Analysis of Cellular Stress Response in Two AUG of Human SND1 Gene
10.3969/j.issn.0253-9896.2014.07.001
- VernacularTitle:针对人SND1基因两个AUG的细胞应激分析
- Author:
Xingjie GAO
;
Jinyan HE
;
Lin GE
;
Yi ZHANG
;
Xue FU
;
Jie YIN
;
Wei ZHANG
;
Xuebin SHI
;
Zheng SU
;
Zhi YAO
;
Jie YANG
- Publication Type:Journal Article
- Keywords:
recombinant fusion proteins;
stress;
plasmids;
gene expression;
SND1;
AUG;
stress granules
- From:
Tianjin Medical Journal
2014;(7):625-629
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct eukaryotic Flag (DYKDDDDK) expressing recombinant plasmids, pCMV-N-Flag-SND1-No1/2, which contain the coding sequence of human SND1-No1(from 1st AUG)or SND1-No2 (from 2nd AUG), and perform the cellular localization analysis of Flag-tagged SND1-No1/2 under stress condition to study the function of the two AUG in the SND1 containing stress granules formation. Methods The gene fragments of SND1-No1/2 were amplified by PCR from the whole SND1 transcript and inserted into pCMV-N-Flag expressing vector through BamHI/EcoRI double en-zyme digestion and T4 DNA Ligase connection. The recombinant pCMV-N-Flag-SND1-No1/2 plasmids were transfected in-to HeLa cells and the expression of Flag-SND1-No1/2 fusion proteins was examined by Western blotting assay. Immunofluo-rescence assay was performed to detect the co-localization of Flag-SND1-No1/2 with endogenous SND1 granule. Results The pCMV-N-Flag-SND1-No1/2 were sequenced and digested correctly by restriction single/double enzyme. The Flag-tagged SND1-No1/2 fusion proteins were also detected in transfected HeLa cell by Western blotting assay. Both of them showed the co-localization with endogenous SND1 granule. Conclusion The recombinant eukaryotic plasmids of pCMV-N-Flag-SND1-No1/2 were constructed successfully and expressed effectively. The depletion of 1st AUG failed to af-fect the formation of SND1 containing stress granules.
