To study the regulation of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanine nucleotide biosynthesis, we examined the effects of nucleosides, nucleotides, nucleotide analogs, or the IMPDH inhibitor mycophenolic acid (MPA) on the steady-state levels of IMPDH mRNA. The results indicated that IMPDH gene expression is regulated inversely by the intracellular level of guanine ribonucleotides. We have shown that treatment with guanosine increased the level of ceHlular guanine ribonucleotides and subsequently reduced IMPDH steady-state mRNA levels in a time-and dose-dependent manner. Conversely, MPA treatment diminished the level of guanine ribonucleotides and increased IMPDH mRNA levels. Both of these effects on the steady-state level of IMPDH mRNA could be negated by cotreatment with guanosine and MPA. The down regulation of IMPDH gene expression by guanosine or its up regulation by MPA was not due to major changes in transcriptional initiation and elongation or mRNA stability in the cytoplasm but rather was due to alterations in the levels of the IMPDH mRNA in the nucleus. These results suggest that IMPDH gene expression is regulated by a posttranscriptional, nuclear event in response to fluctuations in the intracellular level of guanine ribonucleotides.Cellular systems have mechanisms to regulate the expression of genes that mediate the synthesis of necessary metabolites. These mechanisms must respond to internal and external factors in such a way that an appropriate supply of these metabolites is provided for the cell or organism. In prokaryotic systems, some genes are regulated at the transcriptional level by metabolic end products. In Bacillus subtilis, the pur operon encodes enzymes involved in purine biosynthesis, 11 of which catalyze the formation of IMP from 5-phosphoribosyl-1-pyrophosphate (13). This operon is regulated by the purine end products, adenine and guanine nucleotides. Although end-product regulation of genes is common in prokaryotic systems, no parallel has been reported in the regulation of eukaryotic nucleotide biosyntheSiS. The steady-state mRNA level of human IMP dehydrogenase (IMPDH; EC 1.1.1.205), the rate-limiting enzyme in de novo guanine nucleotide biosynthesis, is elevated in human HL-60 and CEM leukemia and HO melanoma cells after they are treated with the competitive IMPDH inhibitor mycophenolic acid (MPA) (11,20,21). This induction is preceded by a decrease in the level of cellular guanine nucleotides as a consequence of MPA-mediated inhibition of IMPDH enzyme activity. These observations suggest that the level of purine end products may influence the expression of the IMPDH gene, perhaps in a manner similar to that observed for the pur operon genes in B. subtilis. To examine the control of IMPDH gene expression, we treated human and Chinese hamster cell lines with nucleosides, nucleotides, nucleotide analogs, or MPA and analyzed the effect on the expression of the IMPDH gene. We provide evidence that the expression of this gene is indeed regulated by guanine ribonucleo...

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High-throughput approaches for gene cloning and expression require the development of new nonstandard tools for molecular biologists and biochemists. We introduce a Web-based tool to design primers specifically for the generation of expression clones for both laboratory-scale and high-throughput projects. The application is designed not only to allow the user complete flexibility to specify primer design parameters but also to minimize the amount of manual intervention needed to generate a large number of primers for the simultaneous amplification of multiple target genes.

Addition of mycophenolic acid to cultures of HL-60 cells results in a decreased cellular level of guanine nucleotides and the induction of cell differentiation. During the early stages of this induction, steady- state levels of cellular IMP dehydrogenase (IMPDH), messenger RNA (mRNA), and protein are increased, perhaps because of cellular compensation for the inhibition of IMPDH activity. The subsequent decrease in IMPH mRNA and protein levels after several days of treatment suggests a change in the control of IMPDH expression. In contrast to the pattern of increased IMPDH expression observed in the mycophenolic acid-treated cells, treatment of HL-60 cells with two other inducers of differentiation, namely retinoic acid and phorbol 12- myristate 13-acetate, resulted in stable or decreased levels of cellular IMPDH mRNA and protein. However, the kinetics of this expression were different. These results suggest that a number of factors influence the regulation of IMPDH expression during the induction of HL-60 cell differentiation, including the nature of the inducer. A decrease in the cellular IMPDH activity was observed for all of the inducers, suggesting that this decreased activity may be a determining factor in the acquisition of a mature phenotype in the HL- 60 cells.

The regulation of IMP dehydrogenase (IMPDH) was analyzed in Chinese hamster V79 cell variants that exhibit different degrees of resistance to the cytotoxic effect of mycophenolic acid, a specific inhibitor of IMPDH. Western blot (immunoblot) analysis with an IMPDH antiserum revealed a 14-to 27-fold increase in the amount of enzyme in the mycophenolic acid-resistant cells. The antiserum was also used to screen for a phage containing the IMPDH cDNA sequence from a Agtll expression library. Northern blot (RNA blot) analyses of total cellular and poly(A)+ RNA showed that an IMPDH cDNA probe hybridized to a 2.2-kilobase transcript, the amount of which was associated with increased resistance. Southern blotting with the probe indicated an amplification of the IMPDH gene in the mycophenolic acid-resistant cells. Our findings suggest that the acquired mycophenolic acid resistance of the V79 cell variants is associated with increases in the amount and activity of IMPDH and the number of IMPDH gene copies.IMP dehydrogenase (IMPDH) (EC 1.2.1.14), an enzyme that regulates guanine nucleotide biosynthesis, catalyzes the reaction of the branch point in the synthesis of adenine and guanine nucleotides. In both normal and malignant cells, the activity of this enzyme is positively correlated with cellular growth rate (26,27). Furthermore, in tumor cells IMPDH inhibitors cause a dose-dependent reduction in growth (10,13,22) and in the human promyelocytic HL-60 leukemia cell line also cause a dose-dependent induction of cell maturation (12,20). These observations suggest that IMPDH activity and the production of guanine nucleotides are involved in the regulation of growth and differentiation in mammalian cells.To study the control of IMPDH in mammalian cells, we isolated from the Chinese hamster V79 cell line, variants with altered IMPDH activity. The variants were obtained by treating the V79 cells with 0.5 ,ug of N-methyl-N-nitro-Nnitrosoguanidine and then selecting colonies of cells in the presence of 1 ,ug of mycophenolic acid (MPA), a cytotoxic IMPDH inhibitor (4), per ml as described previously (6). The resistance level of one of these cell variants was further increased by a stepwise selection in the presence of increasing concentrations of MPA. After adaptation to the higher concentration of MPA, the cells were seeded in medium containing an increased concentration of MPA at 200 cells per 60-mm petri dish, and MPA-resistant colonies were isolated 8 days later. This procedure, which was repeated a number of times, resulted in four cell clones, VM1 through VM4, which were resistant to 5, 10, 25, and 50 jig of MPA per ml, respectively, whereas the parental V79 cells were resistant to only 0.1 ,ug of MPA per ml (Table 1). The increased resistance to MPA cytotoxicity in the variant cells was associated with an increased IMPDH activity in their cell homogenates, with VM1 cells exhibiting about a 6-fold increase in IMPDH activity over the parental cells and VM2, VM3, and VM4 cells expressing about 7-, 9-, and 11-fold increases, re...