Excision products of TCR V{alpha} recombination contain in-frame rearrangements: evidence for continued V(D)J recombination in TCR+ thymocytes

Cortical thymocytes that express a TCR on their surface continueto express mRNA for the recombination activating genes RAG-1and RAG-2. As the expression of these genes appears to be necessaryand sufficient for V(D)J recombination to occur, this findingsuggests that productive TCR gene rearrangement may not by itselfterminate TCR recombination. To further study this question,we have utilized a polymerase chain reaction-based strategyto isolate and sequence excision products of secondary TCR generearrangement events. Our data shows that TCR excision productsfrom TCR⁺ thymocytes contain in-frame V–J joints and thussupport the concept that V(D)J recombinatorial activity maycontinue even in thymocytes which have undergone productiveTCR gene rearrangement.     

Re-expression of RAG-1 and RAG-2 genes and evidence for secondary rearrangements in human germinal center B lymphocytes

V(D)J recombination occurs in immature B cells within primary lymphoid organs. However, recent evidence demonstrated that the recombination activating genes RAG-1 and RAG-2 can also be expressed in murine germinal centers (GC) where they can mediate secondary rearrangements. This finding raises a number of interesting questions, the most important of which is what is the physiological role, if any, of secondary immunoglobulin (Ig) gene rearrangements. In the present report, we provide evidence that human GC B cells that have lost surface immunoglobulin re-express RAG-1 and RAG-2, suggesting that they may be able to undergo Ig rearrangement. Furthermore, we describe two mature B cell clones in which secondary rearrangements have possibly occurred, resulting in light chain replacement. The two clones carry both κ and λ light chains productively rearranged, but fail to express the κ chain on the cell surface due to a stop codon acquired by somatic mutation. Interestingly, the analysis of the extent of somatic mutations accumulated by the two light chains might suggest that the λ chain could have been acquired through a secondary rearrangement. Taken together, these data suggest that secondary Ig gene rearrangements leading to replacement may occur in human GC and may contribute to the peripheral B cell repertoire.     

Transposition mediated by RAG1 and RAG2 and the evolution of the adaptive immune system

The RAG1 and RAG2 proteins together initiate V(D)J recombination by performing cleavage of chromosomal DNA adjacent to antigen receptor gene segments. Like the adaptive immune system itself,RAG1 andRAG2 are found only in jawed vertebrates. The hypothesis thatRAG1 andRAG2 arose in evolution as components of a transposable element has received dramatic support from our recent finding that the RAG proteins are a fully functional transposase in vitro. This result strongly suggests that antigen receptor genes acquired their unusual structure as a consequence of the insertion of a transposable element into an ancestral receptor gene by RAG1 and RAG2 approx 450 million years ago.     

抗人C1q轻链可变区的序列结构分析

将抗人Ｃ１ｑ轻链可变区（ｐＣ１ｑＶＬ）氨基酸序列与Ｓｗｉｓｓ蛋白质库的序列进行同源性检索及与已知的ＩｇＶ功能区相关分子进行对准比较，检索了同源性积分子的统计学意义，结果表明ｐＣ１ｑＶＬ归属鼠ＩｇＶＫ，符合Ｉｇｋ轻链可变区的基本特征。同时进行了亲水性、可及性分析及二级结构预测，这为利用构象分析方法研究ｐＣ１ｑＶＬ构象特点打下了基础。     

小鼠与人重排活化基因2启动子的比较

目的 通过比较小鼠与人重排活化基因2(RAG2)启动子，试图寻找与小鼠RAG2启动子特异性结合的转录因子。方法 采用表达luciferase的报告基因载体检测启动子的活性。采用：EMSA(electrophoresis mobility shift assay)检测与启动子结合的转录因子。结果小鼠：RAG2启动子-60／-41区域存在富G的GA盒子，而人RAG2启动子在相应位置却是富A区。突变实验结果显示，GA盒子是小鼠RAG2启动子完整活性所必须的。EMSA结果显示，Spl／Sp3结合在小鼠RAG2启动子-60／-41区域，并且Spl能够协同Pax-5、c-Myb活化小鼠RAG2启动子。结论 尽管小鼠与人RAG2启动子同源性很高，但它们结合的转录因子和功能有所不同。     

