变铅青链霉菌66中对噬菌ΦHAU3显示抗性的基因（ψHAU3￣R）的核苷酸定序和分析

SEQUENCEANALYSIS OF ΦHAU3￣R GENE OF STREPTOMYCES LIVEKANS 66

杂合质粒ｐＩＪ８３１０是链霉菌低拷贝质粒ｐＩＪ９２２１携带了１０．８ｋｂ来自变铅青链霉菌ＪＴ４６菌株，编码对噬菌体ΦＨＡＵ３抗性的ＤＮＡ片段。已知Ｔｎ４５６０插到该克隆中３ｋｂ的ＥｃｏＲＩ或３．３ｋｂ的ＢａｍＨＩ片段（这两个片段之间有２．５ｋｂ的重叠区）上，中断了ΦＨＡＵ３抗性基因的表达。已将３．０ｋｂ的ＢｃｏＡＩ片段分别以两种不同的取向克隆到ｐＢｌｕｅｓｃｒｉｐｔＳＫ（＋）上。核苷酸序列分析揭示出一个１．３ｋｂ的ＯＲＦ的存在，其所编码的４７８个氨基酸组成的蛋白与λ噬菌体的ｅａ５９基因所编码的氨基酸序列显示了高度的同源性，其氨基酸序列的同一性高达３７％，相似性达５８％，而λ噬菌体的ｅａ５９基因是编码赖ＡＴＰ和单键ＤＮＡ的核酸内切酶Ｉ的，该区域与λ的复制无关。此外，这段具有高度同源性的ＤＮＡ区域的Ｇ＋Ｃ％只有６０％，低于变铅青霉菌ＤＮＡＧ＋Ｃ％的平均值（７４％）。

ecombinant plasmid, plasmid,pIJ8310,is the low-copy number plasmid pIJ992 carrying 10.8kb insert from Streptomyces lividans JT46 that confers ΦHAU3-resistance on ZXl.Transposon Tn4560 inserted into 3kb EcoRI or 3.2kb BamHI fragments(with 2.5kboverlapping region)inactivated the ΦHAU3-resistance.The 3kb EcoRI fragment frompIJ8310 was subcloned into pBluescrept SK(+)in both orientations.The FRAME plot ot the DNA sequence shows a 1.3kb open reading frame.The amino acid sequence deduced fromthe DNA sequence of ΦHAU3 resistance gene has a strong similarity with the hypotheticalEa59 protein that was predicted from the DNA sequence of phage λ.The alignment of the two amino acid sequences revealed 37% amino acid identity and 58% similar amino and residues.The ea59 gene of lambda is situated in the dispensable b2 region.It encodedendonuclease I that is ATP and single-strand DNA-dependent but specificly degrades CCC DNA. In addition,the G+C content of the gene encoding the ΦHAU3 resistance openreading frame that has a strong similarity with the ea59 gene of lambda is 60%, but lowthat Streptomyces lividans DNA which has on average about 74%G+C.