| Summary: | 碩士 === 長庚大學 === 基礎醫學研究所 === 86 === Abstract
Cells respond to environmental stresses such as heat, heavy metal ions, oxidative
radicals, cytotoxins, and interferons by preferential accumulation of heat shock
proteins. Some of the heat shock proteins of which molecular weight fall in 12 kDa to
42 kDa are considered to small heat shock proteins. Small heat shock proteins have
more conserved C-terminal domain, but variable N-terminal domains. In cells, these
proteins form oligomer and may protect cells from stresses by maintaining the
stability of cytoskeleton. In human, some of small heat shock proteins include Hsp27,
α-crystallin, p20 and Hsp2B have been found.
A cDNA clone encoding a novel Rab-like protein was isolated from a human
hippocampus library in our library. For resolving the function of the Rab-like protein,
we found a new cDNA clone from fhuman fetal brain library by using yeast two-
hybrid system. The new cDNA clone with 590 bases was predicted to have a open
reading frame encoding a polypeptide of 150 amino acids residues. The sequence of
polypeptide has 40% identity compared with human Hsp27 andα-crystallin. The new
cDNA is then named small heat shock protein like protein cDNA (smhsp-like cDNA)
and the translated polypepteide is called smhsp-like protein.
We confirmed the sequence of the smhsp-like cDNA by screening a human heart
cDNA library and by using 5'-RACE. We subcloned smhsp-like cDNA into suitable
vector and overexpressed smhsp-like protein. For making antibody, we purified the
overexpressed smhsp-like protein as antigen. On another experiment, we found the
mRNA of smhsp-like protein only at human tissues of heart and skeleton muscle. The
same result could not be found on the BALA/c mouse tissues, so we predict that
smhsp-like cDNA expressing only at human specific tissues. We also found the
amount of mRNA could not be detected in our cells, until using the ribonuclease
protection assay (RPA). By the results of RPA, we found that heat and cadmiun could
not change the amount of the smhsp-like mRNA of Hela cells, but the mRNA may be
induced by some unknown components of DMEM. In the final experiment, we found
the expressed smhsp-like protein locate on the cytoplasm of Hela as small granular
particles by transient transfection and immuon-stain.
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