GENETICS OF HUMAN METAL BINDING PROTEINS

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This is a proposal to characterize the cloned DNAs specifying human genes which code for a family of iron binding proteins that modulate iron metabolism, exert controls on hemopoietic cell precursors and trigger the initiation of immunological reactions. Transferrin, lactotransferrin and ovotransferrin are homologous iron binding proteins which appear to be evolutionary products of gene duplication. They are comprised of two similar domains each of which binds iron. Transferrin is the major serum iron binding protein in vertebrates. It transports iron absorbed by the intestine to the bone marrow where it is required by reticulocytes for hemoglobin biosynthesis. Transferrin is required by all proliferating cells. Transferrin's amino acid sequence is homologous to lactotransferrin, an iron binding protein in milk. In chickens, transferrin is identical to ovotransferrin, a glycoprotein synthesized in chicken oviduct. The fourth member of the iron binding glycoproteins is of great biological interest since it is one of the first tumor antigens sequenced. Antigen p97 is unique to human melanoma cell membranes. The antigen p97 is structurally and functionally related to transferrin and is capable of binding iron. Oligonucleotide probes constructed according to the amino acid sequences of transferrin, lactotransferrin and antigen p97 will be used to screen cDNA libraries. The cDNA inserts specific for the genes will be cloned and sequenced. Sequence analysis of the chromosomal genes should reveal mechanisms involved in the generation of repeated genes by duplication of ancestral genes. The chromosomal locations of these genes will be investigated by in situ hybridization and by blot hybridization and synteny analysis of somatic cell hybrids. The flanking regions of the genomic p97 gene will ultimately be examined in melanoma cells and compared to homologous regions in non-malignant cells in order to determine why p97 is expressed specifically in melanoma cell membranes. This study will provide information about (1) the amino acid sequence and the degree of homology among the human proteins: transferrin, lactotransferrin and melanoma antigen p97, (2) the basis for duplication and modification of iron binding glycoprotein genes, (3) the chromosomal locations of the genes of transferrin, lactotransferrin and melanoma p97 antigen and (4) the expression of melanoma p97 antigen in melanoma cells.