CLONING, CHARACTERIZATION, AND DNA-SEQUENCE OF A HUMAN CDNA-ENCODING NEUROPEPTIDE TYROSINE
CLONING, CHARACTERIZATION, AND DNA-SEQUENCE OF A HUMAN CDNA-ENCODING NEUROPEPTIDE TYROSINE
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DOI：
10.1073/pnas.81.14.4577
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
DIXON, JE
中科院分区：
文献类型：
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作者：
MINTH, CD;BLOOM, SR;DIXON, JE
从人嗜铬细胞瘤中分离的RNA的体外翻译表明，该肿瘤含有编码10.5-kDa [千道尔顿]蛋白的mRNA，该蛋白用抗猪神经肽Y的抗血清免疫沉淀。从总RNA合成双链c[互补]DNA并插入pUC 8的Pst I位点。使用混合杂交探针d[A-（A，G）-（A，G）-T-T-（A，G，T）-A-T-（A，G）-T-A-（A，G）-T-G]鉴定含有神经肽Y cDNA的转化体。探针序列基于在猪神经肽Y中发现的已知氨基酸序列His-Tyr-Ile-Asn-Leu。测定了该cDNA的核苷酸序列，在5“-和3”-非翻译区分别含有86和174个碱基。编码序列由291个碱基组成，表明神经肽Y的前体长97个氨基酸（10，839 Da）。氨基酸序列分析表明，该前体蛋白至少有2个蛋白水解位点，分别产生28个（信号肽）、36个（人神经肽Y）和30个（羧基端肽）氨基酸残基的3个肽段。通过Edman降解免疫沉淀的体外翻译产物获得的部分NH 2-末端序列鉴定了Met和Leu在前原肽的前30个残基中的位置。利用噬菌体SP 6启动子制备了一种高灵敏度的特异于神经肽Y mRNA的单链互补mRNA杂交探针。该探针用于鉴定对应于神经肽Y的mRNA。800个基地
In vitro translation of the RNA isolated from a human pheochromocytoma demonstrated that this tumor contained a mRNA encoding a 10.5-kDa [kilodalton] protein, which was immunoprecipitated with antiserum raised against porcine meuropeptide Y. Double-stranded c[complementary]DNA was synthesized from total RNA and inserted into the Pst I site of pUC8. Transformants containing the neuropeptide Y cDNA were identified using the mixed hybridization probe d[A-(A,G)-(A,G)-T-T-(A,G,T)-A-T-(A,G)-T-A-(A,G)-T-G]. The probe sequences were based on the known amino acid sequence, His-Tyr-Ile-Asn-Leu, found in porcine neuropeptide Y. The nucleotide sequence of the cDNA was determined and contained 86 and 174 bases in the 5''- and 3''-untranslated regions, respectively. The coding sequence consisted of 291 bases, suggesting a precursor to neuropeptide Y that was 97 amino acids long (10,839 Da). The deduced amino acid sequence of the precursor suggested that there were at least 2 sites of proteolytic processing, which would generate 3 peptides having 28 (signal peptide), 36 (human neuropeptide Y) and 30 (COOH-terminal peptide) amino acid residues. A partial NH2-terminal sequence obtained by Edman degradation of the immunoprecipitated in vitro translation product identified the positions of Met amd Leu in the first 30 residues of the prepropeptide. A highly sensitive single-stranded complementary mRNA hybridization probe specific for neuropeptide Y mRNA was prepared using the bacteriophage SP6 promoter. This probe was used to identify a mRNA corresponding to neuropeptide Y of .apprxeq. 800 bases.