A novel interferon-gamma regulated human melanoma-associated antigen, gp33-38, defined by monoclonal antibody Me14-D12. II. Molecular cloning of a genomic probe.
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.
Audette
M
M
Ludwig Institute for Cancer Research, Lausanne Branch, Switzerland.
Carrel
S
S
Hayoz
D
D
Giuffrè
L
L
Mach
J P
JP
Kühn
L C
LC
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm / Q000235:genetics*; D003001:Cloning, Molecular; D015342:DNA Probes; D005814:Genes, Viral; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D008545:Melanoma / Q000276:immunology*; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins / Q000032:analysis*; D018139:Simplexvirus / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics
D000911:Antibodies, Monoclonal; D000951:Antigens, Neoplasm; D015342:DNA Probes; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D007371:Interferon-gamma; D013937:Thymidine Kinase