| Summary: | 博士 === 國立清華大學 === 生命科學研究所 === 79 === Ferredoxin is an iron-sulfur protein which serves as an electron transport
intermediae for all mitochondrial cytochromes P450 involved in
steroid,vitamin D, and bile acid metabolism. To study the regulation of
ferredoxin, we have cloned and characterized the human ferredoxin gene
family which included at least one expressed gene and two pseudogenes.
The authentic gene spans more than 20 kb, containing four exons and three
introns. Only one protein product is encoded by this gene family. The
first exon encodes the 60-amino-acid signal peptide, directing transport
of the protein into the inner mitochondrial matrix. The mature peptide of
124 amino acids is encoded by the other three exons. The third exon
encodes the portion of the protein containing the iron-sulfur center and a
domain which binds other components of the electron transport chain. The
transcriptional start sites were determined by primer extension and S1
nuclease mapping. The 5-flanking region of the authentic gene contains
sequences of a TATA box at nucleotide position -30 to -27 and two GC boxes
at -68 to -54 and -103 to -94. Sequence at -1103 to -933 represents a
half Alu repeat.
The pseudogenes lack introns and contain numerous mutations including
insertion, deletion, and substitution which rendered them inactive. They
are 96% and 85% homologous to the expressed gene, yet there is only 78%
homology between themselves. Just upstream and downstream of the
h-3pseudogene, there are two Alu sequences flanking in opposite
orientation. The intronless nature, higher diversity among themselves,
and distinct chromosomal location of the pseudogenes
人類含鐵氧化還原蛋白是種含有兩個鐵原子及兩個硫原子所形成的電子傳遞中間物，
其在粒線體內之作用是接受由NADPH 依性的含鐵氧化還原酵素傳的電子，然後再將電
子傳給細胞色素P450，以完成隨後之氧化還原反應。含鐵氧化還原蛋白在不同組織中
參類固醇荷爾維他命D 之合成和膽汁之代謝。為了研究其基因的表現及調控機轉，我
們將此基因家族選殖出來。此家族包括至少一個會表現的真基因和兩個不會表現的假
基因。真基因長度超過20kb，含有四個表現序列和三個插入序列，此基因只表現出一
種蛋白質。第一個表現序列有60個胺基酸序列之密碼，此為引導胜可協助此蛋白質
進入粒線體中。成熟蛋白分子結合之作用區。轉錄的起始+1，以S1核酸酵素圖譜法和
弔子延伸法決定出來。此會表現之真基因，其上游區域在-30至-27核酸序列位置上
有TATA序列，在-68至-54和-103至-94 位置有GC序列。兩個假基因，缺少插入序列，
並有許多突變，包括插入，缺失和替代如此使得此兩基因不具表現功能。假基因和真
基因之核酸序列有96% 和85%的相似性，而兩假基因間僅有78%的相同。這種沒有插
入序列，高度序列變異，以及位於不同染色體的特性，暗示這類假基因是經由獨立之
反轉錄過程形成的。以序列的變異關係，可推算出此事件大約是在五百萬年及一千五
百萬年前發生的。採用活體外轉錄，或構築一系列含5 端上游之基因片段於氯素乙醯
轉移之之媒介，來分析此基因之起動子活性。這些媒介被暫時引入JEG-3，Y-1，
COS-1和HeLa 細胞中表現，結果發現一個GC和TATA序列可完全表現起動子活性，但僅
含TATA 序列則不行。核酸酵素DNase I和甲基化劑DMS之足跡試驗，証明出兩個GC序
列受到Y-1 和HeLa細胞核萃取物之保護。由此可推測出此基因之GC序列可能和細胞核
內的SP1活性因子相結
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