Isolation and characterization of a glycine transport mutant in an established mammalian cell line, CHO(PEOT/1).

Glycine uptake in CHO(PEOT/1) cells is mediated by at least three systems, of which two have been identified and partially characterized in this study: (1) a low affinity "A" system that transports a number of small neutral amino acids including glycine and methylaminoisobutyric acid (MeAIB), and (2) a high-affinity system, specific for glycine and sarcosine. By a combination of tritium suicide and replica plating, we have isolated a mutant (CHY-3) with a 47% decrease in glycine transport at the standard test concentration of 2.5 microM. Uptake studies with radioactive glycine, MeAIB, and sarcosine revealed that the mutant lacks the glycine-sarcosine system, but has undergone a compensatory 30-50% increase in the A system. Thus, there appears to be a regulatory interaction between these two systems for glycine uptake by CHO cells.

Isolation and characterization of a CHO amino acid transport mutant resistant to melphalan (L-phenylalanine mustard).

A mutant of Chinese hamster ovary cells, CHY-2, was isolated on the basis of its reduced ability to grow on a limiting concentration of leucine and was found to be defective in uptake of leucine via the sodium-independent L system. Consistent with published reports that the L system can mediate melphalan uptake, the D10 of the mutant for melphalan was increased threefold under conditions designed to limit drug uptake to the L system (brief exposure in sodium-free medium). Unlike a previously described melphalan-resistant CHO mutant (CHr), CHY-2 displays no cross-resistance to colchicine or puromycin. It differs from a second melphalan-resistant CHO mutant, melr, in its sensitivity to melphalan in the presence of high Na+, and from a melphalan-resistant mouse leukemic cell in possessing normal levels of intracellular glutathione. Thus, CHY-2 represents a new melphalan-resistant mutant class. The effect of the CHY-2 mutation is pleiotropic, involving significant reductions in amino acid uptake via the L, A and Ly+ (but not ASC) systems. The primary defect is unknown; however, the mutant possesses normal intracellular concentrations of Na+ and K+ and normal membrane fluidity. The growth rate of the mutant in standard medium is greatly reduced (generation time of 60 h vs. 24 h), although it can be improved by the addition of a supplement containing high concentrations of leucine, proline, and peptides.