Isolation and characterization of the cDNA encoding the channel catfish (Ictalurus punctatus) form of cytochrome P450arom.
Isolation and characterization of the cDNA encoding the channel catfish (Ictalurus punctatus) form of cytochrome P450arom.
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编码斑点叉尾鮰 (Ictalurus punctatus) 细胞色素 P450arom 形式的 cDNA 的分离和表征。
DOI：
10.1006/gcen.1994.1113
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发表时间：
1994
影响因子：
2.7
通讯作者：
J. Trant
中科院分区：
文献类型：
--
作者：
J. Trant
细胞色素 P450arom（芳香酶）负责将雄激素转化为雌激素。从斑点叉尾鮰 (Ictalurus punctatus) 卵巢中分离出的富含 Poly(A) 的 mRNA 构建了 cDNA 文库，并将其连接到 EcoRI 切割的 lambda 臂中。使用人类芳香酶 DNA 探针筛选扩增的文库。分离的最长克隆 (2.1 kb) 包含 20 bp 的 5'-非翻译区、1572 bp 开放阅读框和 509 bp 3'-非翻译区。 Northern 印迹分析表明有一个 3.4-kb 的转录本。推定的聚腺苷酸化信号 (AATAAA) 距克隆 3' 端 13 bp。推导的鲶鱼形式芳香酶的氨基酸序列与虹鳟鱼形式有 65% 的同一性。鲶鱼形态与哺乳动物（大鼠、人类、小鼠、牛）和鸡形态有 50% 至 53% 的相同性。所有形态都共享大片具有极高同一性的区域。推导的鲶鱼芳香酶蛋白的长度为 524、517 或 502 个残基，具体取决于翻译起始位点。性腺组织的类固醇生成活性与 mRNA 和蛋白质含量相关。用鲶鱼芳香酶 cDNA 转染的非类固醇生成 COS 细胞，将雄烯二酮转化为雌酮，从而验证了其身份。对鲶鱼 P450arom 进行了修饰，以促进转化大肠杆菌中的生产及其随后通过单柱技术的纯化。这种纯化的蛋白质用于产生抗血清用于蛋白质印迹分析。蛋白质印迹分析表明，鲶鱼形态的大小 (57 kDa) 与其他脊椎动物形态相似。
Cytochrome P450arom (aromatase) is responsible for the conversion of androgens to estrogens. A cDNA library was constructed from poly(A)-enriched mRNA isolated from channel catfish (Ictalurus punctatus) ovary and ligated into EcoRI-cut lambda arms. The amplified library was screened using a human aromatase DNA probe. The longest clone isolated (2.1 kb) contained 20 bp of the 5'-untranslated region, a 1572-bp open reading frame, and a 509-bp 3'-untranslated region. Northern blot analysis indicated a single 3.4-kb transcript. A putative polyadenylation signal (AATAAA) is 13 bp from the 3'-end of the clone. The deduced amino acid sequence of the catfish form of aromatase is 65% identical to the rainbow trout form. The catfish form is 50 to 53% identical to mammalian (rat, human, mouse, bovine) and chicken forms. There are large regions of extremely high identity shared among all the forms. The deduced catfish aromatase protein is 524, 517, or 502 residues in length, depending on the translation initiation site. Steroidogenic activity of gonadal tissue is correlated with mRNA and protein content. Nonsteroidogenic COS cells, transfected with the catfish aromatase cDNA, converted androstenedione to estrone, thus verifying its identify. The catfish P450arom was modified to facilitate production in transformed Escherichia coli and its subsequent purification by a single column technique. This purified protein was used to raise antisera for Western blot analysis. Western blot analysis indicates that the catfish form is similar in size (57 kDa) to other vertebrate forms.