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，即46-6、46-11、46-12和46-13，该克隆是由由Abelson Murine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。克隆的所有成员基本上成为表面II链阳性（MUM^+）B细胞，这是V_H基因在非允许温度下进行培养时的V_H基因的结果。通过使用该系统，我们已经观察到在高温下培养的46-6克隆中产生了各种肉体内圆形DNA。 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 progressy derived from a common precursor cell tr​​ansformed with a ts A-MuLV indicates that内源性V_H基因在体外的替代……更多的自然Ig基因关节不同于通常V_H到DJ_H连接的v_h产生的关节。这样的关节使5mer Caaga处于供体V_H节段的3端，并且缺乏可识别的D段，在体内也可以看到。结果表明，V_H基因更换参与了如前所述的体内生成V_H区域多样性。在加入过程中，在所有后代细胞中选择了一个唯一的V_H基因，以及单个A核苷酸主要添加到连接边界中。A先前未报告的B细胞特异性基因（指定为8HS-20）均通过按下和差异杂交从Pre-B细胞克隆的CDNA库中分离出来。该基因被选为前B和骨髓的B细胞系中的0.7KB转录本，并且在骨髓中也发现了相同的大小转录本，尽管在脾脏中，但在低水平中也发现了相同的转录本。推导的8HS-20 cDNA的氨基酸序列显示了B细胞特异性基因，VPREB-L和免疫球蛋白超级基因家族的成员，包括Vlambda，Vkappa，VH，VH，TCRValpha，VBETA，VBETA和CD8。使用纯化的抗血清对8HS-20寡肽的生化分析表明，该基因用13.5、14、15、5、5和16KDA编码蛋白质，这些蛋白质与前B细胞系中的MU链相结合，并且这些分子与VPREB-L和LAMBDA和LAMBDA 5基因产品同时表达。较少的

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)

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

Tsunetsgu-Yokota,Y.,Minekawa,T.,Shigemoto,K.,Shirasawa,T.and Takemori,T.: "Characterization of a new subgroup of human lgVλ cDNA clone and its expression," Mol.Immunol.(1992)

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.

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