Isolation and properties of Drosophila melanogaster ferritin - Molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome
Isolation and properties of Drosophila melanogaster ferritin - Molecular cloning of a cDNA that encodes one subunit, and localization of the gene on the third chromosome
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DOI：
10.1111/j.1432-1033.1997.00470.x
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发表时间：
1997-07-15
期刊：
影响因子：
--
通讯作者：
Kafatos, FC
中科院分区：
文献类型：
--
作者：
Charlesworth, A;Georgieva, T;Kafatos, FC
通过在溴化钾梯度中离心，从喂铁的果蝇提取物中纯化铁蛋白。在聚丙烯酰胺凝胶电泳上，产物显示两条对应于铁蛋白单体和二聚体的蛋白条带。用 SDS 和 2-巯基乙醇解离后的电泳显示出大约 25、26 和 28 kDa 的三个强条带。 25-kDa 和 26-kDa 亚基的 N 端氨基酸序列相同，但 28-kDa 亚基的 N 端氨基酸序列不同。使用保守铁蛋白 PCR 引物扩增 360 bp cDNA 产物。它用于从黑腹果蝇 cDNA 文库中分离出一个克隆，该文库包含铁蛋白亚基的完整编码序列。通过 RACE 方法获得的额外 5' 序列揭示了推定的铁调节元件的存在。 PCR产物还用于定位第三染色体右臂99F区域的铁蛋白亚基基因的位置。推导的黑腹果蝇铁蛋白亚基的氨基酸序列包含信号序列并且与埃及伊蚊的铁蛋白最相似。讨论了铁蛋白序列的演变。
Ferritin was purified from iron-fed Drosophila melanogaster extracts by centrifugation in a gradient of potassium bromide. On polyacrylamide gel electrophoresis, the product showed two protein bands corresponding to the ferritin monomer and dimer. Electrophoresis following dissociation with SDS and 2-mercaptoethanol revealed three strong bands of approximately 25, 26, and 28 kDa. N-terminal amino acid sequences were identical for the 25-kDa and 26-kDa subunits, but different for the 28-kDa subunit. Conserved ferritin PCR primers were used to amplify a 360-bp cDNA product. which was used to isolate a clone from a D. melanogaster cDNA library that contained the complete coding sequence for a ferritin subunit. Additional 5' sequence obtained by the RACE method revealed the presence of a putative iron regulatory element. The PCR product was also used to locate the position of the ferritin subunit gene at region 99F on the right arm of the third chromosome. The deduced amino acid sequence of the D. melanogaster ferritin subunit contained a signal sequence and resembled most closely ferritin of the mosquito Aedes aegypti. The evolution of ferritin sequences is discussed.