Isolation and characterisation of the human lung NK-1 receptor cDNA.
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.
Hopkins
B
B
Biotechnology Department, ICI Pharmaceuticals, Macclesfield, Cheshire, UK.
Powell
S J
SJ
Danks
P
P
Briggs
I
I
Graham
A
A
D000368:Aged; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D003360:Cosmids; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015723:Gene Library; D006801:Humans; D008168:Lung / Q000502:physiology*; D008297:Male; D008969:Molecular Sequence Data; D015288:Neurokinin A / Q000494:pharmacology; D015287:Neurokinin B / Q000494:pharmacology; D009838:Oligodeoxyribonucleotides; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D017981:Receptors, Neurotransmitter / Q000235:genetics* / Q000502:physiology; D018039:Receptors, Tachykinin; D014981:Xenopus
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D017981:Receptors, Neurotransmitter; D018039:Receptors, Tachykinin; D015288:Neurokinin A; D015287:Neurokinin B; D004247:DNA