Cloning and Characterization of theCDKN2Aandp19ARFGenes fromMonodelphis domestica

The tumor suppressor gene, CDKN2A (p16), encodes a cyclin-dependent kinase inhibitor and functions as a negative regulator in the retinoblastoma pathway that blocks cell cycle progression from the G1 phase. The gene has been found to be deleted, truncated, mutated, or silenced by promoter methylation in a wide range of tumor types. Where melanoma CDKN2A mutations have been characterized, C → T and CC → TT transitions were found, indicating a direct role for ultraviolet radiation (UVR)-induced pyrimidine dimers in the formation of some tumors. The South American opossum, Monodelphis domestica, has been shown by our group and others to be susceptible to the induction of melanoma on chronic exposure to UVR alone. The CDKN2A gene and its exon 1β alternate transcript p19ARF were cloned and sequenced from M. domestica to investigate the role of these genes in the development of UVR-induced melanoma and non-melanoma tumors. Both genes were first amplified by polymerase chain reaction (PCR) using cDNA from an opossum cornealtumor cell-line library and degenerate primers based on human, mouse, and rat CDKN2A gene sequences. To verify these as normal sequences, both genes were then RT-PCR amplified from cultured normal opossum melanocyte mRNA. When comparing the tumor and melanocyte sequences, we found a UVR signature point mutation, a C → T transition, within exon 2 in the corneal tumor cell line. The same mutation at this site in other tumors has been shown to alter the CDKN2A protein's ability to bind CDK4 kinase, which may lead to uncontrolled cell cycling. A comparison of the amino acid sequence of opossum CDKN2A showed identities relative to human, mouse, and rat between 57% and 63%, and when conserved amino acid substitutions are considered (similarity), the range is 63% to 67%. The amino acid identity and similarity for p19ARF ranged from 39% to 49%.