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差减杂交技术成功地从Abelson鼠白血病病毒（A-MuLV）转化的前B淋巴瘤中分离到了新基因8HS-15和8HS-20。8HS-20以0.75kb表示。在B谱系细胞中表达1.5kb转录本，并且与脾脏中正常B细胞相比，前B淋巴瘤中的信使水平显著增加。8HS-15在A-MuLV转化的前B细胞中表达1.5kb转录本，在T谱系细胞、成纤维细胞和组织中表达3.0k B转录本。8HS-20 cDNA克隆的序列分析显示其具有一个长的开放阅读框架，编码123个氨基酸，未经处理的分子量为13 k。8HS-20蛋白与人Vlambda蛋白的同源性为42%，与另一个前B特异性基因Vpre B-1的同源性为38%。我们制备了8HS-20合成肽的多克隆抗血清，并将其用于检测细胞总RNA。 关于我们 成熟B细胞克隆WEHI 231的裂解物，其在表达载体中用或不用8HS-20 cDNA克隆转染。抗血清特异性免疫沉淀10和16 kd的分子在WEHI 231转染8HS-20，而不是在野生型WEHI 231。免疫沉淀分析的一个处女B细胞克隆，CYG 34显示，8HS-20编码的分子似乎与一小部分的μ链，但不与那些连接到k轻链。如在WEHI 231中观察到的，8HS-20编码的分子与CYG 34.2中未知的16 kd分子相关。与IL-1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鉴定了前B细胞系IL-1链相关多肽的复合物。这些多肽中的大多数是连续合成的，并与IL链在处女B细胞系，虽然他们中的一些几乎没有结合到IL 21 c 2二聚体或四聚体伴随存在于同一克隆或人口。然而，除了极少数例外，它们在成熟的B细胞中不再检测到。前B细胞表面的lam链的交联导致细胞内游离Ca 2的增加，表明前B细胞系表面的itm链复合物充当信号转导分子。然而，前B细胞系的受体交联没有诱导增加肌醇磷脂代谢通常在处女和成熟B细胞系中观察到。这些结果表明，在前B细胞向成熟B细胞分化的过程中，前B细胞表达两种不同结构的Ii链复合物，它们具有不同的信号转导能力。这些多肽中的大多数是连续合成的，并与原始B细胞系中的μ链相关，尽管其中一些几乎不结合于在同一克隆或群体中伴随存在的μ 2 κ二聚体或μ 2 κ四聚体。然而，除了极少数例外，它们在成熟的B细胞中不再检测到。前B细胞表面的母链交联导致细胞内游离Ca^<2+>的增加，表明前B细胞系表面的母链复合物充当信号转导分子。然而，前B细胞系的受体交联没有诱导增加肌醇磷脂代谢通常在处女和成熟B细胞系中观察到。这些结果表明，在前B细胞向成熟细胞分化的过程中，细胞表达两种类型的μ链复合物，它们在整体上表现出不同的结构，具有不同的信号转导能力.分化诱导造血细胞克隆的分离：我们已经建立了未成熟的造血细胞克隆从胎儿胸腺转化与温度敏感（ts）突变体的A-MuLV在体外。当胸腺内注射其中一个克隆（B6-24）时，一小部分细胞分化为带有T或B淋巴细胞标记的细胞。而重组白细胞介素-1体外刺激可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，我们还建立了其他表现出Thyl-/Sca-1^+/谱系标记（lin^-）、Thy 1 ^-/Sca 1 ^+/CD^<4+>/B220^+、Thy 1 ^+/Sca-1^+/CD 4 ^+/B220^+表型的未成熟造血细胞克隆。我们正在分析它们分化成多谱系细胞的潜力。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