The analysis of B cell differentiation and maturation

我们建立了未成熟的B细胞克隆46-6、46-11、46-12和46-13，这些克隆是由用Abelson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。在非允许温度下培养时，由于V_H基因的替换，所有克隆成员基本上都变成了表面Ii链阳性（mum^+）的前B细胞。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。分离的环状DNA克隆的结构分析提供了证据，即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。在来自用ts A-MuLV转化的共同前体细胞的后代中，由VH基因置换产生的多样性和连接复合物表明内源性V_H基因置换体外基因工程 关于我们 产生不同于通常的V_H到DJ_H连接所产生的IG基因连接。这种连接使5聚体CAAGA保持在供体V_H片段的3 '端，并且缺乏可识别的D片段，如在体内也可以看到的。结果表明，V_H基因置换参与了体内V_H区多样性的产生。通过消减杂交和差异杂交，从前B细胞克隆的cDNA文库中分离到一个未报道的B细胞特异性基因8HS-20。该基因在前B和骨髓来源的B细胞系中选择性地表达为0.7kb的转录物，并且在骨髓中也发现了相同大小的转录物，尽管在脾脏中的水平较低。推导的8HS-20 cDNA的氨基酸序列显示与B细胞特异性基因VpreB-1以及免疫球蛋白超基因家族的成员包括V λ、V κ、VH、TCR α、V β和CD 8具有同源性。使用针对8HS-20寡肽的纯化抗血清的生化分析表明，该基因编码具有13.5、14、15、5和16 kDa的MW的蛋白质，其与前B细胞系中的μ链缔合，并且这些分子与VpreB-1和Vpreda 5基因产物在相同细胞系中伴随表达。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