Isolation and characterization of the nuclear gene encoding the Rieske iron-sulfur protein (RIP1) from Saccharomyces cerevisiae.
Isolation and characterization of the nuclear gene encoding the Rieske iron-sulfur protein (RIP1) from Saccharomyces cerevisiae.
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DOI：
10.1016/s0021-9258(18)47500-x
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发表时间：
1987-06
期刊：
影响因子：
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通讯作者：
J. Beckmann;P. Ljungdahl;J. L. Lopez;B. Trumpower
中科院分区：
文献类型：
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作者：
J. Beckmann;P. Ljungdahl;J. L. Lopez;B. Trumpower
从酿酒酵母中分离并鉴定了线粒体呼吸链细胞色素bc1复合体Rieske铁硫蛋白的核基因。我们使用了来自粗神经孢子虫（Harnisch， U, Weiss， H., and Sebald， W., 1985）的铁硫蛋白基因片段。用Southern分析法检测酵母菌的基因。生物化学学报，19(4),95-99。利用菌落提升杂交技术对酵母基因组文库进行检测，分离出5个不同但重叠的克隆。几种方法证实了分离的DNA含有Rieske铁硫蛋白的基因。酵母基因不含内含子，可在大肠杆菌中表达。一个900碱基对的HindIII-EcoRI片段被亚克隆到pUC19中，并指导免疫检测蛋白的合成。该基因还通过同源整合破坏其染色体拷贝来鉴定。在pUC18中克隆了一个与HIS3标记相邻的400碱基对的PstI-EcoRI片段作为整合载体。获得了在不可发酵的碳源甘油上不能生长的HIS+转化体。对呼吸缺陷（gly-）菌株的Southern分析证实，该基因的染色体拷贝被破坏，转化体提取物的免疫印迹显示缺乏铁硫蛋白。通过一个2千碱基对的HindIII-BglII片段将呼吸缺陷整合体转化为GLY+，该片段包括该基因的完整拷贝，并携带在多拷贝episal载体上。铁硫蛋白单克隆抗体的免疫印迹显示，互补菌株中铁硫蛋白的过量产生，并显示表达了大约等量的成熟铁硫蛋白和一个比互补菌株中成熟蛋白大约3kda的蛋白。对来自克隆的1.2千碱基对DNA片段进行测序，发现包含645个核苷酸的开放阅读框，能够编码21,946道尔顿蛋白。该基因两侧有起始和终止的一致信号序列，这在酵母中很常见，并且在其之前可能有上游激活序列。成熟铁硫蛋白氨基末端的氨基酸序列分析与从Lys-31开始的核苷酸序列预测完全一致。（摘要删节为400字）
The nuclear gene encoding the Rieske iron-sulfur protein of the cytochrome bc1 complex of the mitochondrial respiratory chain has been isolated and characterized from Saccharomyces cerevisiae. We used a segment of the iron-sulfur protein gene from Neurospora crassa (Harnisch, U., Weiss, H., and Sebald, W. (1985) Eur. J. Biochem. 149, 95-99) to detect the yeast gene by Southern analysis. Five different but overlapping clones were then isolated by probing a yeast genomic library carried on YEp 13 by colony lift hybridization. Several approaches confirmed that the isolated DNA contained the gene for the Rieske iron-sulfur protein. The yeast gene, which contains no introns, can be expressed in Escherichia coli. A 900-base pair HindIII-EcoRI fragment was subcloned into pUC19 and directed the synthesis of immunodetectable protein. The gene was also identified by disruption of its chromosomal copy by homologous integration. A 400 base pair PstI-EcoRI fragment cloned adjacent to a HIS3 marker in pUC18 was used as an integrating vector. HIS+ transformants were obtained which were unable to grow on the nonfermentable carbon source glycerol. Southern analysis of the respiration deficient (gly-) strains confirmed that the chromosomal copy of the gene was disrupted, and immunoblots of extracts of the transformants indicated a lack of iron-sulfur protein. A respiration-deficient integrant was transformed to GLY+ by a 2-kilobase pair HindIII-BglII fragment, including a complete copy of the gene, carried on a multicopy episomal vector. Immunoblots with monoclonal antibodies to the iron-sulfur protein indicated overproduction of the protein in the complemented strain and revealed expression of approximately equal amounts of mature iron-sulfur protein and of a protein approximately 3 kDa larger than the mature protein in the complemented strain. A 1.2-kilobase pair segment of DNA from the clone which complemented the disrupted strains was sequenced and found to contain an open reading frame of 645 nucleotides, capable of encoding a 21,946-dalton protein. The gene is flanked by consensus signal sequences for initiation and termination which are common in yeast and is preceded by a possible upstream activating sequence. Amino acid sequence analysis of the amino-terminal end of the mature iron-sulfur protein agreed exactly with that predicted by the nucleotide sequence starting at Lys-31.(ABSTRACT TRUNCATED AT 400 WORDS)