Takkinen K, Pettersson R F, Kalkkinen N, Palva I, Söderlund H, Kääriäinen L
J Biol Chem. 1983 Jan 25;258(2):1007-13.
We have isolated by molecular cloning the gene coding for the alpha-amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1.) from Bacillus amyloliquefaciens and determined its complete nucleotide sequence. The gene cloned in the plasmid pUB110 using Bacillus subtilis as a host, was contained in a 2.3-kilobase insert. Starting from an ATG initiator codon, an open reading frame comprising a total of 514 amino acids (1542 base pairs) was found within the cloned DNA fragment. The gene region encoding the COOH terminus of alpha-amylase was located by direct COOH-terminal analysis of the purified exoenzyme. The NH2-terminal portion of the gene encodes a 31 amino acid-long signal peptide (Palva, L., Pettersson, R. F., Kalkkinen, N., Lehtovaara, P., Sarvas, M., Söderlund, H., Takkinen, K., and Kääriäinen, L. (1981) Gene 15, 43-51). Since the signal peptide is correctly cleaved in the new host, as shown here by direct NH2-terminal sequence analysis, the exoamylase consists of 483 amino acid residues, corresponding to a molecular weight of 54,778. The reading frame used to deduce the amino acid sequence was found to be correct by comparison with partial amino acid sequence data published previously (Detera, S. D., and Friedberg, F. (1979) Int. J. Peptide Protein Res. 14, 364-372; Chung, H., and Friedberg, F. (1980) Biochem. J. 185, 387-395). Several differences between the sequence presented here and the partial ones published previously, however, were found. The nucleotide sequences both 5' and 3' to the alpha-amylase gene revealed palindromic structures including a stretch of six T-residues, suggesting transcription termination signals on both sides of the gene. Thus, it appears that alpha-amylase is translated from a monocistronic mRNA.
我们通过分子克隆从解淀粉芽孢杆菌中分离出编码α-淀粉酶（1,4-α-D-葡聚糖葡聚糖水解酶，EC 3.2.1.1.）的基因，并确定了其完整的核苷酸序列。以枯草芽孢杆菌为宿主，克隆在质粒pUB110中的基因包含在一个2.3千碱基的插入片段中。从ATG起始密码子开始，在克隆的DNA片段中发现了一个由总共514个氨基酸（1542个碱基对）组成的开放阅读框。通过对纯化的胞外酶进行直接的COOH末端分析，确定了编码α-淀粉酶COOH末端的基因区域。该基因的NH2末端部分编码一个31个氨基酸长的信号肽（帕尔瓦，L.，彼得松，R.F.，卡尔基宁，N.，莱托瓦拉，P.，萨尔瓦斯，M.，索德伦德，H.，塔基宁，K.，卡里亚宁，L.（1981年）《基因》15卷，43 - 51页）。如这里通过直接的NH2末端序列分析所示，由于信号肽在新宿主中被正确切割，胞外淀粉酶由483个氨基酸残基组成，分子量为54,778。通过与先前发表的部分氨基酸序列数据比较（德特拉，S.D.，和弗里德伯格，F.（1979年）《国际肽与蛋白质研究杂志》14卷，364 - 372页；钟，H.，和弗里德伯格，F.（1980年）《生物化学杂志》185卷，387 - 395页），发现用于推导氨基酸序列的阅读框是正确的。然而，这里呈现的序列与先前发表的部分序列之间存在一些差异。α-淀粉酶基因5'和3'端的核苷酸序列显示出回文结构，包括一段六个T残基的序列，表明该基因两侧存在转录终止信号。因此，α-淀粉酶似乎是从单顺反子mRNA翻译而来的。
