A long-term goal of this project is to better understand the molecular basis of immunoglobulin (Ig) gene rearrangement and expression. The human Ig lambda light chain gene complex offers two advantages: it is a system where the molecular regulation of DNA expression of closely linked, very similar genes may be studied. In addition, lambda accounts for about 40% of human serum Igs and therefore study of its expression is of importance for further knowledge of the human immune system. Lambda light chains occur as one of four isotypes which are expressed in human myeloma proteins in the percentages: 15%Mcg, 61% Ke-Oz-, 18% Ke-Oz+ and 6%Ke+Oz-. In addition to these genes for serum proteins the lambda locus encodes gene products expressed only early in B cell ontogeny (14.1, 16.1 and our new 16.2, which are equivalent to C-lambda-5 in mouse) which may be involved in B cell development. This proposal is studying the expression and the molecular regulation of the expression of these human C-lambda genes. This project will complete the characterization (sequence of the human lambda constant region genes. It will use isotype-specific probes to determine the total level of expression of the particular lambda isotypes (Mcg, Ke-Oz-, Ke-Oz+ encoded respectively by genes C-lambda-1, 2, 3 in various cell compartments (blood, lymphnode, spleen) and the fraction of lambda human hybridomas (HuHy) with the particular isotypes. It will also determine the representation of the newest C-lambda gene, C-lambda-7, which we have recently isolated and sequenced, in the above cell populations. The lambda genes 14.1, 16.1 and 16.2 the last being one which we have cloned and sequenced, will also be studied in similar fashion. The techniques to be used include the polymerase chain reaction (PCR) on primer extensions (cDNA) of lambda mRNA and hybridization to excess isotype-specific oligonucleotides. The level of nascent transcripts (run-on transcription), steady state level and RNA half life of the lambda isotypes in permanent cell lines (HuHy) and normal lymphoid populations will be determined. Potential transcriptional enhancer sequences will be located by transfecting permanent lymphoid lines (both lambda-producing and non- producing) via electroporation with CAT (chloramphenicol acetyl transferase) vectors containing our test sequence. Differential enhancer activity will be measured for lambda gene segments and will allow us to test the hypothesis that increased enhancer function is correlated with increased gene rearrangement and hence gene expression. The DNase hypersensitivity of rearranged and unrearranged gene segments will be determined which will help us localize potential enhancer regions and will establish whether both homologous chromosomes are in an open configuration.
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