The Inosinic Acid Pyrophosphorylase Activity of Mouse Fibroblasts Partially Resistant to 8-Azaguanine

Abstract

Resistance to the analogue 8-azaguanine has generally been found to correlate with loss of guanylic acid-inosinic acid (IMP) pyrophosphorylase activity in several biological systems' including 3 mammalian cell lines.2-4 From 2 of these lines mutants selected for even a small degree of resistance contained very little or no pyrophosphorylase activity.2 4 From the other cell line a partially resistant mutant was also isolated in which no change in pyrophosphorylase activity occurred, as well as another mutant (resistant to 8-azaguanosine) which "displayed about onehalf of this activity."3 To investigate further the mechanism of partial resistance we have now isolated several lines of mouse fibroblasts (L cells) which show a small degree of resistance to 8-azaguanine, and have found them to contain [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] per cent of the IMP pyrophosphorylase activity of wild-type L cells. Culture methods: An inoculum of L cells was kindly supplied by Dr. L. Siminovitch in 1960, at which time the chromosome number was reported to be approximately 60. This cell line, designated "wild-type," has been maintained in suspension culture in Eagle's "spinner" medium plus 10% calf serum as previously described.6 It is free of pleuropneumonia-like organisms. Clonal lines were obtained by isolating individual colonies7 and have been stored at -70'C in medium containing 10% glycerol when not in use. 8 Determinations of drug resistance or mutant frequency were performed by inoculating an appropriate number of cells (2 X 102 to 5 X 106) from a suspension culture into replicate 60 mm

Tight Control of Respiration by NADH Dehydrogenase ND5 Subunit Gene Expression in Mouse Mitochondria

“…All the cell lines used in the present work were grown in monolayer culture. The cell line A9 (ATCC CCL-1.4) is a derivative of the L mouse fibroblast cell line deficient in hypoxanthine-guanine phosphoribosyl transferase and is, thus, resistant to 8-azaguanine and 6-thioguanine (34) and incapable of growing in hypoxanthine-aminopterin-thymidine (HAT) medium (19). This cell line was grown in Dulbecco modified Eagle medium with 4.5 mg of glucose/ml (DMEM) supplemented with 10% calf serum and 3 g of 8-azaguanine per ml.…”

Transformation of Cultured Mammalian Cells by Viable Herpes Simplex Virus Subtypes 1 and 2

“…The basis for the selection of transformed cells on HAT is that those cells in which the information for HSV-specific TK synthesis is stably incorporated will be able to utilize thymidine and multiply, whereas the XCTK-cells will not (13). Alternative pathways that would enable cells to bypass the aminopterin block are conceivable, so we tested the transformed lines for TK activity and determined the migration rate of TK activities by gel electrophoresis as described by Munyon et al (7).…”

Mutagenesis in Cultured Human Diploid Cells

“…A great difference was found in the sensitivities to SAG of wild type cells and 8AG-resistant cells. In the presence of 1 pg/ml of 8AG no wild type cells of mouse L strain survived, whereas the plating efficiency of 8AG-resistant clones was not affected (Littlefield 1963). A similar difference in sensitivities was found with human diploid cells: the growth of normal diploid strain cells was completely inhibited by 2.7x 10-6M of 8AG, whereas inhibition of 8AG-resistant cells, derived from a patient with the Lesch-Nyhan syndrome, was not detected with concentrations of less than 2.2 x 10-4M 8AG and was still incomplete with 2.0 x 10-3M 8AG (Albertini and DeMars 1970).…”