Anoxia-induced gene expression in turtle heart - Upregulation of mitochondrial genes for NADH-ubiquinone oxidoreductase subunit 5 and cytochrome c oxidase subunit 1
Anoxia-induced gene expression in turtle heart - Upregulation of mitochondrial genes for NADH-ubiquinone oxidoreductase subunit 5 and cytochrome c oxidase subunit 1
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DOI：
10.1111/j.1432-1033.1996.0083t.x
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发表时间：
1996-10-01
期刊：
影响因子：
--
通讯作者：
Storey, KB
中科院分区：
文献类型：
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作者：
Cai, Q;Storey, KB
用P-32标记的对照和缺氧动物心脏的单链cDNAs探针差异筛选了缺氧暴露的成年龟(Trachemys Cripta Elegans)心脏的cDNA文库，克隆了缺氧应激诱导的基因。获得了4个克隆pBTaR20、pBTaR34、pBTaR63和pBTaR914，证实它们在缺氧浸泡(7℃氮气泡腾20h)后表达上调。对两个克隆pBTaR20和pBTaR63进行了序列分析和体内表达。克隆pBTaR20全长1597bp，克隆pBTaR63全长1837bp。PBTaR20序列含有一个非常接近全长的开放阅读框，可能编码508个氨基酸的多肽。推导的多肽序列与线粒体DNA基因Cox1编码的细胞色素c氧化酶亚单位1(COI)序列有大约83%的同源性。克隆pBTaR63含有一个潜在的全长开放阅读框，可编码591个残基的多肽。这与线粒体基因Nad5编码的另一种线粒体蛋白NADH-泛醌氧化还原酶亚单位5(ND5)相似。Northern杂交分析了Cox1和Nad5的表达时程，结果表明，Cox1和Nad5的mRNA转录产物在缺氧条件下迅速积累(在1h内)。缺氧1h后，Nad5和Cox1的转录含量均有不同程度的升高，但随着缺氧时间的延长(5h或20h)，Nad5mRNA的表达水平仍然较高，而Cox1mRNA的含量有所下降。Northern-Blot杂交还发现，这两个基因在缺氧暴露的另外五个器官(脑、肾脏、肝脏、红色和白色骨骼肌)中存在差异表达，其中在缺氧红色肌肉中这两个基因的mRNA转录水平都有很大的提高。对这些基因在耐寒龟种中的器官特异性分析也表明，这些基因的差异表达发生在血浆冷冻诱导的缺血反应中。
A cDNA library constructed from heart of anoxia-exposed adult turtles (Trachemys scripta elegans) was differentially screened with P-32-labeled single-stranded cDNA probes from heart of control versus anoxic animals to clone genes induced by anoxia stress. Four cDNA clones, pBTaR20, pBTaR34, pBTaR63 and pBTaR914 were obtained and confirmed to be upregulated in response to anoxic submergence (20 h in N-2-bubbled water at 7 degrees C). Two clones, pBTaR20 and pBTaR63, were characterized by sequence analysis and in vivo expression. The clone pBTaR20 had a 1597-bp cDNA sequence and pBTaR63 contained a 1837-bp sequence. The pBTaR20 sequence contained a single open reading frame that was very close to full length and could potentially encode a polypeptide with 508 amino acids. The deduced polypeptide sequence showed approximately 83% of the residues identical with the sequence of cytochrome c oxidase subunit 1 (COI) that is encoded by a mtDNA gene Cox1. The clone pBTaR63 contained a single potentially full-length open reading frame that could encode a polypeptide of 591 residues. This was similar to another mitochondrial protein, NADH-ubiquinone oxidoreductase subunit 5 (ND5), which is encoded by mtDNA gene Nad5. Analysis of the lime course of expression of Cox1 and Nad5 by northern hybridization analysis showed that mRNA transcripts for both accumulated rapidly (within 1 h) in response to anoxia exposure. Both showed similar increases in their transcript content after 1 h of anoxia but with longer anoxia exposures (5 or 20 h) Nad5 mRNA levels remained high whereas Cox1 mRNA content declined somewhat. Northern-blot hybridization also revealed differential expression of these two genes in five other organs of T. s. elegans during anoxia exposure (brain, kidney, liver, red and white skeletal muscle), with a particularly large increase in mRNA transcript levels of both genes in anoxic red muscle. Organ-specific analysis of these genes in a freeze-tolerant turtle species (Chrysemys picta marginata) also showed that differential expression of these genes occurred in response to the ischemia induced by plasma freezing.