CHARACTERIZATION OF THE DOPA DECARBOXYLASE GENE OF MANDUCA-SEXTA AND ITS SUPPRESSION BY 20-HYDROXYECDYSONE
CHARACTERIZATION OF THE DOPA DECARBOXYLASE GENE OF MANDUCA-SEXTA AND ITS SUPPRESSION BY 20-HYDROXYECDYSONE
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DOI：
10.1006/dbio.1995.1137
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发表时间：
1995-05-01
影响因子：
2.7
通讯作者：
RIDDIFORD, LM
中科院分区：
文献类型：
--
作者：
HIRUMA, K;CARTER, MS;RIDDIFORD, LM
Manduca dopa decarboxylase（DDC）cDNA被分离、测序，并发现与果蝇DDC最密切相关（72%氨基酸同一性）。用20-羟基蜕皮激素（20 E）培养第2天的4龄幼虫表皮表明，20 E是确定该基因后期表达所必需的，但其去除是发生这种表达所必需的。用蛋白质合成抑制剂放线菌酮和茴香霉素以及mRNA合成抑制剂α-鹅膏蕈碱进行的实验表明，20 E诱导抑制DDC基因转录的蛋白质。使用表皮提取物和DDC基因5'侧翼区的前1.1 kb的各种片段进行的凝胶迁移率变动分析显示，仅在5'起始位点上游87至167 bp处有一个DNA片段，该片段结合具有预期发育特异性的核蛋白。当没有DDC mRNA时，蛋白质在高蜕皮激素滴度时是丰富的，但在蜕皮激素滴度上升之前（没有DDC mRNA）和下降之后（最大DDC mRNA）都是低的。因此，该蛋白质是蜕皮甾类诱导的转录因子的候选物，其作用是抑制DDC转录。通过使用特异性寡核苷酸证实的DNA酶I足迹分析显示，该蛋白质与序列5 '-GGCTTATGCGCTGCA-3'结合。(C)出版社：Academic Press
Manduca dopa decarboxylase (DDC) cDNA was isolated, sequenced, and found to be most closely related to Drosophila DDC (72% amino acid identity). Culture of Day 2 fourth instar larval epidermis with 20-hydroxyecdysone (20E) showed that 20E was necessary to determine the later expression of the gene, but its removal was required for this expression to occur. Experiments with the protein synthesis inhibitors, cycloheximide and anisomycin, and the mRNA synthesis inhibitor, alpha-amanitin, showed that 20E induced a protein(s) which suppressed transcription of the DDC gene. Gel mobility shift assays using epidermal extracts and various fragments of the first 1.1 kb of the 5' flanking region of the DDC gene showed only one DNA fragment 87 to 167 bp upstream of the 5' initiation site that bound a nuclear protein(s) with the expected developmental specificity. The protein was abundant at the time of high ecdysteroid titer when no DDC mRNA was present, but low both before the rise (no DDC mRNA) and after the decline of ecdysteroid titer (maximal DDC mRNA). Thus, this protein(s) is a candidate for an ecdysteroid-induced transcription factor which acts to suppress DDC transcription. DNase I foot-printing assays confirmed by use of a specific oligonucleotide showed that this protein(s) bound to the sequence 5'-GGCTTATGCGCTGCA-3'. (C) 1995 Academic Press,Inc.