Analysis of lymphoid cell differentiation

1.B系细胞表达新基因的分离和鉴定我们利用消减和差异杂交技术成功地从Abelson小鼠白血病病毒(A-MuLV)转化的前B细胞淋巴瘤中分离到新的基因8HS-15和8HS-20。8HS-20表达为0.75kb。8HS-15在A-MuLV转化的Pre-B细胞中以1.5kb的转录形式表达，在T细胞、成纤维细胞和组织中以3.Okb的转录形式表达。8HS-20克隆的序列分析表明，8HS-20有一个较长的开放阅读框，编码123个氨基酸的蛋白质，未加工的相对分子质量为13k。预测的8HS-20蛋白与人Vlambda的同源性为42%，与另一前B特异性基因VpreB-1的同源性为38%。我们制备了8HS-20多肽的多克隆抗血清，并用它们探测全细胞L…经8HS-20基因克隆后的成熟B细胞克隆WEHI231在表达载体中获得了更多的克隆。该抗血清在转8HS-20的WEHI231中特异性地免疫沉淀10kd和16kd分子，而在野生型WEHI231中不表达。免疫沉淀分析表明，8HS-20编码的分子似乎与一小部分u链有关，但与k轻链无关。在WEHI 231中观察到，8HS-20编码的分子与CYG34.2中未知的16kd分子相关联。与II链相关的小分子多肽的分析及其在信号转导中的作用我们已经鉴定了与前B细胞的IL链相关的多肽复合体。大多数这些多肽是在原始B细胞系中持续合成并与IL链结合的，尽管其中一些多肽几乎不与同一克隆或群体中伴随存在的MK二聚体或IL2lc2四聚体结合。然而，除了极少数例外，在成熟的B细胞中不再检测到它们。前B细胞表面LAM链的交联导致细胞内游离钙的增加，提示前B细胞表面的ITM链复合体是一种信号转导分子。然而，前B细胞系的受体交联性并不能引起未成熟和成熟B细胞系中常见的肌醇磷脂代谢的增加。这些结果表明，在前B细胞向成熟B细胞分化的过程中，细胞表达两种结构不同的II链复合体，它们具有不同的信号转导能力。这些多肽大部分是在原始B细胞系中连续合成并与Mu链结合的，尽管其中一些多肽几乎不与同一克隆或群体中伴随存在的MK二聚体或mu2kappa四聚体结合。然而，除了极少数例外，在成熟的B细胞中不再检测到它们。Pre B细胞表面UM链的交联导致细胞内游离Ca~(2+)和Gt~(2+)的增加，提示Pre B细胞表面的MU链复合体是一种信号转导分子。然而，前B细胞系的受体交联性并不能引起未成熟和成熟B细胞系中常见的肌醇磷脂代谢的增加。这些结果提示，在Pre B向成熟细胞分化的过程中，细胞表达两种不同结构的Mu链复合体，它们具有不同的信号转导能力。通过A-MuLV温度敏感(Ts)突变体的体外转化，我们从胎儿胸腺建立了未成熟的造血系克隆。当其中一个克隆(B6-24)胸腺内注射时，一小部分细胞分化为带有T或B淋巴细胞标志的细胞。而重组白介素1的体外刺激使该克隆分化为巨噬细胞样细胞。这种作用基本上被cAMP类似物及其诱导剂所取代。除B6-24外，我们还建立了其他具有Thy1-/Sca-1^+/谱系标记(lin^-)、Thy1^-/Sca1^+/Cd^&lt；4+&gt；/B220^+、Thy1^+/Sca-1^+/Cd^+/B220^+表型的未成熟造血细胞克隆。我们正在分析它们分化为多系细胞的潜力。较少

1. Isolation and characterization of new genes expressed In B lineage cells.We have succeeded in isolating new genes, 8HS-15 and 8HS-20, from Abelson murine leukemia virus (A-MuLV) transformed pre-B lymphoma using subtraction and differential hybrydization techniques. 8HS-20 is expressed as 0.75kb. transcript in B lineage cells and the level of the message is significantly augmented in pre-B lymphoma when compared to that in normal B cells in the spleen.8HS-15 is expressed as 1.5kb transcript in A-MuLV transformed pre-B cells and 3.Okb transcript in T lineage cells, fibroblast, and tissues. Sequence analysis of 8HS-20 cDNA clone reveals a long open reading frame, capable of encoding a protein of 123 amino acids with an unprocessed molecular weight of 13k. The predicted protein of 8HS-20 shares 42% homology with human Vlambda and 38% homology with another pre B specific gene, Vpre B-1. We prepared polyclonal antisera for synthesized peptides of 8HS-20 and used them to probe total cell l … More ysates of a mature B cell clone WEHI 231 transfected with or without 8HS-20 cDNA clone in expression vector. The antisera specifically immunoprecipitated 10 and 16kd molecules in WEHI 231 transfected with 8HS-20, but not in a wild type of WEHI 231. The analysis of immunoprecipitation of a virgin B cell clone, CYG34 revealed that the molecule encoded by 8HS-20 appeared to be associated with a small fraction of mu chains, but not with those linked to k light chains. As observed in WEHI 231, the molecule encoded by 8HS-20 was associated with unknown 16kd molecule in CYG34.2. Analysis of small polypeptides