Wild-type and mutant cells

Fig. 6.3. Cutoff fluorescence selection for screening. Instrumentation, labeling, and biological noise introduce spreading into a fluorescence measurement, such that the fluorescence probability distributions for wild-type and mutant cells overlap. The logarithm of single-cell fluorescence as measured by flow cytometry is generally well-approximated by a symmetrical Gaussian curve. A cutoff fluorescence value is selected for screening, with all cells above that value sorted out. The enrichment factor forthe mutants is the ratio of (dotted + striped areas)/(striped area), and the probability of retention of a given mutant clone at a single pass is the (striped + dotted area)/(all area under mutant curve).

Incorporation of a stable isotopic tag into proteins/peptides in metabolically active cells was first described to quantify protein abundance in yeast (43). Wild-type and mutant cell populations were grown in media containing the naturally abundant isotopes of nitrogen and enriched in 15N, respectively, followed by trypsin digestion and LC-ESI-MS/MS analysis to identify and quantify relative phosphopeptide levels in both populations (43). 

Miesfeld, R., Okret, S., Wikstrom, A.C., Wrange, O., Gustafsson, J.A. and Yamamoto, K.R. (1984) Characterization of a steroid hormone receptor gene and mRNA in wild-type and mutant cells. Nature, 312, 779-781. 

Growth conditions Wild type and mutant cells of Synechocyst ts... 

Figure 1. Growth Sensitivity of Wild-Type and Mutant Cells to...

Since mycophenolic acid is an inhibitor of the penultimate enzyme in guanyl nucleotide synthesis (10), the effects of mycophenolic acid on the guanine nucleotide levels in wild-type and mutant cells were examined. Figure 2. Whereas, a profound depletion of GTP occurred in wild-type cells incubated 4 hr with 1 uM mycophenolic acid, a similar GTP depletion was seen only at 5 jiM mycophenolic acid in MYCO-IA cells (Figure 2). No depletion of intracellular GTP was observed in MYCO-lA-20 cells incubated with exogenous mycophenolic acid at concentrations as high as 25 pM. 

Figure 2. Ribonucleoside Triphosphate Concentrations in Wild-Type and Mutant Cells Before and After Incubation with Mycophenolic...

Since, the sensitivities of the IMP dehydrogenase activities to mycophenolic acid inhibition were altered in mutant cell extracts, we compared several other kinetic parameters of the IMP dehydrogenase activities from wild-type and mutant cells. Table 1 indicates that for the IMP dehydrogenase activities in both mutant lines the Vmax and apparent K j s for IMP are increased considerably over those found for wild-type enzyme activity. The apparent Kn, values for NAD were also determined. Table 1. 

Reports of purine overproduction induced by pharmacologic inhibitors of IMP dehydrogenase (8) led us to examine the effects of mycophenolic acid on the rates of purine synthesis and excretion in wild-type and mutant cells. At the same concentrations (1-25 uM) of mycophenolic acid that depleted... 

Figure 3. Sensitivities of Wild-Type and Mutant Cell IMP Dehydrogenase Activities to Inhibition by Mycophenolic Acid.

Figure 4. Effect of Mycophenolic Acid on the Rates of Purine Synthesis and Excretion in Wild-Type and Mutant Cells. The effects of either a 1 uM, 5 uM. or 25 uM mycophenolic acid incubation for 4 hr on the rates of purine synthesis were performed as described previously (7).

These studies with wild-type and mutant cells defective in IMP dehydrogenase and the previous data with the adenylosuccinate synthetase-deficient cell line suggest that among the clinical population with dominantly inherited hyperuricemia, patients with partial deficiencies in these enzymes exist. It is hoped that these pharmacogenetic cell culture models for overproduction hyperuricemia will lead to the initiation of a search for hyperuricemia patients with either of these deficiencies. If such patients are found it may be possible to design chemotherapeutic regimens by which effectors (inhibitors) of purine synthesis might ameliorate the overproduction of purines by the de novo pathway. 

Let us say that we have performed experiments to measure the protein expression rates of wild-type and mutant cell strains. 

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