ILG1:在纽约实验室大肠杆菌菌株中发现一种与噬菌体整合酶相似的新p4(英文)

ILG1: A new p4 bacteriophage-like integrase found in a New York laboratory Escherichia coli strain

目的 免疫球蛋白类别转换在生发中心 B细胞中进行 ,从而 B细胞由产生 Ig M转换到产生其他类别的免疫球蛋白如 Ig G,Ig A,Ig E.目前导致类别转换的机制还不清楚 .我们试图克隆参与类别转换的基因 .方法 原发性免疫缺陷患者 B细胞不能进行类别转换 ,而正常细胞则可以 .据此 ,我们将患者与正常人的 B细胞在同一条件下分别培养以诱导类别转换 .从培养的细胞中分别提取 RNA,并反转录成 c DNA,由此制备扩增物 (amplicon) .最后将来源于患者 B细胞的扩增物与正常人 B细胞的扩增物混合在一起 ,在一定条件下进行剔除杂交 .经 5轮剔除杂交以后 ,将剔除共同基因以后的剩余物进行克隆 .经筛选以后将阳性克隆物进行测序及序列分析 .结果 在所有克隆中 ,我们发现一个新基因 ,命名为整合酶样基因 - 1.后证实 ,该基因来源于实验室细菌株 TOP10 F'序列分析表明 ,IL G1具有原核基因特性 ,但缺乏真核细胞的基因特性 .在 DNA水平 ,IL G1与任何基因都不具有同源性 ,但在蛋白水平 ,它与来源于溶原性 p4噬菌体的整合酶具有高达 5 8%的同源性 ,我们认为 IL G1定居于一个新的溶原性 p4原噬菌体 Φ- TOP10 F.IL G1的催化活性区具有高度保守特性 ,因为活性区的关键氨基酸残基是保守的 ,这包括高度保守的 R- H- R- Y结构 .

AIM Immunoglobulin (Ig) class switch recombination (CSR) in germinal center B cells results in the switch in expression from one Ig isotype (IgM) to another isotype (IgG, IgA, or IgE). Mechanisms involved in this event are not understood. To understand this, we have begun to clone and characterize gene (s) involved in CSR by PCR based cDNA representational difference analysis (cDNA RDA). METHODS B cells from common variable immuodeficiency (CVI) patients which can not undergo CSR due to B cells intrinsic defects, but those from normal control can. These two types of B cell were cultured at the same condition where Ig class switching was induced. RNA from both CVI patient B cells (Driver) and normal control B cells (Tester) was isolated and reverse transcripted into cDNA respectively. After amplicons were prepared, cDNA RDA was applied by subtracting tester amplicon with excess amount of driver amplicon. After five rounds of cDNA RDA, the subtract was cloned and sequenced. RESULTS Among the clones isolated, one novel gene, which we named integrase like gene 1 (ILG1), was isolated from a common laboratory Escherichia coli strain (TOP10F'), which contaminated samples isolated from human normal and common variable immunodeficient patient B lymphocytes. ILG1 was demonstrated to come from this bacteria by its presence in bacterial rather than human DNA. Furthermore, the sequence of ILG1 exhibits prokaryotic, but not eukaryotic features. At the DNA level, ILG1 is not similar to any known genes. However, at the protein le vel, ILG1 has 58% similarity to integrases from cryptic p4 bacteriophages. Indeed, ILG1 is most similar to the bacteriophage p4 family (S clade) of integrases. Because of these similarities, we propose that ILG1 resides on a new cryptic p4 prophage Φ TOP10F found in this bacterium. Finally, the catalytic domain of ILG1 contains the conserved features found in site specific recombinases. The critical residues that form the catalytic active site pocket are conserved, including the highly conserved R H R Y hallmark of these recombinases. CONCLUSION ILG1 is likely an active site specific recombinase and a new member of the bacteriophage p4 family integrases.