The analysis of B cell differentiation and maturation

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，它们是用Abelson小鼠白血病病毒（a - mulv）的温度敏感（ts）突变体转化的COMM'on前体细胞产生的。在不允许的温度下培养时，由于V_H基因的替换，克隆的所有成员基本上都成为表面Ii链阳性（mum^+）的前B细胞。利用该系统，我们观察到在高温培养的克隆46-6中产生了多种染色体内环状dna。对分离的环状DNA克隆的结构分析表明，V_H基因的替换是通过分子内DNA缺失发生的，如V-(D)- j连接。对ts a - mulv转化的共同前体细胞后代中大量含有VH基因替代产生的功能性重链变量、多样性和连接复合体的DNA克隆的序列分析表明，体外内源性V_H基因替代产生的Ig基因连接与通常的V_H - DJ_H连接产生的Ig基因连接不同。这种关节将5mer CAAGA保持在供体V_H段的3'端，缺乏可识别的D段，在体内也可以看到。结果表明，V_H基因替换参与了体内V_H区域多样性的产生，这与之前的假设一致。在连接过程中，所有子代细胞都选择了一个独特的V_H基因，并在连接边界上显性地添加了单个a核苷酸。从B细胞前克隆的cDNA文库中，通过减法和差异杂交分离到一个未见报道的B细胞特异性基因8HS-20。该基因在B前和骨髓来源的B细胞系中选择性表达为0.7kb的转录本，在骨髓和脾脏中也发现了相同大小的转录本，尽管水平较低。8HS-20 cDNA的氨基酸序列与B细胞特异性基因vpreb - 1和免疫球蛋白超基因家族成员Vlambda、Vkappa、VH、TCRValpha、Vbeta和CD8同源。利用纯化的抗8HS-20寡肽血清进行生化分析表明，该基因编码的分子量为13.5、14、15、5和16kDa的蛋白与前b细胞系的mu链相关，这些分子在同一细胞系中与vpreb - 1和lambda5基因产物同时表达。少

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