Genomic cloning and promoter analysis of the GAHSP40 gene

The new heat shock protein (GAHSP40), which binds to Gadd34, is a member of the Hsp40 family gene and has a J domain, which is similar to bacterial DNAJ. We have isolated and sequenced the mouse GAHSP40 gene including 1.6 kb of the 5'-flanking region. Primer extension analysis revealed that the transcription initiation site was located 36-bp upstream of the ATG translation initiation codon. In order to identify the heat-responsive regions in the GAHSP40, NIH3T3 cells were transiently transfected with a series of 5' terminus-truncated mutants of the GAHSP40 promoter linked to the luciferase reporter gene. We found that the region of -284 to -184 bp from initiation start site responded to heat shock treatment. By the gel shift analysis, we found the heat shock elements (HSEs) located in this region from -257 to -225. This HSEs has five 5 bp motifs. The transfection studies using HSEs mutant vectors revealed that those 3' two 5 bp motifs are essential for heat responsive transcription.