FIGURE 1.

ISX protein binds with BCMO1 promoter fragments. A, genomic DNA was isolated from mouse intestine and, using primer pairs listed in Table 1, the full-length Bcmo1 promoter was PCR amplified into six overlapping fragments (F1–F6) as indicated. Individual fragment sizes and positions are relevant to the Bcmo1 ATG start site. B, Western blot analysis for ISX protein expression in BL21 cells (± isopropyl β-d-thiogalactopyranoside (IPTG) induction), using the anti-His antibody. P, pellet fraction; S, supernatant/soluble fraction. C, in EMSAs, individual ³²P-labeled Bcmo1 promoter fragments were incubated with crude ISX protein. Bcmo1 promoter fragments F2 and F3, each capable of interacting with ISX protein are indicated by the complex/shift (arrow) and faster migrating unbound [³²P]DNA probes are shown as free probes within a bracket. D and E, ISX protein binds specifically to Bcmo1 promoter fragments 2 and 3. ³²P-Labeled Bcmo1-promoter fragment 2 (D) and fragment 3 (E) were incubated with the supernatant fraction of ISX protein. Protein-DNA complexes were resolved on 6% PAGE gels, and results were obtained by autoradiography. Shifts of the ISX protein bound Bcmo1-probe complexes are indicated by arrows. Although the free unbound probe migrated rapidly upon electrophoreses, the retarded, shifted ISX-Bcmo1 bound probe migrated more slowly and above the labeled free DNA probe. The figures show representative autoradiographies of images from at least two independent experiments using different ISX protein crude extract preparations.