The structure and expression of a human Ly-6-related gene homologous to mouse TSA-1 /

Mouse Ly-6 antigens belong to a family of structurally related GPI-anchored cell surface proteins encoded by tightly-linked, but independently regulated genes on chromosome 15. Functional data so far suggest roles in intercellular communication and trafficking of cells during hematopoiesis and immune responses. High sequence divergence between species has hampered homology cloning of human Ly-6 genes. The major part of this thesis reports the characterization of the first of two human Ly-6 homologues which have been discovered recently. Also reported is work on the generation of recombinant forms of Ly-6 proteins for use in Ly-6 ligand studies and in raising antibodies to the novel human Ly-6-related proteins. cDNA clone 9804, provided by collaborators at CytoMed (Cambridge Mass.), encoded a human Ly-6 sequence most like mouse TSA-1. It was used as a probe to isolate genomic fragments containing 9804 exons. Further cloning and characterization of overlapping fragments revealed that the 9804 gene consists of 4 exons, arranged like other Ly-6 genes, and spanning approximately 5 kb. Screening of human-hamster hybrid cell DNA by PCR associated the 9804 gene with chromosome 8. Fluorescence in situ hybridization, performed by collaborator D. Well, refined the mapping to 8q24.3, a region showing synteny with mouse chromosome 15 in the region containing Ly-6 genes. Northern blot analyses demonstrated a broad tissue distribution of 9804 expression, similar to that of TSA-1. While present in many different human tumor cell lines, 9804 mRNA was not detected in the B cell lines Raji and Daudi. 9804 mRNA levels were inducible in vitro by IFN-alpha and ATRA. The response to ATRA was restricted to myeloid leukemia cell lines and was a late event (36--48 hrs), whereas IFN-alpha produced a stronger and more rapid (8--12 hrs) enhancement in normal PBMC and a broad range of cell lines. The induction required protein synthesis, and was apparently at the level of transcription. Solu