| Description |
Two mutant clones of Chinese hamster ovary (CHO-K1) cells were obtained by selection schemes designed to favor cells with defective amino acid transport through the cationic amino acid transporter, system y+. One of the mutants, CH-K r, was selected for resistance to chronic exposure to high concentrations of lysine and arginine in the growth medium. The other, C2.2, was selected using a suicide protocol that employed a brief exposure to tritiated lysine. As might be expected, transport activity by system y+ was decreased in both mutants. However, the phenotypes of CH-Kr and C2.2 were more complicated and several transport systems were affected. Transport through systems A, L, and y+ was reduced in both mutants, with the effect extending to system ASC in CH-Kr cells. Transport of aspartate through system X[AG-] was not affected in either cell line. Despite the different selection schemes, these mutants appeared initially to be very similar. Other metabolic parameters were measured and compared in both mutants. These results revealed that the mutants differ in many important respects. For example, CH-Kr maintains a lower intracellular sodium concentration than parental cells, whereas no difference in intracellular sodium was detected in C2.2. This was surprising, considering the generally lower transport rates observed in CH-Kr, and was especially puzzling when intracellular amino acid measurements revealed that CH-Kr also maintained reduced intracellular amino acid pools compared to parental cells or C2.2. Reduced internal sodium represents a steeper sodium gradient that would be expected to accelerate instantaneous amino acid uptake and to maintain higher amino acid pools. Taken together, the physiological data presented here imply the existence of a complex regulatory scheme governing the activity of these transporters. To address this issue, an initial comparison of gene expression in the mutants was made by the Differential Display technique. An analysis of approximately 20% of the mRNAs in these cells revealed eight differences between wild type and the two mutants. None of the alterations were shared by both mutants. These data reveal that the mutations underlying the similar phenotypes observed in CH-Kr and C2.2 are, in fact, dramatically different, and demonstrate the applicability of the Differential Display technique to the study of such mutations. |
