Novel gene sequences expressed by human melanoma cells identified by molecular subtraction

Despite the variety of approaches used, only a limited number of tumor-associated antigens have been described for each histological type of tumor. In this report, we present a new strategy involving molecular subtraction to identify novel melanoma-associated gene sequences. Toward this end, 156 complementary DNA clones were isolated with a subtracted melanoma complementary DNA probe (melanoma minus lung carcinoma) after screening 2 x 10(4) independent recombinants of a melanoma expression library by in situ plaque hybridization. These clones were then polymerase chain reaction amplified, screened for duplication, and categorized into 53 discrete genes. By applying poissonian distribution to the numbers of duplicate isolates, we found most of the genes to be rare messages, present at less than 1 copy/200 molecules of mRNA in a typical somatic cell. Messages specific for a type of tissue are usually expressed in this range. The expression of the 53 genes was further studied in human tumor cell lines and normal tissues. Partial sequence data obtained for 20 complementary DNA clones revealed 8 novel human genes. The mRNA transcripts for 5 of the novel genes were identified by Northern blot analysis. Thus, molecular subtraction appears to be applicable for the identification of novel tumor-associated sequences. Some of the potential advantages and limitations of this technology are discussed, including its application to the molecular characterization of immunogenic melanoma-associated antigens.