Noyes B E, Mevarech M, Stein R, Agarwal K L
Proc Natl Acad Sci U S A. 1979 Apr;76(4):1770-4. doi: 10.1073/pnas.76.4.1770.
We have used a specific deoxyoligonucleotide probe to detect gastrin mRNA in poly(A)-enriched RNA preparations from hog antrum. The nucleotide sequence of the oligonucleotide, d(C-T-C-C-T-C-C-A-T-C-C-A), was deduced from the unique amino acid sequence Trp-Met-Glu-Glu of gastrin. When used with hog antral RNA, the dodecanucleotide is an effective primer for the synthesis of gastrin-specific cDNA as judged by nucleotide sequence analysis of cDNA isolated by polyacrylamide gel electrophoresis. We have determined an 81-nucleotide sequence corresponding to the region of the gastrin mRNA that codes for the known amino acid sequence of the G34 progastrin intermediate species, and we have demonstrated the presence of two consecutive basic residues preceding the G34 sequence in the prohormone. Hybridization of gastrin cDNA or synthetic dodecanucleotide to hog antral RNA separated by gel electrophoresis on agarose gels in the presence of methylmercuric hydroxide indicates that the mRNA coding for gastrin is about 620 nucleotides long. These results suggest that the gastrin precursor peptide contains 110-140 amino acids. This method should be of general application for detection and characterization of mRNAs corresponding to proteins of known amino acid sequence.
我们使用了一种特异性脱氧寡核苷酸探针，来检测来自猪胃窦富含多聚腺苷酸（poly(A)）的RNA制剂中的胃泌素mRNA。该寡核苷酸的核苷酸序列d(C-T-C-C-T-C-C-A-T-C-C-A)是根据胃泌素独特的氨基酸序列色氨酸-甲硫氨酸-谷氨酸-谷氨酸推导出来的。当与猪胃窦RNA一起使用时，通过对聚丙烯酰胺凝胶电泳分离得到的cDNA进行核苷酸序列分析判断，该十二核苷酸是合成胃泌素特异性cDNA的有效引物。我们确定了一个81个核苷酸的序列，它对应于胃泌素mRNA中编码G34前胃泌素中间物种已知氨基酸序列的区域，并且我们已经证明在该前体激素的G34序列之前存在两个连续的碱性残基。在氢氧化甲基汞存在的情况下，胃泌素cDNA或合成的十二核苷酸与在琼脂糖凝胶上通过凝胶电泳分离的猪胃窦RNA杂交，表明编码胃泌素的mRNA约620个核苷酸长。这些结果表明胃泌素前体肽含有110 - 140个氨基酸。该方法对于检测和鉴定与已知氨基酸序列的蛋白质相对应的mRNA应具有普遍适用性。
