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Fig. 4. Binding of the WER to CPC promoter. (A) EMSA using the purified
WER protein and two 20 bp long DNA fragments (WBSI and WBSII) from the
CPC promoter. Lane 1 of each gel contains the free DNA probe without
the WER protein, and lane 2, 3 and 4 of each gel contain increasing amount of
the WER protein (1x, 3x and 6x). (B) Sequences of mutated
WBSI and WBSII used in this experiment. Each double-stranded probe contains a
single base substitution as indicated. Lines indicate no change. (C) EMSA
using the purified WER protein and the mutated probes as shown in B. (D)
Competitive EMSA. Competitions were performed using increasing amounts of
wild-type DNA fragments or some mutated derivatives as shown in B. Lane 1 of
each gel contains the free DNA probe without the WER protein and lane 2
contains the WER protein and the radiolabeled wild-type DNA fragment (WBSI or
WBSII) as a probe without a competitor. Increasing amounts (1x,
10x and 50x) of the unlabeled wild type DNA fragments (lanes 3, 4
and 5), and the unlabeled mutated versions of WBSI or WBSII (lanes 6, 7, 8 and
lanes 9, 10, 11) were added as a competitor. (E) Binding competition between
WBSI and WBSII. Increasing amounts of the wild-type or mutated derivative
(m10) of WBSII, and increasing amounts of the wild-type or mutated derivative
(m6) of WBSI were used as an unlabeled competitor for the binding of the WER
protein to radiolabeled WBSI and to radiolabeled WBSII, respectively. Lane 1
contains only free probe (radiolabeled WBSI or WBSII) and lane 2 contains the
WER protein and the radiolabeled wild-type DNA fragment (WBSI or WBSII) as a
probe without a competitor. Lanes 3, 4 and 5 of each gel contain increasing
amounts of the unlabeled wild-type DNA fragments (unlabeled WBSI for
radiolabeled WBSII, and unlabeled WBSI for radiolabeled WBSII) as a competitor
(1x, 10x and 50x). Lanes 6 and 7 contain the same components
as lane 3, 4 and 5, except that they contain increasing amounts of unlabeled
mutated versions of WBSII (m10) or WBSI (m6) instead of the unlabeled
wild-type DNA fragments as a competitor.
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