associated with Ii chain and their role in signal transduction We have identified the complexes of polypeptides associated with IL chains of pre B cell lines. Most of these polypeptides were continuously synthesized and associated with IL chains in virgin B cell lines, although some of them scarcely bound to the mk dimer or IL2lc2 tetramer concomitantly present in the same clone or population. However, they were no longer detectable in mature B cells with rare exceptions. Cross-linking of lam chains on the surface of pre B cells resulted in an increase in intracellular free Ca2, indicating that the itm chain complex on the surface of pre B cell lines acted as a signal transduction molecule. However, the receptor cross linkage of pre B cell lines did not induce the increased inositol phospholipid metabolism usually observed in virgin and mature B cell lines. These results suggest that, during the differentiation from,pre B to mature B cells, the cells express two types of Ii chain complexes which exhibit different structures as a whole and possess different signal transducing capacities.We have identified the complexes of polypeptides associated with mu chains of pre B cell lines. Most of these polypeptides were continuously synthesized and associated with mu chains in virgin B cell lines, although some of them scarcely bound to the mk dimer or mu2kappa tetramer concomitantly present in the same clone or population. However, they were no longer detectable in mature B cells with rare exceptions. Cross-linking of mum chains on the surface of pre B cells resulted in an increase in intracellular free Ca^<2+>, indicating that the mum chain complex on the surface of pre B cell lines acted as a signal transduction molecule. However, the receptor cross linkage of pre B cell lines did not induce the increased inositol phospholipid metabolism usually observed in virgin and mature B cell lines. These results suggest that, during the differentiation from pre B to mature cells, the cells express two types of mu chain complexes which exhibit different structures as a whole and possess different signal transducing capacities.3. Isolation of differentiation-inducible hematopoletic cell clones.We have established immature hematopoietic clones from fetal thymus by transforming with a temperature sensitive (ts) mutant of A-MuLV in vitro. When one of the clone (B6-24) was intrathymically injected, a small fraction of the cells differentiated into the cells bearing T or B lymphocytes markers. Whereas, the stimulation by recombinant interleukin-1 in vitro made this clone differentiate into macrophage-like cells. This effect is essentially replaced by CAMP analogue and its inducing reagents. In addition to B6-24, we have established other immature hematopoietic cell clones exhibiting the phenotypes of Thyl-/Sca-1^+/lineage marker (lin^-), Thy1^-/Sca1^+/CD^<4+>/B220^+, Thy1^+/Sca-1^+/CD4^+/B220^+. We are analyzing their potentiality to differentiate into multi lineage cells. Less