Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli
Cloning and expression of the ApaLI, NspI, NspHI, SacI, ScaI, and SapI restriction-modification systems in Escherichia coli
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DOI：
10.1007/s004380050890
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发表时间：
1998-11-01
期刊：
影响因子：
--
通讯作者：
Benner, J
中科院分区：
文献类型：
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作者：
Xu, SY;Xiao, JP;Benner, J
编码ApaLI（5 ′-GTGCA-C-3 ′）、NspI（5 ′-RCATG Y-3 ′）、NspHI（5 ′-RCATG Y-3 ′）、SacI（5 ′-GAGT C-3 ′）、SapI（5 ′-GCTCTTCN 1 - 3 ′、5 ′-N4 GAAGAGC-3 ′）和ScaI（5 ′-AGT ACT-3 ′）限制性修饰系统的基因已在E.杆菌ApaLI、M. NspI，M. NspHI和M. SacI与已知的甲基化酶表明，它们含有C5胞嘧啶甲基化酶的10个保守基序特征。NspI和NspHI限制修饰系统在氨基酸序列上高度同源。NspI和NlaIII（5 '-CATG-3'）限制性内切酶的C-末端具有显著的相似性。在SacI位点的内部C5的5 mC修饰使其对SacI消化具有抗性。SacI位点的外部5 mC修饰对SacI消化没有影响。序列5 '-GCTCTTC-3'中第二个碱基的N4 mC修饰阻断SapI消化。在SapI位点的其他胞嘧啶的N4 mC修饰不影响SapI消化。Sca I位点的N4 mC修饰阻断Sca I抑制。DNA转化酶同源物被发现邻近ApaLI限制修饰系统。在SapI限制性内切酶基因的上游发现了一个DNA转座酶亚基同源物。
The genes encoding the ApaLI (5'-G TGCA-C-3'), NspI (5'-RCATG Y-3'), NspHI (5'-RCATG Y-3'), SacI (5'-GAGT C-3'), SapI (5'-GCTCTTCN1 -3', 5'- N4GAAGAGC-3') and ScaI (5'-AGT ACT-3') restriction-modification systems have been cloned in E. coli. Amino acid sequence comparison of M .ApaLI, M. NspI, M. NspHI, and M. SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5'-CATG-3') restriction endonucleases share significant similarity. 5mC modification of the internal C5 in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5'-GCTCTTC-3' blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.