AMPLIFICATION OF THE TRANSKETOLASE GENE IN DESENSITIZATION-RESISTANT MUTANT Y1 MOUSE ADRENOCORTICAL TUMOR-CELLS

As shown previously, mutants of the Y1 mouse adrenocortical tumor cell line that resist agonist-induced desensitization of adenylyl cyclase have elevated levels of a 68-kDa protein (designated p68), suggesting a possible relationship between p68 and the regulation of adenylyl cyc lase activity. In the present study, cDNA cloning and sequencing were used to identify p68 as mouse transketolase. Cells overexpressing p68 exhibited a 17.4-fold increase in transketolase enzymatic activity rel ative to parental Y1 cells and a 28-fold amplification of the transket olase gene as determined by Southern blot hybridization analysis, Usin g fluorescent in situ hybridization analysis, the transketolase gene w as mapped to mouse chromosome 16B1 and to human chromosome 3p21.2, Tra nsketolase gene amplification was associated with telomeric fusion of the chromosome 16 pair together with the appearance of multiple copies of the transketolase gene throughout a different chromosome, The rela tionship between overexpression of transketolase and desensitization r esistance was evaluated in somatic cell hybrids formed between a desen sitization-resistant adrenal cell line and a desensitization-sensitive rat glial cell line. In these hybrids, transketolase overexpression b ehaved dominantly, whereas desensitization resistance behaved recessiv ely, These results dissociate the desensitization resistance phenotype from overexpression of transketolase and suggest that desensitization resistance may have resulted from disruption of an essential regulato ry gene in conjunction with the amplification event.