Abstract
Mouse neuroblastoma cells (NB) selected for 10 000-fold increased resistance to mycophenolic acid (NB-Myco) showed a 200–500-fold increase in IMP dehydrogenase protein, and the enzyme (IMP: NAD + oxidoreductase, EC 1.1.1.205) also exhibited a 2400-fold increased k i for mycophenolic acid and reduced catalytic efficiency (Hodges, S.D., Fung, E., McKay, D.J., Renaux, B.S., and Snyder, F.F. (1989) J. Biol. Chem. 264, 18137–18141). The molecular basis of these changes is the subject of the present study. The nucleotide sequence of IMP dehydrogenase cDNA from NB-Myco cells revealed four nucleotide changes. One of these changes did not result in a codon change, and a second one corresponding to methionine-483 was present in the parental NB mouse line. The remaining two nucleotide substitutions and deduced residue changes are: the C to T transition at base 998 relative to initiation of translation, altering threonine-333 to isoleucine; and the C to A transversion at base 1052, altering serine-351 to tyrosine. Evidence was also obtained for IMP dehydrogenase having undergone gene amplification. IMP dehydrogenase mRNA levels were 500-fold increased in NB-Myco cells as compared to parental NB cells. DNA dot blot analysis showed a 25-fold increase in IMP dehydrogenase gene copy number and restriction enzyme analysis revealed similar gene structure for NB and NB-myco cells.
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