Genetic studies on the role of the nucleoside transport function in nucleoside efflux, the inosine cycle, and purine biosynthesis.

Abstract

A mutant clone (AU-100) which is 90% deficient in adenylosuccinate synthetase activity was characterized from wild-type murine S49 T-lymphoma cells. This AU-100 cell line and its hypoxanthine-guanine phosphoribosyltransferase-deficient derivative, AUTG-50B, overproduce purines severalfold and excrete massive amounts of inosine into the culture medium (Ullman et al., Proc. Natl. Acad. Sci. U.S.A. 79:5127-5131, 1982). We introduced a mutation into both of these cell lines which make them incapable of taking up nucleosides from the culture medium. The genetic deficiency in nucleoside transport prevents the adenylosuccinate synthetase-deficient AU-100 cells from excreting inosine. Because of an extremely efficient intracellular inosine salvage system, the nucleoside transport-deficient AU-100 cells also no longer overproduce purines. AUTG-50B cells which have been made genetically deficient in nucleoside transport still overproduce purines but excrete hypoxanthine rather than inosine. These studies demonstrate genetically that nucleoside transport and nucleoside efflux share a common component and that nucleoside transport has an important regulatory function which profoundly affects the rates of purine biosynthesis and purine salvage.

Expression of the high-affinity purine nucleobase transporter in mutant mouse S49 cells does not require a functional wild-type nucleoside-nucleobase transporter.

“…Kinetic (7,18,21,29) and genetic (5,8,10,31,33) evidence support the existence of a single nucleoside transporter which mediates the permeation of all purine and pyrimidine ribonucleosides and deoxyribonucleosides across the mammalian plasma membrane. Mutant S49 T lymphoblasts have been generated and characterized which are genetically deficient in nucleoside transport capability (5,8,10,31,33). Most nucleoside transport-deficient S49 cell lines also have a markedly impaired capacity to transport purine nucleobases (6), although a single nucleoside transpor,t-deficient cell line has been described with augmented hypoxanthine transport capability (3).…”

Cloning and characterization of the cDNA encoding human adenylosuccinate synthetase

“…A select population of previously unclassified idiopathic, overproducing, hyperuricemic gout patients would be prime candidates for such a study. Evidence supporting a possible deficiency of AS underlying some of these cases comes from the genetic model developed with A-100 cells [23,24]. This cell line is a mutated mouse T cell lymphoma cell which is 80% deficient in AS activity.…”

“…No information is available regarding the human isoforms and few mutations in AS are known in humans suggesting that the enzyme is important for growth and development. A mutated murine lymphoma cell line has been identified with a partial defiency in AS [23,24]. These cells synthesized large amounts of iitiP which is secreted as inosine, thus suggesting a link between the AS deficiency and hyperuricemic states.…”

Expression of a novel high-affinity purine nucleobase transport function in mutant mammalian T lymphoblasts.

“…The conditions for the mutagenesis of S49 cells with MNNG have been described elsewhere (2,26,27). The JPA2 cell line was isolated by plating mutagenized wild-type cells over mouse embryo fibroblast feeder layers (7) in semisolid (0.3%) agarose containing 50 ,uM hypoxanthine, 11 ,uM azaserine, and 10 puM Growth rate determinations.…”

“…Hypoxanthine salvage was determined in the presence of azaserine, an inhibitor of the purine-biosynthetic enzyme phosphoribosylformylglycineamide synthetase (14). The growth sensitivities of wild-type and JPA2 cells to nucleosides were compared as described previously (2,26,27 Enzyme assays. Cell-free extracts were prepared and gel sieved over Sephadex G25, and hypoxanthine-guanine phosphoribosyltransferase and adenine phosphoribosyltransferase activities were measured as described by lovannisci et al (11).…”