Grimm B, Ish-Shalom D, Even D, Glaczinski H, Ottersbach P, Ohad I, Kloppstech K
Institut für Botanik, Universität Hannover, Federal Republic of Germany.
Eur J Biochem. 1989 Jul 1;182(3):539-46. doi: 10.1111/j.1432-1033.1989.tb14861.x.
A cDNA clone, pCHS62, was isolated using poly(A)-rich RNA from heat-shocked Chlamydomonas reinhardtii cells. The clone has a length of 1.1 kb and codes for the complete heat-shock protein which was reported to be associated with the grana region of the thylakoid membranes and ascribes protection against photoinhibition during heat-shock. An expression vector prepared in the pUC19 plasmid was used to obtain a fusion protein against which rabbit polyclonal antibodies have been raised. The antibodies react specifically with the heat-shock protein of 22 kDa synthesized in vivo during heat-shock, which is localized in the grana thylakoids, with the in vitro translated product using poly(A)-rich RNA from heat-treated cells as well as with the hybrid release translation product of the pCHS62 clone. The clone was sequenced. It contains a 5' region consisting of 85 nucleotides, an open reading frame of 471 nucleotides and a non-coding 3' region of 600 nucleotides. Northern hybridization indicates a length of 1.7 kb for the messenger RNA of heat-shock protein 22. Analysis of similarity between the derived amino acid sequence of this protein and other heat-shock proteins demonstrates that this protein belongs to the small-molecular-mass plant heat-shock protein family and also shows similarities with animal heat-shock proteins including the presence of a short region possessing similarity with bovine alpha-crystalline as reported for other heat-shock proteins. The molecular mass of the protein as determined from the sequence is 16.8 kDa. Despite its localization in the chloroplast membranes, it does not seem to include a transit peptide sequence, in agreement with previous data. The sequence contains only a short hydrophobic region compatible with its previously reported localization as a thylakoid extrinsic protein.
利用来自热激莱茵衣藻细胞的富含多聚腺苷酸（poly(A)）的RNA分离出一个cDNA克隆pCHS62。该克隆长度为1.1 kb，编码完整的热激蛋白，据报道该蛋白与类囊体膜的基粒区域相关，并赋予热激期间对光抑制的保护作用。使用在pUC19质粒中构建的表达载体获得了一种融合蛋白，并以此制备了兔多克隆抗体。这些抗体与热激期间体内合成的22 kDa热激蛋白特异性反应，该蛋白定位于基粒类囊体中，与使用来自热处理细胞的富含多聚腺苷酸的RNA进行体外翻译的产物以及pCHS62克隆的杂交释放翻译产物也发生反应。对该克隆进行了测序。它包含一个由85个核苷酸组成的5'区域、一个471个核苷酸的开放阅读框和一个600个核苷酸的非编码3'区域。Northern杂交表明热激蛋白22的信使RNA长度为1.7 kb。对该蛋白推导的氨基酸序列与其他热激蛋白之间的相似性分析表明，该蛋白属于小分子质量植物热激蛋白家族，并且与动物热激蛋白也显示出相似性，包括存在一个与牛α-晶体蛋白具有相似性的短区域，这与其他热激蛋白的报道一致。根据序列确定的该蛋白的分子量为16.8 kDa。尽管它定位于叶绿体膜中，但与先前的数据一致，它似乎不包含转运肽序列。该序列仅包含一个短的疏水区域，与其先前报道的作为类囊体外周蛋白的定位相符。
