| Co-Investigator(Kenkyū-buntansha) |
TOKUHISA Takeshi Kobe Univ., Center for Int. Cor., 国際交流センター, 教授 (20134364)
MIZUGUCHI Junichiro NIH, Dep. Immunol., Head, 体液性免疫部, 室長 (20150188)
OHNISHI Kazuo NIH, Dep. Cell. Immunol., Researcher, 細胞免疫部, 研究員 (90169011)
KIMOTO Hiroshi NIH, Dep. Cell. Immunol., Researcher, 細胞免疫部, 研究員 (20225080)
重本 和宏 国立予防衛生研究所, 細胞免疫, 研究官 (00192104)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1991: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1990: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Research Abstract |
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
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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
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