Critical roles of activation-induced cytidine deaminase in the homeostasis of gut flora

Activation-induced cytidine deaminase (AID) plays an essential role in class switch recombination (CSR) and somatic hypermutation (SHM) of immunoglobulin genes. We report here that deficiency in AID results in the development of hyperplasia of isolated lymphoid follicles (ILFs) associated with a 100-fold expansion of anaerobic flora in the small intestine. Reduction of bacterial flora by antibiotic treatment of AID-/- mice abolished ILF hyperplasia as well as the germinal center enlargement seen in secondary lymphoid tissues. Because an inability to switch to immunoglobulin A on its own does not lead to a similar phenotype, these results suggest that SHM of ILF B cells plays a critical role in regulating intestinal microflora.     

Antibody class switching mediated by yeast endonuclease-generated DNA breaks

Antibody class switching in activated B cells uses class switch recombination (CSR), which joins activation-induced cytidine deaminase (AID)-dependent double-strand breaks (DSBs) within two large immunoglobulin heavy chain (IgH) locus switch (S) regions that lie up to 200 kilobases apart. To test postulated roles of S regions and AID in CSR, we generated mutant B cells in which donor Smu and accepter Sgamma1 regions were replaced with yeast I-SceI endonuclease sites. We found that site-specific I-SceI DSBs mediate recombinational IgH locus class switching from IgM to IgG1 without S regions or AID. We propose that CSR evolved to exploit a general DNA repair process that promotes joining of widely separated DSBs within a chromosome.     

Strand-biased spreading of mutations during somatic hypermutation

Somatic hypermutation (SHM) is a major means by which diversity is achieved in antibody genes, and it is initiated by the deamination of cytosines to uracils in DNA by activation-induced deaminase (AID). However, the process that leads from these initiating deamination events to mutations at other residues remains poorly understood. We demonstrate that a single cytosine on the top (nontemplate) strand is sufficient to recruit AID and lead to mutations of upstream and downstream A/T residues. In contrast, the targeting of cytosines on the bottom strand by AID does not lead to substantial mutation of neighboring residues. This strand asymmetry is eliminated in mice deficient in mismatch repair, indicating that the error-prone mismatch repair machinery preferentially targets top-strand uracils in a way that promotes SHM during the antibody response.     

Uracil DNA glycosylase activity is dispensable for immunoglobulin class switch

Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step in immunoglobulin class switch recombination (CSR). AID is proposed to deaminate cytosine to generate uracil (U) in either mRNA or DNA. In the second instance, DNA cleavage depends on uracil DNA glycosylase (UNG) for removal of U. Using phosphorylated histone gamma-H2AX focus formation as a marker of DNA cleavage, we found that the UNG inhibitor Ugi did not inhibit DNA cleavage in immunoglobulin heavy chain (IgH) locus during CSR, even though Ugi blocked UNG binding to DNA and strongly inhibited CSR. Strikingly, UNG mutants that had lost the capability of removing U rescued CSR in UNG-/- B cells. These results indicate that UNG is involved in the repair step of CSR yet by an unknown mechanism. The dispensability of U removal in the DNA cleavage step of CSR requires a reconsideration of the model of DNA deamination by AID.     

Generation and analysis of interleukin-4 deficient mice

Interleukin-4 (IL-4) promotes the growth and differentiation of many hematopoietic cells in vitro; in particular, it directs the immunoglobulin (Ig) class switch to IgG1 and IgE. Mice homozygous for a mutation that inactivates the IL-4 gene were generated to test the requirement for IL-4 in vivo. In the mutant mice T and B cell development was normal, but the serum levels of IgG1 and IgE were strongly reduced. The IgG1 dominance in a T cell-dependent immune response was lost, and IgE was not detectable upon nematode infection. Thus, some but not all of the in vitro properties of IL-4 are critical for the physiology of the immune system in vivo.     

鸡Igλ轻链基因克隆、序列分析及其在COS7细胞膜上的定位表达

为了研究鸡B细胞膜免疫球蛋白(mIg)的功能,深入了解鸡免疫系统抗体基因的特点,从鸡法氏囊B细胞cDNA中扩增出Igλ轻链基因,对其核苷酸序列和氨基酸序列进行了分析和比较。该基因cDNA全长873bp,编码含226个氨基酸的Igλ轻链,N-端21个氨基酸构成轻链信号肽,随后是2个Ig样结构域。运用融合PCR的方法将该基因与牛IgG Fc受体γRⅡ跨膜区序列嵌合形成重组跨膜分子,构建真核表达载体pcDNA-λR2T,转染COS7细胞,荧光抗体染色及流式细胞术检测到重组鸡Igλ轻链在细胞膜上的表达。所扩增的鸡Igλ轻链基因以及构建表达于细胞膜上的重组鸡Igλ轻链跨膜分子,为研究鸡免疫系统中的抗体轻链基因,探索Igλ轻链和B细胞膜免疫球蛋白的功能奠定技术基础。     

