Kobayashi K, Kanno S, Smit B, van der Horst G T, Takao M, Yasui A
Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, 980 8575 Sendai, Japan.
Nucleic Acids Res. 1998 Nov 15;26(22):5086-92. doi: 10.1093/nar/26.22.5086.
We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s).
我们分离并鉴定了小鼠光解酶样基因mCRY1（mPHLL1）和mCRY2（mPHLL2），它们属于包括植物蓝光受体在内的光解酶家族。mCRY1和mCRY2基因分别位于第10号染色体的C区和第2号染色体的E区，在所检测的所有小鼠器官中均有表达。我们利用其C端序列制备了针对每个基因产物的特异性抗体，这两个基因的C端序列完全不同。对培养的小鼠细胞进行免疫荧光染色显示，mCRY1定位于线粒体，而mCRY2主要存在于细胞核中。通过对分离的小鼠肝细胞提取物进行免疫印迹分析，证实了CRY蛋白的亚细胞分布。利用绿色荧光蛋白融合肽，我们发现小鼠CRY2蛋白的C端区域含有一个独特的核定位信号，而CRY1蛋白中不存在该信号。CRY1的N端区域显示含有线粒体转运信号。来自小鼠和人类细胞的重组CRY1蛋白以及天然CRY1蛋白均显示出与DNA琼脂糖有紧密的结合活性，而在与CRY1相同的条件下，CRY2蛋白根本不与DNA琼脂糖结合。哺乳动物光解酶同源物不同的细胞定位和DNA结合特性表明，尽管这两种蛋白质在序列上相似，但它们具有不同的功能。
