Molecular Cloning and Characterization of Rat 86-kDa Heat Shock Protein

• Main Authors: Chia Wei Li, 李家偉
• Other Authors: Margaret Dah-Tsyr Chang
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/42705201967743465834

碩士 === 國立清華大學 === 生物技術研究所 === 89 === HSP90 is the most abundant cytosolic HSPs in eukaryotic cells. In vertebrates, HSP90s are encoded by two distinct genes designated as hsp86 and hsp84. Here we report the molecular cloning and characterization of the rat hsp86. Based on the homology among human hsp90-α, porcine hsp90, and mouse hsp86, the conserved region in rat hsp86 was amplified by PCR and used as the probe. The full-length rat hsp86 was obtained from genomic library screening, followed by subcloning and Exonuclease III nested deletion. The assembly result revealed that a 2.6 kb cDNA spreading over about 7.3 kb genomic region contained 11 exons and 10 introns (GeneBank accession number AJ 297736). All splice sites confirmed to the GT/AG rule, except for the splice donor site of intron 3. 5’-RACE was used to map the transcription start site to the position lying 641 base pairs upstream of the ATG codon. The deduced amino acid sequence of rat HSP86 is 97%, 98%, and 98% homologous to human hsp90-α, porcine hsp90, and mouse hsp86 respectably. Moreover, two HSP90s of similar molecular weights were distinguished by SDS-PAGE with a special buffer condition. To detect the difference in gene expression of hsp86 and hsp84, [35S] metabolic labeling and RT-PCR was performed. These results suggest that rat HSP86 displays a higher level of induction than HSP84 upon treatment with 60 mM CdCl2.