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A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.
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The human Me14-D12 antigen is a cell surface glycoprotein regulated by interferon-gamma (IFN-gamma) on tumor cell lines of neuroectodermal origin. It consists of two non-convalently linked subunits with apparent mol. wt sizes of 33,000 and 38,000. Here we describe the molecular cloning of a genomic probe for the Me14-D12 gene using the gene transfer approach. Mouse Ltk- cells were stably cotransfected with human genomic DNA and the Herpes Simplex virus thymidine kinase (TK) gene. Primary and secondary transfectants expressing the Me14-D12 antigen were isolated after selection in HAT medium by repeated sorting on a fluorescence activated cell sorter (FACS). A recombinant phage harboring a 14.3 kb insert of human DNA was isolated from a genomic library made from a positive secondary transfectant cell line. A specific probe derived from the phage DNA insert allowed the identification of two mRNAs of 3.5 kb and 2.2 kb in primary and secondary L cell transfectants, as well as in human melanoma cell lines expressing the Me14-D12 antigen. The regulation of Me14-D12 antigen by INF-gamma was retained in the L cell transfectants and could be detected both at the level of protein and mRNA expression.
Antibodies, Monoclonal/diagnostic use, Antigens, Neoplasm/genetics, Cloning, Molecular, DNA Probes, Genes, Viral, Humans, Interferon-gamma/pharmacology, Melanoma/immunology, Melanoma-Specific Antigens, Neoplasm Proteins/analysis, Simplexvirus/genetics, Thymidine Kinase/genetics
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