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ANALYSIS OF MOLECULAR STRUCTURE AND REACTION MECHANISM OF RESTRICTION ENDONUCLEASE FROM ACIDOPHILIC BACTERIA

嗜酸菌限制性内切酶分子结构及反应机制分析

基本信息

批准号：
07680688
负责人：
INAGAKI Kenji
金额：
$ 1.15万
依托单位：
OKAYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

1) Cloning and sequencing of the retriction-modification system gene from acidophilic bacteria.The Afa22MI restriction-modification system from Acidocella facilis 22M recognizes the nucleotide sequence CGATCG.The M.Afa22MI gene was cloned into E.coli XL-1 Blue MRF^1 and the nucleotide sequence was determined. The homologous regions observed in 5-methyl cytosine methyltransferases (C5-MTase) were identified in the deduced amino acid sequence of M.Afa22MI.These conserved regions and the carboxyl terminal sequence of the M.Afa22MI showed high similarity with those of M.XorII which recognized same nucleotide sequence. The ORF located upstream of MTase gene showed high homology with the very short patch repair endonuclease of XorII and other vsr gene found in C5-MTase-endonuclease system.2) Purification and characterization of DNA methylases.Two DNA methyltransferases, M.Afa22MI and M.Afa16RI,were purified and characterized from same species Acidocella facilis 22M and 16R,respectively.3) Determination of methylation specificity of sequence-specific DNA methylase using MALDI-TOF MS.Time-dependent phosphodiesterase digestion of methylated oligonucleotide coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has enabled a sensitive and straightforward method for determing the methylation specificity of type II DNA methyltransferase. The mass deference between the peaks corresponded to the individual nucleotide released by the enzymatic cleavage, and the methylation site can be explicitly identified in reference to the know sequence of the substrate DNA.4) Crystallization of type II restriction endonuclease.We prepared large amounts of the homogeneous R.Aor 13HI,and crystallized it with an oligonucleotide containing it's recognition site (5'-TCCGGA-3') by hanging drop vapor diffusion method. A small crystal was obtained by precipitation with polyethylene glycol 4000 or 6000,100 mM HEPES (pH8.0), 100mM NaCl.

1)嗜酸菌逆转录酶修饰系统基因的克隆和测序：利用Acidocella facilis 22M的Afa22MI限制性内切酶修饰系统识别CGATCG核苷酸序列，将其克隆到E.coliXL-1 Blue MRF^1中，并测定了其核苷酸序列。5-甲基胞嘧啶甲基转移酶(C5-MTase)在Afa22MI推导的氨基酸序列中发现了同源区域，这些保守区和羧基末端序列与识别相同核苷酸序列的M.XorII高度相似。位于MTase基因上游的ORF与C5-MTase-核酸内切酶系统中发现的XorII和其他VSR基因的极短片段修复内切酶具有很高的同源性。2)DNA甲基酶的纯化和性质。从同一物种Acidocella facilis 22M和16R中纯化并鉴定了两种DNA甲基转移酶M.Afa22MI和M.Afa16RI，3)用MALDI-TOF MS测定序列特异性DNA甲基化酶的甲基化特异性。甲基化寡核苷酸的时间依赖性磷酸二酯酶消化与基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)相结合，为检测II型DNA甲基转移酶的甲基化特异性提供了一种灵敏而直接的方法。4)II型限制性内切酶的结晶。我们制备了大量均一的rAor 13HI，并用悬滴气相扩散法将其与含有识别位点的寡核苷酸(5‘-TCCGGA-3’)结晶。用聚乙二醇4000或6000,100 mM HEPES(pH8.0)，100 mM NaC l沉淀得到小晶体。