Systematic isolation of genes encoding candidate proteins for human melanoma antigens using serial analysis of gene expression (SAGE) and EST database

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To identify genes preferentially expressed in melanocyte / melanoma, we have applied two methods; comparison of cDNA profiles generated by serial analysis of gene expression (SAGE) and of EST databases from various tissues. AcDNA profile of highly pigmented melanoma cell line SKme123 was first evaluated using SAGE. 25,997 tags consisting of 10,382 transcripts were sequenced. This melanoma SAGE library was compared to SAGE databases from normal tissues including brain, colon and testis, brain and colon cancers, and also compared to the melanocyte cDNA library. Two tags possibly encoding new melanocyte specific genes with relatively high frequency were identified, and their specific expression was confirmed by RT-PCR and Northern blot analysis. Full length clones of these sequences were isolated from the SKme123 λ phage. One was a splicing variant of TRP2; the other was a novel gene with unknown function. In the next study, by comparing EST databases from various tissues using computer, 80 possibly melanocyte specific genes were selected, then, their expression was evaluated using RT-PCR. Seven sequences were found to express only in melanocytes or melanoma, and the isolation of the full length cDNA were attempted. M43 was a splicing variant of AMI, previously identified melanoma antigen recognized by T cells. M44 encoded a novel melanocyte specific molecule. M125 was a splicing variant of Melastatin 1, a possible tumor marker for melanoma. M40, M109 and M674 were found to be a3'-sequence of the novel melanocyte specific gene. M216 may also have a novel melanocyte specific gene at its 5'-upstream. Therefore, melanocyte / melanoma specific genes can be systematically isolated using SAGE and EST databases. These melanocyte specific genes may be useful for research on biology of melanocytes as well as development of diagnostic and therapeutic methods for disorder of pigmentation and melanoma.