Evolution of autoantibody responses via somatic hypermutation outside of germinal centers

Somatically mutated high-affinity autoantibodies are a hallmark of some autoimmune diseases, including systemic lupus erythematosus. It has long been presumed that germinal centers (GCs) are critical in autoantibody production, because they are the only sites currently believed to sustain a high rate of somatic hypermutation. Contrary to this idea, we found that splenic autoreactive B cells in autoimmune MRL.Fas(lpr) mice proliferated and underwent active somatic hypermutation at the T zone-red pulp border rather than in GCs. Our results implicate this region as an important site for hypermutation and the loss of B cell self-tolerance.     

AID-driven deletion causes immunoglobulin heavy chain locus suicide recombination in B cells

Remodeling of immunoglobulin genes by activation-induced deaminase (AID) is required for affinity maturation and class-switch recombination in mature B lymphocytes. In the immunoglobulin heavy chain locus, these processes are predominantly controlled by the 3' cis-regulatory region. We now show that this region is transcribed and undergoes AID-mediated mutation and recombination around phylogenetically conserved switchlike DNA repeats. Such recombination, which we term locus suicide recombination, deletes the whole constant region gene cluster and thus stops expression of the immunoglobulin of the B cell surface, which is critical for B cell survival. The frequency of this event is approaching that of class switching and makes it a potential regulator of B cell homeostasis.     

The need for central and peripheral tolerance in the B cell repertoire

The immune system normally avoids producing antibodies that react with autologous ("self") antigens by censoring self-reactive T and B cells. Unlike the T cell repertoire, antibody diversity is generated within the B cell repertoire in two phases; the first occurs by gene rearrangement in primary lymphoid organs, and the second phase involves antigen-driven hypermutation in peripheral lymphoid organs. The possibility that distinct cellular mechanisms may impose self tolerance at these two different phases of B cell diversification may explain recent findings in transgenic mouse models, in which self-reactive B cells appear to be silenced both by functional inactivation and by physical elimination.     

TAPHS1基因序列多态性与小麦生殖发育稳定性的相关分析

TAPHS1基因是MFT-like基因,可调控小麦成熟期的籽粒休眠(影响穗发芽抗性)且与开花的发育控制相关,位于小麦3A染色体短臂上。为探究该基因序列多态性与小麦生殖发育稳定性的关系,设计PCR引物扩增TAPHS1基因的2个高频变异区,获得86个品种相应基因区段的DNA序列信息;以不同播期之间抽穗期相差时间为指标调查评价各品种的生殖发育稳定性。结果表明:在TAPHS1基因的高频变异区存在5种多态性,将其命名为A类、B类、C类、D类、E类;A类、B类、C类属非编码区多态性,D类及E类同时涉及编码区和非编码区多态性;D类及E类序列多态性与生殖发育稳定性相关,并根据D类序列信息开发了分子标记。     

Chromosomal locations of the murine T-cell receptor alpha-chain gene and the T-cell gamma gene

Two independent methods were used to identify the mouse chromosomes on which are located two families of immunoglobulin (Ig)-like genes that are rearranged and expressed in T lymphocytes. The genes coding for the alpha subunit of T-cell receptors are on chromosome 14 and the gamma genes, whose function is yet to be determined, are on chromosome 13. Since genes for the T-cell receptor beta chain were previously shown to be on mouse chromosome 6, all three of the Ig-like multigene families expressed and rearranged in T cells are located on different chromosomes, just as are the B-cell multigene families for the Ig heavy chain, and the Ig kappa and lambda light chains. The findings do not support earlier contentions that genes for T-cell receptors are linked to the Ig heavy chain locus (mouse chromosome 12) or to the major histocompatibility complex (mouse chromosome 17).     

