Cloning, nucleotide sequence and expression of rat heat inducible hsp70 gene

In rat cells hyperthermia induces two hsp70 transcripts of 2.5 kb and 2.7 kb. We have cloned and determined the nucleotide sequence of a gene (named hsp70.1) encoding the 2.5 kb transcript as shown by Northern blot analysis using the 5' end and 3' end specific hybridization probes. It contains an uninterrupted open reading frame of 1926 bp, it encodes a protein of approx. 70,100 Da and the predicted amino acid sequence of its product shows 98% similarity to the mouse hsp70.1 protein. The transcription start site was localized 224 bp upstream the ATG codon by RNase protection and primer extension mapping. Upstream the transcription initiation site several potential regulatory motifs including a TATA box, two Sp1 binding sites, one inverted and one direct CCAAT box and three HSEs (heat shock elements) were found. Transfection experiments with constructs in which the CAT reporter gene was fused to fragments of the 5' end flanking sequences of the isolated gene confirmed that the promoter of the rat hsp70.1 gene is functional and heat inducible.