA ((34) using anti-nucleolin antiserum at 1:50 dilution. The mAb 12CA5, specific for the HA epitope was prepared as high titer ascites fluid. DNA Mobility-Shift Analysis. Binding to a synthetic duplex oligonucleotide transporting the LR1 S1 site was assayed by DNA mobility-shift, as explained (1)..