CLONING AND ANALYSIS OF THE PLASMID-BORNE GENES ENCODING THE BSP6I RESTRICTION AND MODIFICATION ENZYMES
CLONING AND ANALYSIS OF THE PLASMID-BORNE GENES ENCODING THE BSP6I RESTRICTION AND MODIFICATION ENZYMES
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DOI：
10.1016/0378-1119(94)00795-t
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发表时间：
1995-05-19
期刊：
影响因子：
3.5
通讯作者：
JANULAITIS, A
中科院分区：
文献类型：
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作者：
LUBYS, A;JANULAITIS, A
Bsp6I 限制和修饰 (R-M) 系统已定位于天然存在于芽孢杆菌属的质粒 pXH13 上。菌株 RFL6。编码 Bsp6I R-M 系统（识别序列 GCNGC 的 Fnu4HI 同裂酶）的基因已通过两步在大肠杆菌中克隆。含有限制性内切酶（ENase；bsp6IR）和DNA甲基转移酶（MTase；bsp6IM）基因的2126 bp区域的核苷酸序列已被确定。这些基因相距 99 bp，并串联排列，bsp6IR 位于 bsp6IM 之前。 DNA 序列预测 ENase 为 174 个氨基酸 (aa) (19.9 kDa)，MTase 为 315 个氨基酸 (aa) (36.3 kDa)。米。 Bsp6I 包含 m(5)C-MTase 特征的所有保守 aa 序列基序。此外，其可变区与 M 的 5'-GCNGC-3' 特异性靶标识别结构域 (TRD) 略有相似。 Φ3T。 R 之间没有发现 aa 序列相似性。 Bsp6I 和 M 。 Bsp6I，也不在R之中。 Bsp6I 和其他已知的 ENase。我们测试了携带完整 R-M 系统的重组质粒转化天然和预甲基化大肠杆菌宿主的能力。结果表明，预甲基化提高了完整R-M系统的建立效率。此外，我们还获得了转化效率的方向依赖性差异。
The Bsp6I restriction and modification (R-M) system has been localized on the plasmid pXH13, naturally occurring in the Bacillus sp. strain RFL6. The genes coding for the Bsp6I R-M system, a Fnu4HI isoschizomer recognizing the sequence GCNGC, have been cloned in Escherichia coli by two steps. The nucleotide sequence of a 2126-bp region containing the genes for restriction endonuclease (ENase; bsp6IR) and DNA methyltransferase (MTase; bsp6IM) has been determined. The genes are separated by 99 bp and are arranged tandemly with bsp6IR preceding bsp6IM. The DNA sequence predicts an ENase of 174 amino acids (aa) (19.9 kDa) and a MTase of 315 aa (36.3 kDa). M . Bsp6I contains all the conserved aa sequence motifs characteristic for m(5)C-MTases. In addition, its variable region exhibits a slight similarity to the 5'-GCNGC-3'-specific target-recognition domain (TRD) from M . phi 3T. No aa sequence similarity was found between R . Bsp6I and M . Bsp6I, nor among R . Bsp6I and other known ENases. We have tested recombinant plasmids carrying the complete R-M system for their ability to transform native and pre-methylated Escherichia coli hosts. The results indicate that pre-methylation increases the efficiency of establishment of the complete R-M system. In addition, we have obtained orientation-dependent differences in transformation efficiency.