Relative KD for Rox1 binding to mutant sites

Original base in Rox1 consensus sequence
A190 (4.7)20 (1)290 (15)250 (13)150 (7.5)160 (8)100 (5)
C40 (1)250 (13)580 (29)360 (18)180 (9)170 (8.5)70 (3.5)
G490 (12)160 (8)480 (24)260 (13)20 (1)180 (9)150 (7.5)
T25 (0.6)75 (3.8)20 (1)20 (1)270 (14)20 (1)20 (1)
  • The KD values are presented in nanomolars. The bases inthe Rox1 consensus sequence usedin this experiment are numbered as follows: C1C2C3A4T5T6G7T8T9C10T11C12. The single base pair mutants of the core sequence ATTGTT (positions 4 to 9) were cloned into pCY4 (see materials and methods), and the fragments used in gel retardation experiments were 420-bp HindIII fragments. The mutations in C3 were generated using 32-bp synthetic DNA (see materials and methods). Each binding reaction contained 15,000 c pm of DNA (equivalent to 1 ng) and 5-800 ng of MPB-Rox1(HMG) protein. The numbers in parentheses are the fold increase in the KD values (fold weakening of the binding) compared to the consensus sequence. The values for the consensus sequence are presented in boldface type. The KD for the consensus sequence of the 32-bp synthetic DNA was twofold higher than for the 420-bp fragment as seen in the C3 column.