Lisowska K, Krawczyk Z, Widłak W, Wolniczek P, Wiśniewski J
Department of Tumor Biology, Institute of Oncology, Gliwice, Poland.
Biochim Biophys Acta. 1994 Sep 13;1219(1):64-72. doi: 10.1016/0167-4781(94)90247-x.
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.
在大鼠细胞中，热激诱导产生两种大小分别为2.5 kb和2.7 kb的hsp70转录本。我们已经克隆并测定了一个编码2.5 kb转录本的基因（命名为hsp70.1）的核苷酸序列，这是通过使用5'端和3'端特异性杂交探针进行Northern印迹分析所证实的。它包含一个1926 bp的不间断开放阅读框，编码一种约70,100 Da的蛋白质，其预测的氨基酸序列与小鼠hsp70.1蛋白显示出98%的相似性。通过RNA酶保护和引物延伸定位法，转录起始位点位于ATG密码子上游224 bp处。在转录起始位点上游发现了几个潜在的调控基序，包括一个TATA盒、两个Sp1结合位点、一个反向和一个正向CCAAT盒以及三个热休克元件（HSE）。用构建体进行转染实验，其中CAT报告基因与分离基因5'端侧翼序列的片段融合，证实大鼠hsp70.1基因的启动子具有功能且可被热诱导。
