博士 / 國立海洋大學 / 水產生物技術研究所 / 90 / Heat shock proteins (HSPs) with different molecular weights are rapidly synthesized within stressed cells after exposure to environmental stresssors. Among the stress-inducible HSPs, the members of HSP70 function as a molecular chaperone. Besides the stress-inducilbe HSP70, a form of HSP70 called heat shock cognate protein 70 (HSC70), which is constitutively expressed in unstressed cells under physiological conditions. A spontaneous high expression of heat shock protein 70 (HSP70) was detected in zebrafish (Danio rerio) at early larval stage (80-84 hr after fertilization), but HSP70 level was either low or barely detectable in 12, 24, 36, 60, and 108-hr-old zebrafish. The level of HSP70 in 84-hr-old larvae was estimated to be 6 to 8-fold that of 12-hr-old embryos. The spontaneous HSP70 expression was a transient process, as the level of HSP70 in 108-hr-old larvae dropped to that in 12-hr-old embryos. Although the transient increase of a 62-kDa HSF1-like polypeptide in 72 to 96-hr-old larvae seemed to correlate with the onset of the spontaneous HSP70 production, an anti-HSF1 antibody cocktail supershifted the stress-induced heat shock element (HSE) binding complex but not the unstressed zebrafish extracts, indicating that the spontaneous HSP70 expression was not triggered by a HSF1-like factor. Northern blot analysis indicated no apparent change in total hsp70 mRNA expression in zebrafish at different stages. Using gene-specific primers, quantitative RT-PCR analysis provided supporting evidence that normal zebrafish at all stages had barely detectable levels of stress-dependent hsp70 mRNA and these zebrafish expressed similar levels of hsc70 mRNA after normalization against b-actin expression, thus excluding the possible involvement of stress-related hsp70 mRNA in producing the spontaneously expressed HSP70. Because no increase in hsc70 or hsp70 mRNA accumulation could be detected in 80-and 84-hr-old larvae, the spontaneous high expression of HSP70 appears to be controlled by a mechanism different from that regulates the expression of stress-inducible HSP70. The mRNAs from heat-treated zebrafish were successfully translated in rabbit reticulocytes in the presence of [35S]-methionine including the stress-inducible HSP70, whether hsc70 mRNA from 36 to 108-hr-old zebrafish had different translatability was not revealed by this in vitro translation assay. In order to examine if the spontaneous HSP70 expression was regulated by the variation in hsc70 mRNA in zebrafish at different developmental stages, five gene specific primers targeting different positions of the 3’ end of hsc70 mRNA were designed. Gene-specific semi-quantitative RT-PCR showed that the level of hsc70 mRNA containing 3’-UTR downstream of the stop codon was 25 to 30% of that in 84-hr-old larvae. No variation in the 3’-end structure of hsc70 mRNA was found between 80 and 84-hr-old larvae. To determine if the spontaneously expressed HSP70 acted as a chaperone during zebrafish development, the extracts of 84-hr-old zebrafish were crosslinke with different kinds of cross-linking agents including EGS, DSS and glutaraldehyde. Three crosslinked complexes containing HSP70 with molecular weights estimated to be 75 kDa, 280 kDa and 300 kDa, respectively, detected by Western blot analysis. When the crosslinked complexes were probed with the anti-dj2 antibody, the cochaperone dj2 the zebrafish dj2-like factor was also present in the 300-kDa complex. Moreover, the expression of dj2-like factor in developing zebrafish seemed to be regulated by a mechanism similar to that controlling the spontaneous HSP70 expression, since similar levels of dj2 mRNA was detected in 12 to 108-hr-old zebrafish by Northern blot analysis when a high expression of dj2-like factor was found in 80 to 84-hr-old zebrafish. To sum up, the spontaneously expressed HSP70 in 80 to 84-hr-old zebrafish should be controlled at the translational level, possibly related to the variation in the 3’-end structure of hsc70 mRNA. The spontaneously expressed HSP70 might bind the cochaperone dj2 in developing zebrafish to act as a molecular chaperone.
| Identifer | oai:union.ndltd.org:TW/090NTOU0613019 |
| Date | January 2002 |
| Creators | Fu-lung Yeh, 葉富隆 |
| Contributors | Todd Hsu, 許濤 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 103 |
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