限制和修饰酶基因LlaBⅢ的克隆及分析

Cloning and Structure Analysis of A Restriction and Modification System,LlaBⅢ from Lactococcus lactis subsp.cremoris W56

pJW5 66是从丹麦乳酪生产菌株Lactococcuslactissubsp .cremorisW5 6中分离到的 ,一个 2 2 4kb、具有限制和修饰作用的质粒。内切酶ClaⅠ对pJW5 66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SphⅠ HindⅢDNA片段上。序列分析表明该片段包含一个 4 5 72bp的开放阅读框架 ,编码一个由 15 76 15 84个氨基酸残基组成的蛋白质 ,该基因命名为LlaBⅢ。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N6 - 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。

A 22.4kb naturally occurring plasmid pJW566,isolated from %L.lactis% W56, was found to encode an R/M system named %Lla%BⅢ [1] .The %Lla%BⅢ R/M system was isolated on a chloramphenicol resistant derivative of plasmid pJW566,resulting in a plasmid pJK1. Subcloning analysis showed that the %Lla%BⅢ determinant was located on a 5 kb %Hin%dⅢ-%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 N+6-adenine methyltransferases. The C-terminus of the deduced amino acid sequence showed no homology to known R/M systems.Therefore, this polypeptide encoded by %Lla%BⅢ is a multifunctional protein possessing putative DNA recognition,methylation and restriction activities.