限制和修饰酶基因Lla BⅢ的克隆及分析

pJW566是从丹麦乳酪生产菌株Lactococcus lactis subsp.cremoris W56中分离到的，一个22.4kb，具有限制和修饰作用的质粒，内切酶ClaⅠ和pJW566不完全消化，所得片段与来自于质粒pVC5的氯霉素抗性基因连接得到一个携带有完整限制和修饰酶基因的质粒pJK1。基因亚克隆分析发现该基因位于约5kb的Sph0Ⅰ-Hin dⅢDNA片段上。序列分析表明该片段包含一个4572bp的开放阅读框架、编码一个由1576／1584个氨基酸残基组成的蛋白质，该基因命名为Lla BⅢ。蛋白质同源性查询发现在该蛋白的N－末端有7个保守区域，与R／M系统Ⅰ型和Ⅲ型内切酶有较高同源性，在蛋白的中间区域有4个代表N^6-腺苷酰甲基转移酶的特征序列，而蛋白的C－末端不同于任何已知蛋白。这种具有限制、修饰和可能的DNA识别作用的多功能蛋白，可能是一新的R／M系统。

Cloning and structure analysis of a restriction and modification system, LlaBIII from Lactococcus lactis subsp. cremoris W56]

A 22.4 kb naturally occurring plasmid pJW566, isolated from L. lactis W56, was found to encode an R/M system named LlaBIII. The LlaBIII R/M system was isolated on a chloramphenicol resistant derivative of plasmid pJW566, resulting in a plasmid pJK1. Subcloning analysis showed that the LlaBIII determinant was located on a 5 kb HindIII-Sph I fragment. The fragment was sequenced. It contained a single open reading frame (ORF), corresponding to a protein of 1584 or 1576 aa. In the deduced amino acid sequence seven helicase motifs characteristic of endonuclease type I and type III and a conserved catalysis motif X in the R subunits of type I R/M systems were located in the N-terminus, followed by four conserved motifs found in DNA N6-adenine methyltransferases. The C-terminus of the deduced amino acid sequence showed no homology to known R/M systems. Therefore, this polypeptide encoded by LlaBIII is a multifunctional protein possessing putative DNA recognition, methylation and restriction activities.