Resistance to 5-fluoroorotic acid and pyrimidine auxotrophy: a new bidirectional selective system for mammalian cells.

We have isolated a clone of murine erythroleukemic cells which will yield a population when a single cell is suspended in a medium composed of dialyzed sera and small molecules. We report that it is feasible--in one experiment--to screen more than 10 of these cells for growth under selective media containing 10(-4) M 5-fluoroorotic acid and 10(-4) M uridine. Cells capable of sustained growth in such media were eventually recovered.

Effects of natural pyrimidines and of certain related compounds on the spontaneous activity of the mouse.

Uracil and perhaps other natural pyrimidines may effect the level of arousal of the mammalian brain since: 1) heterocyclic 6-membered rings, which resemble uracil, form part of the structure of many hypnotics; and 2) 6-azauracil (and its riboside) have shown to be hypnotic for several mammalian species, including man. The parenteral administration of uridine, 6-azauridine, cytidine or thymidine depressed the spontaneous activity of adult male C-57 mice. 6-Azauridine was much less potent than the other ribosides tested.

Evidence for a striking increase in acetylcholinesterase activity in cultured human fibroblasts which are trisomic for chromosome two.

Acetylcholinesterase (AchE) is reported to have a narrowly restricted distribution among human tissues. Three strains of human fibroblasts which are trisomic for chromosome 2 had an average level of AchE activity over 28 times higher than the average level in 19 control strains of human fibroblasts. In contrast, the mean pseudocholinesterase activity of the trisomy-2 strains did not differ appreciably, or significantly, from the mean for the control strains.

Increased intracellular phosphoribosylpyrophosphate and accelerated orotic acid decarboxylation in a mouse cell line resistant to purine and pyrimidine ribonucleosides.

A line of mouse fibroblasts (A9AU-1), originally selected for growth in the presence of 6-azauridine, has been found to be resistant to cytotoxic concentrations of adenosine, guanosine, and thymidine. A9AU-1 cells convert orotic acid to uridine 5'-monophosphate at twice the rate of the A9P line from which the A9AU-1 clone was selected. The resistant cells also excrete purines, synthesized de novo, into the medium at an increased velocity.

Concurrent development of resistance to 6-azauridine and adenosine in a mouse cell line.

A variant of the hypoxanthine-guanine phosphoribosyltransferase deficient, and adenine phosphoribosyltransferase deficient mouse resistant to 6-azauridine. These cells are not only resistant to 6-azauridine (5 X 10(-4) M), but also to adenosine (10(-3) M). Resistance persists indefinitely even in the absence of both compounds.

Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells. I. Effect of substitution of galactose for glucose as the sole hexose in the medium in human diploid cell strains and in a rat hepatoma line.

In human diploid cell strains, the substitution of galactose for glucose as the sole hexose in the medium had no measurable effect on the specific activity of the cell protein for any of the three enzymes of the Leloir pathway. These enzymes are galactokinase, alpha-D-galactose-1-phosphate: UDP glucose uridyl transferase and UDP galactose 4-epimerase. A cell strain from a patient with galactosemia had no detectable activity for the transferase.

Studies on the regulation of the three enzymes of the Leloir pathway in cultured mammalian cells. II. A search for quantitative interrelationships between the three enzyme activities.

Studies on a normal human diploid cell strain revealed that the specific activity of the cell protein, for each of the three enzymes of the Leloir pathway, changed significantly as the cells grew. The kinetics of change in specific activity varied according to the enzyme being studied, and the kinetics for each enzyme varied from experiment to experiment. Within each experiment, there was no consistent correlation between specific activity for any one enzyme and specific activity for the other two.