猪伪狂犬病病毒gD基因的克隆与分析

克隆测定12条PRV不同毒株的gD全序列连同Genebank中登录的9条gD基因全序列共21条基因序列,使用生物软件对它们的基因序列的同源性、突变区域的定位、遗传进化关系、氨基酸序列的同源性、蛋白质亲水性、抗原表位分析、三级结构预测等生物信息学的内容进行预测和分析。结果表明:PRV-gD基因的开放阅读框的核苷酸长度在1197~1215nt之间,氨基酸长度在399~405个之间,核酸同源性在97.3%~100%之间,氨基酸的同源性在89.8%~98.8%之间,在核酸820~837位有个高变重复区。在遗传进化关系上将我国PRV流行分为四川、华北、东南三个区域。该结果说明PRV-gD基因具有很高的保守性。     

Programmed gene rearrangements altering gene expression

Programmed gene rearrangements are used in nature to to alter gene copy number (gene amplification and deletion), to create diversity by reassorting gene segments (as in the formation of mammalian immunoglobulin genes), or to control the expression of a set of genes that code for the same function (such as surface antigens). Two major mechanisms for expression control are DNA inversion and DNA transposition. In DNA inversion a DNA segment flips around and is rejoined by site-specific recombination, disconnecting or connecting a gene to sequences required for its expression. In DNA transposition a gene moves into an expression site where it displaces its predecessor by gene conversion. Gene rearrangements altering gene expression have mainly been found in some unicellular organisms. They allow a fraction of the organisms to preadapt to sudden changes in environment, that is, to alter properties such as surface antigens in the absence of an inducing stimulus. The antigenic variation that helps the causative agents of African trypanosomiasis, gonorrhea, and relapsing fever to elude host defense is controlled in this way.     

Development of the expressed immunoglobulin μ chain repertoire during maturation of mice B cells

In the bone marrow and spleen, the developing B cell populations undergo both negative and positive selections to shape their B cell receptor repertoire. To gain insight into the shift of the immunoglobulin heavy (IgH) chain repertoire during B cell development, we undertook large scale Ig μ chain repertoire analysis of pre-B, immature B and spleen B cell populations. We found that the majority of V_H gene segments, V_H families, J_H and D gene segments, were observed to have significantly different usage frequencies when three B cell populations were compared, but the usage profile of the V_H, D, and J_H genes between different B cell populations showed high correlations. In both productive and nonproductive rearrangements, the length of CDRH3 shortened significantly on average when B cells entered the periphery. However, the CDRH3 length distribution of nonproductive rearrangements did not follow a Gaussian distribution, but decreased successively in the order 3n-2, 3n-1 and 3n, suggesting a direct correlation between mRNA stability and CDRH3 length patterns of nonproductive rearrangements. Further analysis of the individual components comprising CDRH3 of productive rearrangements indicated that the decrease in CDRH3 length was largely due to the reduction of N addition at the 5′ and 3′ junctions. Moreover, with development, the amino acid content of CDRH3 progressed toward fewer positively charged and nonpolar residues but more polar residues. All these data indicated that the expressed Ig μ chain repertoire, especially the repertoire of CDRH3, was fine-tuned when B cells passed through several checkpoints of selection during the process of maturation.     

44例多发性骨髓瘤实验室指标及Ig类型改变的分析

目的探讨骨髓细胞形态学检查结合血清蛋白电泳、免疫固定电泳、免疫球蛋白定量检查诊断多发性骨髓瘤,并探讨Ig类型改变的原因．方法对44例多发性骨髓瘤患者的骨髓细胞形态学检查、血清蛋白电泳、免疫固定电泳及免疫球蛋白的数据进行统计分析．结果44例MM患者中,骨髓瘤细胞〈10％的有40例（90．9％）,且浆细胞具有较高的异质性;血清蛋白电泳检出M蛋白带的比例为88．6％;免疫固定电泳检出率为100％,免疫分型IgG占50％,IgA占20．5％,IgM占2．2％,轻链型占27．3％;免疫球蛋白定量显示同型的免疫球蛋白含量显著升高,但轻链型的各免疫球蛋白含量却正常或降低．其中1例MM患者骨髓移植后出现Ig类型转换．结论综合各实验室指标,可以最大程度减少多发性骨髓瘤的漏诊、误诊,Ig类型的改变对多发性骨髓瘤的临床诊断及治疗有再要意义．     

苏云金芽胞杆菌肠毒素基因克隆与序列分析

根据蜡质芽孢杆菌(Bc)肠毒素基因序列,设计特异PCR引物,对本实验室保存的 16个苏云金芽孢杆菌 (Bt)菌株进行检测.结果显示, 15个菌株含有肠毒素基因片段.克隆了Bt8010肠毒素entS基因的编码框 (ORF)序列, 将该序列测序,用在线的BLAST软件与DNASIS软件进行同源性分析.结果表明,该基因与已知的Bc和炭疽芽孢杆菌肠毒素基因有很高的同源性,但该基因有 12个核苷酸缺失,不能表达完整多肽.