RUI: Secreted Ferritin: Structure and Cloning

9731745 Linder研究人员已经获得证据表明，肝细胞分泌的铁蛋白和血清铁蛋白与细胞内铁蛋白具有不同的结构。他们假设分泌/血清铁蛋白与细胞内铁蛋白只有部分同源性，并且由不同的基因编码。目的是克隆分泌/血清铁蛋白亚基的cdna来验证这一假设。从马血清和大鼠肝癌细胞培养基中分离铁蛋白。将得到n端和内部氨基酸序列。利用氨基酸序列(a)设计用于筛选大鼠肝脏文库的探针，以及(b)设计用于对大鼠肝脏mRNA进行RT-PCR的引物来克隆cdna。将对全尺寸克隆进行测序，并用于确定分泌铁蛋白亚基的初级结构，以便与已知的细胞内铁蛋白进行比较。克隆将通过证明(a)它们编码对铁有转录反应的mrna，以及(b)亚基不同部分的氨基酸序列是由克隆适当区域的核苷酸序列编码的来验证。如果需要，克隆也将被验证，通过杂交克隆选择的大鼠肝脏mRNA编码适当大小的产物，并被抗细胞内铁蛋白的抗体识别。铁蛋白是一种储存铁的分子，在所有细胞中都有大量存在。人们对它的结构、功能和遗传学有很多了解。在血清中也发现了铁蛋白的痕迹，但对其结构、功能和基因几乎一无所知。研究人员有证据表明，血清铁蛋白与细胞中的铁蛋白明显不同，并将测试这一概念。如果他们是正确的，他们的发现将极大地改变人们对什么是铁蛋白的看法，并且血清中铁蛋白的测量结果将变得清晰。

9731745 Linder The investigators have obtained evidence that ferritin secreted by hepatic cells, and serum ferritin, have a different structure than the well-studied intracellular ferritins. They hypothesize that secreted/serum ferritin has only partial homology to intracellular ferritins and is encoded by different genes. The objective is to clone cDNAs for the subunits of secreted/serum ferritin to test the hypothesis. Ferritin from horse serum and the medium of rat hepatoma cells will be isolated. N-terminal and internal amino acid sequence will be obtained. cDNAs will be cloned by using the amino acid sequence (a) to design probes with which to screen a rat liver library, and (b) to design primers with which to perform RT-PCR on rat liver mRNA. Full sized clones will be sequenced and used to determine the primary structure of the subunits of secreted ferritin for comparison with that of known intracellular ferritins. Clones will be validated by demonstrating (a) that they encode mRNAs transcriptionally responsive to iron, and (b) that amino acid sequences for different portions of the subunits are encoded by nucleotide sequence in appropriate regions of the clones. If needed, clones will also be validated by showing that rat liver mRNA selected by hybridization to the clones encodes a product of the appropriate size and is recognized by antibody against intracellular ferritin. Ferritin is an iron storage molecule found in significant amounts inside all cells. A great deal is known about its structure, function and genetics. Traces of a ferritin are also found in blood serum, but almost nothing is known about its structure, function and genetics. The investigators have evidence that blood serum ferritin differs markedly from that in cells and will test this concept. If they are correct, their findings will greatly change views about what is ferritin, and exactly what is being measured as ferritin in serum will become apparent.