Hendy G N, Kronenberg H M, Potts J T, Rich A
Proc Natl Acad Sci U S A. 1981 Dec;78(12):7365-9. doi: 10.1073/pnas.78.12.7365.
We have cloned cDNA copies of human preproparathyroid hormone in Escherichia coli after insertion of double-stranded DNA into the Pst I site of plasmid pBR322 using the poly(dG) . poly(dC) homopolymer extension technique. Recombinant plasmids coding for preproparathyroid hormone were identified by filter hybridization assay using as a probe 32P-labeled bovine preproparathyroid cDNA. Nucleotide sequence analysis of five recombinant plasmids permitted the assignment of 74 nucleotides of the 5' noncoding region, the entire coding region of 345 nucleotides, and the entire 3' noncoding region of 348 nucleotides of the mRNA. The coding sequence predicts the previously unknown "pre" amino acid sequence and clarifies the hormone's amino acid sequence, which has been disrupted. The 5' noncoding region contains an AUG codon followed by a UGA stop codon before the authentic initiator codon. The 3' noncoding region is 120 nucleotides longer than in bovine preproparathyroid mRNA and contains two A-A-U-A-A-A sequences, potential signals for polyadenylation.
我们利用聚（dG）·聚（dC）同聚物延伸技术，将双链DNA插入质粒pBR322的Pst I位点后，在大肠杆菌中克隆了人甲状旁腺激素原前体的cDNA拷贝。使用32P标记的牛甲状旁腺激素原前体cDNA作为探针，通过滤膜杂交试验鉴定出编码甲状旁腺激素原前体的重组质粒。对五个重组质粒进行核苷酸序列分析，确定了mRNA的5'非编码区的74个核苷酸、345个核苷酸的整个编码区以及348个核苷酸的整个3'非编码区。编码序列预测了先前未知的“前导”氨基酸序列，并阐明了已被破坏的激素氨基酸序列。5'非编码区在真正的起始密码子之前包含一个AUG密码子，接着是一个UGA终止密码子。3'非编码区比牛甲状旁腺激素原前体mRNA长120个核苷酸，并包含两个A - A - U - A - A - A序列，这是聚腺苷酸化的潜在信号。
