Cloning and analysis of unique human glutaminase isoforms generated by tissue-specific alternative splicing

Three human glutaminase (hGA) isoforms were identified, two of which repres ent isoforms previously unidentified in any species. One isoform contains a n open reading frame with high homology with the rat kidney-type glutaminas e, suggesting that this isoform represents the human kidney-type glutaminas e, hKGA. A second isoform, termed hGAC, contains an open reading frame that matches hKGA except for a unique COOH-terminal amino acid sequence. In add ition, a third human glutaminase isoform was identified from a computer sea rch and on further analysis was found to represent an additional unique iso form, hGAM. hKGA is expressed predominantly in brain and kidney but not in liver, hGAC is expressed principally in cardiac muscle and pancreas but not in liver or brain, and hGAM is expressed solely in cardiac and skeletal mu scle. hGAC is the predominant isoform expressed by a human breast cancer ce ll line that exhibits a high rate of glutamine utilization and glutaminase activity. Genomic Southern analysis as well as isolation and analysis of fi ve glutaminase genomic clones suggested that all three hGA isoforms origina te from the same locus and therefore represent mRNA species that are produc ed by tissue-specific alternative splicing of a single pre-mRNA. Furthermor e, an RT-PCR assay was developed that can be used to easily differentiate b etween hKGA and hGAC mRNA species.