(A) DNAase I protection of regions 1 and 2 in en DNA. A Clal-Nael fragment from plasmid p615, 5′ end–labeled at the Clal site, was incubated for 30 min at 0°C without protein (0) or with 0.9 (1), 4.4 (2), or 22 μg (3) of bacterial extract containing the En HD fusion protein (construct A in ), partially digested with DNAase I as described in Experimental Procedures and electrophoresed on a 6% sequencing gel. The Ftz lanes represent protection obtained by 1.5 (1), 7.4 (2), or 37 (3) μg of bacterial extract containing the Ftz fusion protein (construct G in ). Protected (−) and enhanced (+) sites of DNAase I cleavage in and around sites 1 and 2 are indicated. The arrowhead indicates an enhanced band present only when the Ftz protein is used. No protection was observed in bacterial extracts producing truncated, inactive fusion proteins. A third protected region contained in this fragment is not visible in this separation. Protection by the En fusion spans 29,18, and 20 bp for regions 1,2, and 3, respectively.
(B) DNAase I protection of a fragment containing six copies of the NP sequence (NP6; see ). Increasing amounts of the En fusion protein extract, no protein (0), 0.35 (1), 1.75 (2), 9 (3), or 44 μg (4) in 25 μl were incubated with the DNA fragment (end-labeled at its HindIII site). Digestion with 1 μg/ml of DNAase I is as in Experimental Procedures. The arrows indicate the positions and orientations of the six NP consensus sequences. The positions of the bands resulting from DNAase I cuts are indicated for the first copy of the NP sequence. These positions of cleavage are repeated in each subsequent copy of the NP sequence having the same orientation (copies 1 to 4). A characteristic pattern of protection/enhancement due to the En protein extract is observed in each of these copies. This pattern changes for copies 5 and 6, which are in the opposite polarity. Calcitonin fusions C and D (see ) exhibit a similar pattern of protection.
(C) Concentration of En HD fusion protein extract required to protect one or three copies of the LP sequence (see ). Fragments LP1 and LP3 were footprinted with increasing amounts of the extract: no extract (0); 1.3 (1); 2.7 (2); 5.5 (3); 11 (4); 22 (5); or 44 μg (6) of total protein. The positions of the DNAase I cuts within the various LP sequences are indicated by comparison with the G/A Maxam–Gilbert sequencing lane (note that the positions of the bands in this ladder are shifted compared with the DNAase I lane because of the difference in the position of cleavage []). Each palindrome and its center is indicated. The dashed lines represent the limits of each copy.