Immunoglobulin mu chains synthesized in murine pre‐B cells are known to be associated with surrogate light chains designated as omega (omega), iota (iota) and B34. In addition to these molecules, we identified the complexes of polypeptides (50, 40, 27 and 15.5 kd) associated with surface or intracellular mu chains of pre‐B cell lines. Most of these polypeptides were continuously synthesized and associated with mu chains in virgin B cells lines, although some of them scarcely bound to the mu kappa dimer or mu 2 kappa 2 tetramer concomitantly present in the same clone or population. However, in mature B cells they were no longer detectable except B34. Cross‐linking of micron chains on the surface of pre‐B cells resulted in an increase in intracellular free Ca2+, indicating that the micron 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 mu chain complexes which exhibit different structures as a whole and possess different signal transducing capacities.

Lymphoid cell lines were isolated that were inducible for the expression of surface immunoglobulin by shift from 35.5 to 39.5 degrees C after infection of mouse bone marrow cells with a mutagen‐treated Abelson murine leukemia virus. Virus produced by one of the cell lines (ts49) transmitted the temperature‐sensitive phenotype to new lymphoid transformants as well as to NIH/3T3 cells. In addition, the tyrosine autophosphorylating activity of the p120gag‐abl protein synthesized in ts49‐transformed cells was found to be temperature‐sensitive. Shift experiments using ts49‐transformed lymphoid cells showed that at 39.5 degrees C they synthesize increased amounts of mu and kappa chain RNA and protein, and that they can be further induced to secrete IgM when treated with lipopolysaccharide.

SummarySequence analysis of a large number of DNA clones containing a functional heavy chain variable, diversity, and joining (V.DJ.) complex generated by V. to V.DJ. joining (V. gene replacement) in the progeny derived from a common precursor cell transformed with a temperature-sensitive (ts) Abelson routine leukemia virus (A-MuLV) indicates that endogenous V. gene replacement in vitro generates immunoglobulin gene joints distinct from those generated by the usual V. to DJ. joining. Such joints keep the pentamer CAAGA at the 3' end of the donor VH segment and lack a recognizable D segment, as can be seen also in vivo. The results suggest that V. gene replacement participates in generating V. region diversity in vivo, as previously postulated. During the joining process, a unique V. gene was selected in all progeny cells, together with a single A nucleotide dominantly added to the junctional boundaries. The basis of these regulatory processes is discussed.T he random joining of Ig heavy chain variable (V.), diversity (D), and joining (J.) segments, and the deletion/insertion of nudeotides at the boundaries of recombination sites, lead to the generation of V. region diversity (1). The flexibility of the joining process, on the other hand, results in a considerable proportion of nonproductive rearrangements. Analysis of pre-B cell lines transformed with Abelson mufine leukemia virus (A-MuLV) strongly indicates that the cells generated after V. to DJ. joining would be null cells with nonproductive V.DJ. rearrangements on both chromosomes (2). However, these cells can also perform a further V. to VaDJ. joining using a 5' V. segment to replace the VH sequence of the nonfunctional V.DJ. complex, leading to the generation of a functional V.DJ. complex. It has been suggested that this V. gene replacement is mediated by a mechanism analogous to V. to DJ. recombination through the signal heptamer embedded in the 3' end of the VH coding region, which is identical to the signal sequence found at the 5' end of D elements (3-5).We have established immature B cell clones 46-6, 46-11, 46-12, and 46-13, generated from a common precursor cell transformed with a temperature-sensitive (ts) mutant of A-MuLV (6). All members of clones essentially became surface # chain-positive (#m+) pre-B cells as a result of V. gene replacement when they were cultured at nonpermissive temperature. By using this system, we have recently observed that various intrachromosomal circular DNAs were generated in clone 46-6 cultured at high temperature (7). The structural analysis of the isolated circular DNA clones provided evidence that V. gene replacement occurs by intramolecular DNA deletion, as seen in V-(D)-Jjoining (8, 9). In the present study, we analyzed the nucleotide sequences of genomic DNA clones of these progenies containing a functional V.DJ. complex generated by V. gene replacement in order to determine the V. region diversity generated by such a recombination process. Materials and MethodsCell Lines. A pre-B cell line, 46, was derived from...

The long-term bone marrow culture system developed by Dexter