Hagiwara H, Matsuoka M, Ohshima M, Watanabe M, Hosokawa D, Ohashi Y
Faculty of Agriculture, Tokyo University of Agriculture and Technology, Japan.
Mol Gen Genet. 1993 Aug;240(2):197-205. doi: 10.1007/BF00277057.
Gel shift mobility analysis, using the proximal 0.3 kb fragment of the tobacco pathogenesis-related protein 1a gene (PR-1a) and nuclear extracts from healthy Samsun NN tobacco leaves, which do not produce PR-1 proteins, showed a broad shifted signal with low mobility. This signal was not detected with nuclear proteins from the interspecific hybrid of Nicotiana glutinosa x Nicotiana debneyi, which constitutively produces the PR-1a protein. Similar shifted signals were detected with both proximal and distal regions of the 0.3 kb fragment using nuclear proteins from healthy Samsun NN tobacco, but not with proteins from the interspecific hybrid. Further experiments, performed using 5' or 3' truncated fragments of the 0.3 kb fragment, identified two independent binding sites: a distal site between -179 and -168 bp from the transcription start site, and a proximal site between -61 and -37 bp. Footprint analysis revealed two protected sequences, a distal region between -184 and -172 bp, and a proximal region between -68 and -51 bp. These results indicate the presence of regulatory factor(s) for expression of the acidic PR-1a gene. The possibility of negative regulation of the gene is discussed.
凝胶迁移率变动分析使用烟草病程相关蛋白1a基因（PR-1a）近端0.3 kb片段以及不产生PR-1蛋白的健康三生NN烟草叶片的核提取物，结果显示出一个迁移率低的宽迁移信号。在用本氏烟草×德氏烟草的种间杂种的核蛋白进行检测时未检测到该信号，该种间杂种组成型产生PR-1a蛋白。使用健康三生NN烟草的核蛋白对0.3 kb片段的近端和远端区域进行检测时均检测到了类似的迁移信号，但使用种间杂种的蛋白则未检测到。使用0.3 kb片段的5'或3'截短片段进行的进一步实验确定了两个独立的结合位点：一个位于转录起始位点上游-179至-168 bp之间的远端位点，以及一个位于-61至-37 bp之间的近端位点。足迹分析揭示了两个受保护序列，一个位于-184至-172 bp之间的远端区域，以及一个位于-68至-51 bp之间的近端区域。这些结果表明存在酸性PR-1a基因表达的调控因子。文中还讨论了该基因负调控的可能性。
