COAMPLIFICATION IN TUMORS OF KRAS2, TYPE-2 INOSITOL 1,4,5-TRIPHOSPHATE RECEPTOR GENE, AND A NOVEL HUMAN GENE, KRAG

Analysis of a region of DNA, coamplified in tumors with KRAS2, resulte d in the identification of the human homologue of the mouse KRAG gene. The gene was widely expressed in a range of cell lines, tumors, and n ormal tissue and demonstrated a high degree of alternate splicing. A h uman KRAG cDNA sequence, with a structure similar to that encoded by t he amplified gene in mouse Y1 adrenal carcinoma cells, was isolated by RT-PCR. The predicted amino acid similarity between the two sequences was 91%, and hydrophobicity plots suggested a structure closely resem bling that of transmembrane 4 superfamily members. Identification of a PCR-based restriction fragment length polymorphism confirmed bialleli c expression of KRAG but suggested allele-specific splicing difference s in tumors. Northern analysis of mRNA derived from a range of tissues suggested high level expression in muscle and confirmed alternate spl icing. To facilitate the analysis of exon junctions, a YAC clone encod ing the genomic sequence was identified. This allowed the localization of KRAG to human chromosome 12p11.2. Isolation of one end of this non chimeric clone demonstrated a perfect match with a 247-bp sequence wit hin the 3' untranslated region of the type 2 1,4,5-inositol triphospha te receptor gene. Multiplex PCR confirmed the inclusion of both genes in the KRAS2 amplicon in human malignancy, suggesting that either may contribute to the malignant phenotype. (C) 1996 Academic Press, Inc.