| Budget Amount *help |
¥7,400,000 (Direct Cost: ¥7,400,000)
Fiscal Year 1989: ¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 1988: ¥3,700,000 (Direct Cost: ¥3,700,000)
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| Research Abstract |
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
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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
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