Hopkins B, Powell S J, Danks P, Briggs I, Graham A
Biotechnology Department, ICI Pharmaceuticals, Macclesfield, Cheshire, UK.
Biochem Biophys Res Commun. 1991 Oct 31;180(2):1110-7. doi: 10.1016/s0006-291x(05)81181-7.
Functional cDNA clones for human NK-1 receptor were isolated from human lung RNA using the polymerase chain reaction (PCR). We have screened a human cosmid library and isolated a clone which appeared to contain the entire NK-1 receptor gene. From the published rat NK-1 receptor cDNA sequence we designed primers within the protein coding sequence, but outwards towards both the 5' and 3' ends of the putative human protein sequence. By this method we derived DNA sequence from the 3' end of the human gene. In order to determine the 5' end of the gene we used a PCR based method called Rapid Amplification of cDNA Ends (RACE). From the derived human sequences amplimers were designed upstream of the ATG initiation codon and downstream of the stop codon. The entire cDNA was obtained by RNA-PCR from human lung RNA. The sequence obtained was 407 amino acids in length, encoding an open-reading frame that was highly homologous to the rat NK-1 receptor cDNA (89%). The entire human cDNA was then cloned into a mammalian expression vector and mRNA was synthesized by in vitro transcription. Applications of tachykinins caused membrane current responses in Xenopus oocytes injected with the in vitro synthesized mRNA. The most potent of the three tachykinin peptides tested was Substance P. The human NK-1 receptor gene has been mapped to chromosome 2 using the polymerase chain reaction to specifically amplify the human sequence in hamster/human hybrid DNA and also in mouse/human monochromosome hybrids.
利用聚合酶链反应（PCR）从人肺RNA中分离出了人NK-1受体的功能性cDNA克隆。我们筛选了一个人黏粒文库，并分离出一个似乎包含完整NK-1受体基因的克隆。根据已发表的大鼠NK-1受体cDNA序列，我们在蛋白质编码序列内设计了引物，但引物朝向假定的人蛋白质序列的5'端和3'端。通过这种方法，我们从人基因的3'端获得了DNA序列。为了确定基因的5'端，我们使用了一种基于PCR的方法，即cDNA末端快速扩增（RACE）。从推导的人序列中，在ATG起始密码子上游和终止密码子下游设计了扩增子。通过RNA-PCR从人肺RNA中获得了完整的cDNA。获得的序列长度为407个氨基酸，编码一个与大鼠NK-1受体cDNA高度同源（89%）的开放阅读框。然后将完整的人cDNA克隆到哺乳动物表达载体中，并通过体外转录合成mRNA。速激肽的应用在注射了体外合成mRNA的非洲爪蟾卵母细胞中引起了膜电流反应。所测试的三种速激肽肽中最有效的是P物质。利用聚合酶链反应在仓鼠/人杂种DNA以及小鼠/人单染色体杂种中特异性扩增人序列，已将人NK-1受体基因定位到2号染色体上。
