The analysis of B cell differentiation and maturation

B细胞分化成熟分析

• 批准号: 02454196
• 负责人: TAKEMORI Toshitada
• 金额: $ 3.97万
• 依托单位: National Institute of Health
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1990
• 资助国家: 日本
• 起止时间: 1990 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-02454196/
• 关键词: B cell maturation; mu chain expression; V_H gene replacement; circular DNA; B cell specific gene; surrogate L chains; lg gene super family; mu chain transport; V_H gene replacement; circular DNA; シグナル伝達; μ鎖複合体; 前B細胞; virgin B細胞

We have estabilished immature B cell clones, 46-6, 46-11, 46-12 and 46-13, generated from a COMM'on precursor cell transformed with a temperature sensitive(ts)mutant of Abelson murine leukemia virus(A-MuLV). All members of clones essentially became surface Ii chain positive (mum^+) pre B cells as a result Of V_H gene replacement when they were cultured at nonpermissive temperature. By using this system, we have observed that various intrachromosomal circular DNAs were generated in a clone 46-6 cultured at high temperature. The structural analysis of the isolated circular DNA clones provided the evidence that V_H gene replacement occurs by intramolecular DNA deletion as seen in V-(D)-J joining.Sequence analysis of a large number of DNA clones containing a functional heavy chain variable, diversity and joining complex generated by VH gene replacement in the progeny derived from a common precursor cell transformed with a ts A-MuLV indicates that endogenous V_H gene replacement in vitro ge … More nerates Ig gene joints distinct from those generated by usual V_H to DJ_H joining. Such joints keep the 5mer CAAGA at the 3'end of the donor V_H segment and lack a recognizable D segment, as can be also seen in vivo. The results suggest that V_H gene replacement participates in generating V_H region diversity in vivo as previously postulated. During the joining process, a unique V_H gene was selected in all progeny cells, together with a single A nucleotide dominantly added to the junctional boundaries.A previously unreported B cell specific gene, designated 8HS-20, was isolated from the cDNA library of a pre-B cell clone by subtraction and differential hybridization. This gene is selectively expressed as a 0.7kb transcript in pre-B and bone marrowderived B cell lines and the same size transcript is also found in bone marrow and, albeit at low levels, in spleen. The deduced amino acid sequence of 8HS-20 cDNA displayed homology to a B cell specific gene, VpreB-l, and members of the immunoglobulin super gene family including Vlambda, Vkappa, VH, TCRValpha, Vbeta and CD8. Biochemical analysis using purified antiserum against 8HS-20 oligopeptides indicates that the gene encodes proteins with MW of 13.5, 14, 15, 5 and 16kDa, which associate with mu chains in pre-B cell lines, and that these molecules are concomitantly expressed with VpreB-l and lambda5 gene products in the same cell lines. Less

我们已经建立了未成熟的 B 细胞克隆 46-6、46-11、46-12 和 46-13，它们是由用 Abelson 鼠白血病病毒 (A-MuLV) 温度敏感 (ts) 突变体转化的 COMM'on 前体细胞产生的。 ），由于 V_H 基因替换，克隆的所有成员在通过使用该系统，我们观察到在高温培养的克隆46-6中产生了各种染色体内环状DNA。对分离的环状DNA克隆的结构分析提供了V_H基因替换是通过分子内DNA缺失发生的证据。如 V-(D)-J 连接中所示。对源自共同前体的后代中含有功能性重链变量、多样性和由 VH 基因替换产生的连接复合物的大量 DNA 克隆进行序列分析用 ts A-MuLV 转化的细胞表明，内源性 V_H 基因替换产生的 Ig 基因接头与通常的 V_H 至 DJ_H 连接产生的接头不同，此类接头将 5mer CAAGA 保留在供体 V_H 片段的 3' 端。且缺乏可识别的 D 片段，正如在体内所见，结果表明，如先前假设的那样，V_H 基因替换参与了体内 V_H 区域多样性的产生。在所有子代细胞中选择了一个独特的 V_H 基因，以及主要添加到连接边界的单个 A 核苷酸。从前 B 细胞克隆的 cDNA 文库中分离出了以前未报道的 B 细胞特异性基因，命名为 8HS-20通过消减和差异杂交，该基因在前 B 细胞系和骨髓来源的 B 细胞系中选择性表达为 0.7kb 转录物，并且在骨髓中也发现了相同大小的转录物，尽管水平较低。使用纯化的生化分析，推导的 8HS-20 cDNA 氨基酸序列与 B 细胞特异性基因 VpreB-1 和免疫球蛋白超基因家族成员（包括 Vlambda、Vkappa、VH、TCRValpha、Vbeta 和 CD8）具有同源性。针对 8HS-20 寡肽的抗血清表明该基因编码 MW 为 13.5、14、15、5 的蛋白质和 16kDa，它们与前 B 细胞系中的 mu 链相关，并且这些分子在相同细胞系中与 VpreB-1 和 lambda5 基因产物同时表达。

项目成果

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Usuda,S.、Takemori,T.、Matsuoka,M.、Shirasawa,T.、Yoshida,K.、Mori,A.、Ishizake,K. 和 Sakano,H.：“通过免疫球蛋白 V 基因替换生成的环状 DNA；

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Shirasawa, T.、Onishi, K.、Hagiwara, S.、Shigemoto, K.、Takebe, Y、Rajewsky, K. 和 Takemori, T.：“与未成熟 B 细胞中 μ 链相关的高贵基因产物”，EMBO J. (1992)

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Tsunetsgu-Yokota, Y.、Minekawa, T.、Shigemoto, K.、Shirasawa, T. 和 Takemori, T.：“人类 lgVλ cDNA 克隆的新亚组的表征及其表达”，Mol.Immunol.

Shirasawa,T.,I.Miyazoe,Hagiwara,S.,Kimoto,H.,Shigemoto,K.,Taniguchi,M.and Takemori.T.: "Mu chain diversity generated by V gene replacement is highly limited in progenies differentiated fromn common precursors transformed with a ts mutant of Abelson murine

Shirasawa,T.,I.Miyazoe,Hagiwara,S.,Kimoto,H.,Shigemoto,K.,Taniguchi,M.and Takemori.T.：“V 基因替换产生的 Mu 链多样性在从

T.Shirasawa,I.Miyazoe,H.Kimoto,S.Hagiwara,K.Shigemoto,M.Taniguchi,T.Takemori: "Mu chain diversity generated by Vgene replacement is highly limited in the progenies differentiated from a common precursor cell transformed with a ts mutant of AーMuLV" Proc.Na

T. Shirasawa、I. Miyazoe、H. Kimoto、S. Hagiwara、K. Shigemoto、M. Taniguchi、T. Takemori：“V 基因替换产生的 Mu 链多样性在由转化的常见前体细胞分化而来的后代中受到高度限制。 A-MuLV 的 ts 突变体"Proc.Na