Identification of a 1.2 Kb cDNA fragment from a region on 9p21 commonly deleted in multiple tumor types.

Chromosome band 9p21 is a frequent target of homozygous deletion in many tumor types. Putative tumor suppressor genes, CDKN2A (p16), p14(ARF) and CDKN2B (p15), were localized to 9p21. However, there have been reports that suggest that there may be other genes targeted for inactivation in the region. We have developed a method to search for transcribed sequences within large genomic regions. We tested our approach in a 100-kilobase region on 9p21, which is 40 kilobases telomeric to CDKN2A. The method, termed expressed sequence selection (ESS), resulted in the isolation of genomic fragments known to be from 9q21 that are homologous to transcribed sequences. One fragment was used to obtain a 1.2 kilobase cDNA. The sequence of the 5' half of the cDNA was almost identical to exons 3-5 of the MTAP gene, which maps to chromosome band 9p21. The 3' portion of the cDNA had sequence homology to the ALA gene, which maps to chromosome arm 9q. Using Northern blot analysis, the 1.2 Kb cDNA identified several widely expressed transcripts ranging from 1 Kb to 8.5 Kb and displayed a complex pattern of alternative splicing in which certain exons of the 1.2 Kb cDNA are excluded from some of the splice products. Using cancer tissue Northern blots, we could show that all of the transcripts are absent from a leukemia cell line and a lung cancer cell line (K562, A549) with homozygous, genomic deletions within chromosome band 9p21. In addition, the 7 Kb transcript is also absent from two additional tumor cell lines (Molt4, a leukemia derived cell line, and in G361, a melanoma derived cell line) with homozygous deletions. Further investigation will determine whether the difference in the expression pattern between the 7 Kb transcript compared with the other sized transcripts could be due to specific targeting for alteration in certain tumor types.