OPAQUE-2 IS A TRANSCRIPTIONAL ACTIVATOR THAT RECOGNIZES A SPECIFIC TARGET SITE IN 22-KD-ZEIN GENES
OPAQUE-2 IS A TRANSCRIPTIONAL ACTIVATOR THAT RECOGNIZES A SPECIFIC TARGET SITE IN 22-KD-ZEIN GENES
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DOI：
10.1105/tpc.4.6.689
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发表时间：
1992-06-01
期刊：
影响因子：
11.6
通讯作者：
HOSCHEK, G
中科院分区：
文献类型：
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作者：
SCHMIDT, RJ;KETUDAT, M;HOSCHEK, G
opaque-2（o2）是玉米中的一个调控位点，其在控制编码22-kD玉米醇溶蛋白的基因的表达中起重要作用。通过DNA酶I足迹法和DNA结合分析，我们已经确定了22 kD玉米醇溶蛋白基因启动子中O2蛋白（O2）的结合位点。22 kD玉米醇溶蛋白基因启动子中被O2识别的序列与其他“碱性/亮氨酸拉链”（bZIP）蛋白识别的靶位点相似，因为它含有DNA结合所必需的ACGT核心。该位点位于相对于翻译起始点的-300区域，位于高度保守的玉米醇溶蛋白基因序列基序（称为“醇溶谷蛋白盒”）下游约20 bp处。“采用凝胶迁移率变动分析，我们使用O2抗体和来自O2无效突变体的核提取物来证明玉米胚乳核中的O2蛋白识别玉米醇溶蛋白基因启动子中的靶位点。流动性变化测定使用核蛋白从O2无效突变体表明，其他胚乳蛋白除了O2可以结合O2的目标网站，O2可能与这些蛋白质之一。我们还表明，在酵母细胞中的O2蛋白可以激活的lacZ基因的表达含有一个多聚体的O2靶序列作为其启动子的一部分，从而确认其作为转录激活因子的作用。计算机辅助搜索表明，O2靶位点不存在于玉米醇溶蛋白基因的启动子以外的22 kD类。这些数据表明一个可能的解释在分子水平上的差异影响o2突变的玉米醇溶蛋白基因家族的某些成员的表达。
opaque-2 (o2) is a regulatory locus in maize that plays an essential role in controlling the expression of genes encoding the 22-kD zein proteins. Through DNase I footprinting and DNA binding analyses, we have identified the binding site for the O2 protein (O2) in the promoter of 22-kD zein genes. The sequence in the 22-kD zein gene promoter that is recognized by O2 is similar to the target site recognized by other "basic/leucine zipper" (bZIP) proteins in that it contains an ACGT core that is necessary for DNA binding. The site is located in the - 300 region relative to the translation start and lies about 20 bp downstream of the highly conserved zein gene sequence motif known as the "prolamin box." Employing gel mobility shift assays, we used O2 antibodies and nuclear extracts from an o2 null mutant to demonstrate that the 02 protein in maize endosperm nuclei recognizes the target site in the zein gene promoter. Mobility shift assays using nuclear proteins from an o2 null mutant indicated that other endosperm proteins in addition to O2 can bind the O2 target site and that O2 may be associated with one of these proteins. We also demonstrated that in yeast cells the O2 protein can activate expression of a lacZ gene containing a multimer of the O2 target sequence as part of its promoter, thus confirming its role as a transcriptional activator. A computer-assisted search indicated that the O2 target site is not present in the promoters of zein genes other than those of the 22-kD class. These data suggest a likely explanation at the molecular level for the differential effect of o2 mutations on expression of certain members of the zein gene family.