MOLECULAR BASIS OF IMMUNOGLOBULIN HEAVY CHAIN SWITCH

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The murine B cell lymphoma, I.29, consists of cells expressing either IgM or IgA with the identical idiotype and with the identical heavy (H) chain variable (V) region sequence. By DNA blotting experiments and by examination of cloned H chain genes, we found that the IgM cells contain one expressed VDJ-CMu gene and one non-expressed DJ-CMu gene segment, and all the other H chain C genes in the germline configuration. The IgA cells have deleted Mu genes from both chromosomes, and have undergone a typical switch recombination with the Alpha gene on the expressed chromosomes, and the Gamma3 gene on the non-expressed chromosome. When purified IgM cells from the tumor are cultured in vitro they can be induced to switch to IgA or to IgE with LPS or with monoclonal anti-IgM or anti-I.29 idiotype (Id). We propose to clone T cells which will help the switch to either IgA or to IgE. We plan to clone DNA fragments bearing the rearranged Alpha and Mu genes about 3 days after induction of switching to examine the earliest DNA recombination events, to determine whether specific sites are involved and whether sister-chromatid exchange may occur. The IgM cells appear to be committed to switch to either IgA or IgE, as the Alphagene is hypomethylated (relative to liver DNA) in the IgM cells, whereas the Gamma2b gene is not. Furthermore, the Alpha and Epsilon genes are being transcribed at a low level in the IgM cells, whereas the Gamma1 and Gamma2b genes are not. We will further examine the mechanism of predetermination of isotype specificity by studies of chromatin structure and by attempting to induce isotype switching in hybrids of the B cell lymphoma, WEHI 279, and I.29Mu cells. The DNA sequences required for isotype switching and the specificity of switching will be studied by transfection of Ig H chain gene constructs which can serve as substrates for switch recombination into I.29Mu cells, which will then be induced to switch. Eventually we will attempt to prepare a cell-free system which can recombine switch region sequences. Our plans also include the cloning of cDNAs for poly(A)+ RNAs which appear when switching is induced. We will attempt to determine what proteins these genes encode.