A novel muscle-specific factor
is responsible for recognition of TGTnACA sequences in all three
5′-distal sub-elements of the rMME of rm-hsc71m. (A) Electrophoretic
mobility shift analysis showing binding specificity of a novel muscle-specific
factor to rm-hsc71mE-related sequences in each sub-region of the rMME
of rm-hsc71m. The rm-hsc71mE-related sequences
in each region (a box, b box and c box, respectively; see panel
D for each sequence) were identified by a computer search. The bm
box is derived from the b box with three base changes. The MCKE-RH
is the right half E-box in the MCKE (see Table 1). Each box was
used as a probe in a gel shift analysis containing Rivulus muscle
or liver S150 extracts. Specific and non-specific bands are marked
by filled and unfilled arrowheads, respectively. (B)
Competitive electrophoretic gel mobility shift analysis showing
the relative binding affinity of the muscle-specific factor to each
box. Increasing amounts (0, 10× and
50×) of a molar excess of cold competitor
were added to the reaction containing the b box probe and Rivulus
muscle S150 extracts. Specific and non-specific bands are marked
by filled and unfilled arrowheads, respectively. (C)
Electrophoretic mobility shift analysis showing that the central TGTnACA
sequences are critical for muscle-specific factor binding. A double-stranded
mutant oligonucleotide (m box; see panel D) in which all of the
central conserved sequences of b box were altered (TGTGACA→ACAGTGT) was used as probe, along with parent b box
probe, containing Rivulus muscle and liver S150 extracts. (D)
Comparison of sequences recognized by a muscle-specific factor (only
a top strand of sequences is shown: a, b and c boxes corresponded
to sequences from –2627 to –2606, from –2296
to –2275 and from –2009 to –1988, respectively).
Consensus binding bases are indicated as a boldface letter in a
box. Boldface letters indicate homologous sequences in the MCKE-RH
and the b box, and the underlined sequences in the m box indicate
mutagenized bases from the b box.