We continue using multiparameter sorting and analysis with the fluorescence activated cell sorter (FACS) to define functionally and clinically relevant lymphocyte subpopulations in mouse and man and select variant and DNA-transformed animal cell lines for recombinant DNA analyses of the genes and genetic mechanisms controlling the structure and production of immunologically relevant molecules. Thus, we plan to: (1) Extend our library of immunoglobulin constant-region (IgCH) """"""""switch-variant"""""""" families derived from hybridoma cell lines by selecting interchromosomal and interspecies (mouse-human) variants producing """"""""chimeric"""""""" antibodies, variants producing modified parental IgCH structures, and regulatory variants showing changes in the expression of parental IgCH genes. We will then investigate the genetic mechanisms underlying these """"""""IgCH switches"""""""" by testing for mutations, rearrangements, somatic cross-overs, or other chromosomal changes; methylation or other DNA modifications affecting transcription; or differential splicing of primary transcripts or other mechanisms affecting translation. (2) Transform with DNA to obtain cell lines secreting or carrying gene products derived from a different species or haplotype. We hope to obtain transformants for genes expressed in differentiated lymphocytes, e.g., mouse Lyt-1 and Lyt-2 and their human homologs (Leu-1 and Leu-2). (3) Define B- and T-cell subpopulations in mouse and man according to correlated surface-marker expression and investigate the genetic mechanisms influencing the expression of surface markers, secreted products, and differentiation-specific mRNAs in FACS-isolated T- and B-cell subpopulations. We have obtained (1) transformants expressing some ten different human antigens or receptor molecules including Leu 1, Leu 2, HLA, Beta 2 microglobulin, nerve growth factor receptor, and transferrin receptor and (2) a number of transformants expressing mouse molecules including Ly-1, Lyt-2, L3T4, several B-cell specific antigens, and IgG-Fc receptor. Using the FACS, we selected amplified cell lines making Leu 2 and its putative mouse homolog, Lyt-2. Using these lines and several molecular biology methods, most notably selected cDNA probes, we cloned the genes for each of these molecules. We have now obtained the nucleotide sequences of these genes, confirmed their genetic homologym, and begun our structure-function correlation studies. An interesting surprise is that these genes are new members of the immunoglobulin gene super family with closest homology to IgV regions. (CS)
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