Genomic cloning and promoter analysis of the GAHSP40 gene

• Published in: Journal of cellular biochemistry 2002, Vol.84 (2), p.401-407
• Main Authors: Hamajima, Fumiyasu, Hasegawa, Tadao, Nakashima, Izumi, Isobe, Ken-ichi
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; Base Sequence; Cloning, Molecular; DNA; DNA Primers; GADD34; heat shock element; heat shock factor; Heat-Shock Proteins - genetics; HSP40 Heat-Shock Proteins; Mice; Molecular Sequence Data; Mutation; Promoter Regions, Genetic; Transcription, Genetic
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.10029

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. J. Cell. Biochem. 84: 401–407, 2002. © 2001 Wiley‐Liss, Inc.