In order to understand the development and functioning of the thymus, both in terms of T cell differentiation and stromal cell environmental support, we have undertaken a molecular approach to identify genes that are uniquely expressed in this organ. We have created an anchored RT-PCR based cDNA library from 14 day fetal thymuses after culturing them in deoxyguanosine and treating them with an anti-CD45 antibody to deplete the cell population of hematopoietic cells. The cDNA library was subtracted with poly A+ RNA prepared first from a fibroblast cell line and then from whole spleen. Three prime sequencing of 225 random cDNAs revealed 139 which were not in the databases of known sequences. The novel cDNAs were then screened by Northern blotting for expression in various tissues and in a set of SV40 transformed thymic epithelial cell lines. Four genes were selected for further analysis because they were limited in their expression to the thymus or to one of the stromal cell lines. All four were completely sequenced by obtaining full length cDNA clones from a SCID thymus library. One gene turned out to code for laminin gamma 2, an extracellular matrix protein produced by epithelial cells; this gene was described in the literature one year after we began our project. The second gene appears to encode a 12 transmembrane domain antiporter, distantly related to members of the known cation antiporter family. The third gene encodes a type II membrane bound serine protease and the fourth a globular protein with weak homology to known transcription factors. During the past year, we have extensively characterized the antiporter protein using two peptide antisera raised against either the C-terminus or the large putative cytoplasmic loop in the middle of the molecule. Both antisera immunoprecipitated a 45kD protein from a coupled in vitro transcription and translation assay. Addition of a microsomal fraction showed that the protein was glycosylated. Biotinylation experiments on a stable rat basophilic leukemia cell line cDNA transfectant showed that the protein was on the cell surface. A competitive quantitative PCR assay showed that the mRNA for this gene was mainly expressed in the thymus, although it was also detected at 10-fold lower levels in heart and brain. Expression in the thymus of mice bearing the SCID mutation was enriched 10-fold. In situ hybridization of tissue sections of adult thymus using sense and antisense probes revealed expression of the gene mainly in the cortex. Currently, we are isolating and sequencing a genomic clone and preparing a targeting vector for the creation of a knockout mutation by homologous recombination.
