Figure 2.

Structure of the mHSF3 gene and DNA-binding activity of its products. (A) Exons and introns are denoted by boxes and lines. Exon 1 and exon 1a encode the translation start sites of mHSF3a and mHSF3b, respectively. The exons coding for the functional domains are indicated at the top. DBD, DNA-binding domain; HR-A/B, heptad repeat of hydrophobic amino acids A and B; DHR, downstream of the heptad repeat. The positions and directions of the primers used to amplify the cDNA fragments I–V by RT-PCR are indicated at the bottom. (B) RT-PCR was performed using total RNA from various tissues of 6-wk-old ICR mice. The plasmid pcDNA3.1-mHSF3a or b was used as a template for the control PCR (mHSF3). cDNA fragments I–V were sequenced. The fragment II and the upper fragment V contain exon 11, whereas the fragment I and the lower fragment V do not. (C) Comparison of structure between mHSF3a and mHSF3b. Numbers of amino acids are indicated. mHSF3b contains a part of the DBD domain of mHSF3a (amino acids 34–114). (D) mHSF3a binds specifically to HSE. GST and mHSF3a, mHSF3b, hHSF1, and hHSF4 fused to GST were expressed in E. coli. Bacterial cell lysate was incubated with ³²P-labeled ideal HSE oligonucleotides and loaded on a 4% native gel (left). HSF, HSF:HSE complex; Free, free probe. Competition assay was performed using cold HSE86 oligonucleotides containing three perfect nGAAn units and its mutants as competitors. (E) DNase I footprinting analysis of GST-mHSF3a binding to the promoter of the human Hsp70 gene. Increasingly large amounts of lysate containing GST-mHSF3a (0, 0.5, 1, 2, and 4 μl of cell lysate) were assayed. Cont., zero microliters of lysate; A+G, A and G ladders. The HSE contains five inverted nGAAn units located at −91 to −115.