Analysis of the Regulatory Region of the Bovine X-Chromosomal Amelogenin Gene

The amelogenin proteins, which are crucial for normal enamel mineral formation, are secreted by ameloblasts during development of tooth enamel. In order to better understand the mechanisms involved in regulation of expression of the amelogenin genes, the bovine X-chromosomal amelogenin gene was cloned and a 3.5 KB fragment upstream of exon 1 was inserted into a βgalactosidase (βgal) expression vector for production of transgenic mice. When tissues from these mice were treated with Xgal, a substrate for βgal, only ameloblasts and some of the adjacent stratum intermedium cells contained blue stain. To obtain further information concerning regulation of expression, the 3.5 KB amelogenin gene fragment was evaluated in transfection experiments. Nonoverlapping 1.9 and 1.5 KB fragments of the upstream region were subcloned separately into a vector that contains the SV40 promoter and the CAT reporter gene. Each amelogenin gene fragment was able to suppress CAT activity driven by the heterologous SV40 promoter in transfected HeLa cells. We theorize that each of these gene fragments contains regulatory elements important for the tissue-specific and developmentally-regulated pattern of expression of the X-chromosomal amelogenin gene.     

pSFV1载体系统的改造

目的 获得具有多种稀有酶位点和CMVIE增强子/启动子及SV40晚期poly(A)加尾信号的pSFV1载体系统。方法 首先将含有多种稀有酶位点的人工接头插入到pSFV1的BamHⅠ和SmaⅠ位点（获得的载体称为mpSFV1)。然后在mpSFV1和辅助载体Helper2的SpeⅠ和SphⅠ位点分别插入SV40晚期poly(A)加尾信号和CMVIE增强子/启动子序列,以间接免疫荧光法检测构建的表达载体mpSFV1-A-CMV表达登革2型病毒PrME基因的效率。并用RT-PCR法鉴定辅助载体Helper2-A-CMV包装形成重组病毒的能力。结果 经PCR和酶切鉴定证明,接头,CMVIE增强子/启动子序列和SV40晚期poly(A)加尾信号序列均已导入pSFV1载体系统中;测序结果也与已知序列一致。间接免疫荧光法证明,PrME蛋白在BHK21细胞中获得了表达,同时,改造后的辅助载体也具有包装形成重组病毒的能力。结论 已将以RNA为基础的pSFV1载体系统构建成以DNA为基础的表达载体系统。     

肾细胞癌RASSF1A基因启动子区甲基化状况与表达水平研究

目的检测19例肾细胞癌患者癌组织及癌旁组织Ras相关区域家族蛋白1A（RASSFlA）基因启动子区甲基化情况并检测其mRNA表达水平，探索两者之间的联系。方法收集肾细胞癌患者癌组织及相应癌旁组织19份，分别提取其基因组DNA，以甲基化特异性PCR（Methylation special PCR，MSP）法检测RASSFlA基因启动子区甲基化情况。并以QPCR法检测了RASSFlA基因的mRNA表达水平。结果19例中有lO例肾细胞癌患者癌组织存在高甲基化，mRNA表达水平表达降低，二者之间存在显著负相关性（r=-0．8734，P〈0．01）。结论肾细胞癌中RASSFlA基因启动子区存在高甲基化，并抑制该基因的表达。     

The roles of the RAG1 and RAG2 “non-core” regions in V(D)J recombination and lymphocyte development

The enormous repertoire of the vertebrate specific immune system relies on the rearrangement of discrete gene segments into intact antigen receptor genes during the early stages of B-and T-cell development. This V(D)J recombination is initiated by a lymphoid-specific recombinase comprising the RAG1 and RAG2 proteins, which introduces double-strand breaks in the DNA adjacent to the coding segments. Much of the biochemical research into V(D)J recombination has focused on truncated or “core” fragments of RAG1 and RAG2, which lack approximately one third of the amino acids from each. However, genetic analyses of SCID and Omenn syndrome patients indicate that residues outside the cores are essential to normal immune development. This is in agreement with the striking degree of conservation across all vertebrate classes in certain non-core domains. Work from multiple laboratories has shed light on activities resident within these domains, including ubiquitin ligase activity and KPNA1 binding by the RING finger domain of RAG1 and the recognition of specific chromatin modifications as well as phosphoinositide binding by the PHD module of RAG2. In addition, elements outside of the cores are necessary for regulated protein expression and turnover. Here the current state of knowledge is reviewed regarding the non-core regions of RAG1 and RAG2 and how these findings contribute to our broader understanding of recombination.