X-ray-induced mutants resistant to 8-azaguanine. II. Cell cycle dose response.

Synchronous Chinese hamster ovary cells were irradiated in G1 or S phase. Colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 mug/ml 8-azaguanine (AG). An expression period of over three generations (multiplicity of 20) was utilized, with expression times ranging from 58 to 114 h. Both G1 and S phase were practically identical in sensitivity to X-ray-induced mutations, with mutant frequency/viable cell/rad ranging from 1 X 10(-7) (75-100 rad) to 8 X 10(-7) (1000 rad). The spontaneous mutation rate, shown by Luria-Delbruck fluctuation analysis, was 5 X 10(-7) per generation. Thirty-three mutants, isolated at random and grown for over 30 generations in the absence of AG, were analyzed for plating efficiency (PE) in different concentrations of AG or in hypoxanthine-aminopterin-thymidine (HAT) medium. Of these, 64% were resistant (PE greater than 0.1) to 7.5 mug/ml AG, 85% to 5.0 mug/ml, and 91% to 3.5 mug/ml. Only 42% showed possible hypoxanthine-phosphoribosyltransferase (hprtase) deficiency as evidenced by HAT sensitivity (PE less than 0.1). Wild type controls exhibited PE's in 3.5 mug/ml AG of less than 0.001 and in HAT of greater than 0.5. Of ten mutants studied, all demonstrated survival response to radiation similar to wild type cells (D0 of approx. 120 rad). For radiation protection standards, the radiation dose required to induce mutations at a rate equal to that occurring spontaneously is called the doubling dose. The doubling dose observed for acute irradiation was about 3 rad and was estimated to be 10-60 rad for chronic irradiation, similar to that often reported for in vivo studies.     

Eremothecium ashbyii mutants resistant to 8-azaguanine. II. Mutants with different degrees of resistance to 8-azaguanine]

Guanine, unlike adenine and hypoxanthine, can not eliminate the inhibitory effect of adenine analogues on the growth and flavinogenesis of Eremothecium ashbyii. Guanine does not restore riboflavin synthesis inhibited with 5-10(-3) M 8-azaguanine. Low adenine concentrations (10(-4)-3-10(-4) M), which do not influence the inhibitory effect of 5.-10(-3) M 8-azaguanine, restore the riboflavin synthesis in combination with guanine. On the basis of the data obtained as well as the data of biochemical analysis it is concluded that the riboflavin producer studied lacks guanosinemonophosphate reductase. The mutants resistant to various concentrations of 8-azaguanine have been obtained. In all mutants resistant to 8-azaguanine the efficiency of the incorporation of 14C-guanine and 14C-adenine into mycelium is decreased as compared with the susceptible strain. The mutant Azg-R 10 resistant to high (3-10(-3) M) concentrations of 8-azaguanine, 8-azaadenine and 2,6-diaminopurine secretes inosine-like compounds when grown in a synthetic medium. The stepwise increase of the mutant resistance to 8-azaguanine from 10(-4) M TO 3-10(-3) M did not result in further enhancement of riboflavin synthesis.     

Anomalies in the selection of mutants of Schizosaccharomyces pombe resistant to 8-azaguanine

Summary Anomalies in selection render 8-azaguanine unsuitable as a selective agent for screening forward mutations in continuous cultures of Sch. pombe. This system may, however, provide the basis for an enrichment method for the simultaneous isolation of mutants at two loci.     

Induction of 8-azaguanine resistant mutants in human cultured cells exposed to 31 MeV protons

We report results on the induction of 8-azaguanine (8-AG)-resistant mutants in cultured human cells (EUE) exposed to 31 MeV protons. The spontaneous frequency of mutants was 5.6 +/- 0.7 x 10(-6) per viable cell. Gamma rays were taken as reference radiation. Expression times giving the highest frequency of mutants after 31 MeV protons and gamma irradiation were found to be about 10 days for both radiations. The dose-response relationship for mutant induction by protons, as determined at the optimal expression time, was compared to that obtained after gamma rays. The relative biological effectiveness (RBE) is 2.4 +/- 0.5, this value being higher than the RBE value determined for cell survival.     

Toxicity of 6-thioguanine and 8-azaguanine to non-dividing liver cell cultures

8-azaguanine and 6-thioguanine were both toxic to non-dividing liver cells in primary cultures. In addition, these agents were toxic to an established line of liver-derived epithelial cells brought to growth arrest by serum deprivation. These observations demonstrate that the toxicity of 8-azaguanine and 6-thioguanine can occur at least in part through mechanisms that do not involve effects on DNA synthesis or incorporation of the analogs into DNA.Abbreviations AG 8-azaguanine - ARL adult rat liver epithelial cell line - HGPRT hypoxanthine-guanine phosphoribosyl transferase - WME Williams Medium E     

The relative effectiveness of 6-thioguanine and 8-azaguanine in selecting resistant mutants from two V79 Chinese hamster cells in vitro

The frequency of surviving colonies in two V79 cell lines exposed to either 6-thioguanine or 8-azaguanine was dependent on initial plating density. Different degrees of metabolic-co-operation were found to occur in the two cell lines and the loss of both spontaneous and added mutants occurred at a lower cell density when 6TG was used for selection than when 8 AZ was used in both cell lines. Both analogues were degraded on incubation in medium plus serum in the absence of added cells. Variation in serum batch had little effect on the rate of degradation or on the frequency of colonies recovered after treatment of V79 cell lines with 8AZ. The reasons for preferring 8AZ to 6TG as a selective agent are discussed.     

Flavinogenesis and regulation of purine biosynthesis de novo in Pichia guilliermondi mutants resiatant to 8-azaguanine]

93 mutants resistant to 8-azaguanine (AGR-mutants) were derived from the strain of Pichia guilliermondii with blocked guanine deaminase (EC 3.5.4.3.) by UV-irradiation. The mutants retained the ability to uptake 8-azaguanine and guanine but could not deaminate guanine. Some of the AGR-mutants were found to accumulate large amounts of hypoxanthine and small amounts of guanine in the cultural medium. The inhibitory effect of guanine and 8-azaguanine but not adenine on the purine biosynthesis de novo was considerably decreased. It was established observing the rates of 5 amino 4-imidazoleribotide accumulation in purine-requiring AGR-mutants in the presence of different purines. The regulation of the activity and biosynthesis of IMP-dehydrogenase (EC 1. 2. 1. 14) with guanine compounds in AGR-mutants was completely preserved. Under cultivating in iron-rich medium all the AGR-mutants accumulated more riboflavin than the strain H-101 and the wild type strain. That occured as a result of the increase of flavinogenesis velocity in AGR-mutants during late logarithmic and negative growth acceleration phases. Some of mutants also synthesized more riboflavin in iron-deficient medium. Depression of riboflavine synthetase was not observed in the iron-rich cells of AGR-mutants.     

Isolation and characterization of an adherent, 8-azaguanine resistant variant of the Burkitt lymphoma cell line, Raji

A substrate adherent, fibroblast-like cell line (Raji-A) has been isolated from a suspension culture of an established Burkitt lymphoma cell line (Raji). Except for the altered morphology associated with substrate attachment, Raji-A is identical to Raji with respect to karyotype, isozyme composition, susceptibility to Epstein-Barr virus (EBV) infection and inducibility of latent EBV. In order to facilitate fusion experiments with Raji-A, drug resistant variants were induced by treating cells with ethylmethane sulfonate followed by selection in growth medium which contains 8-azaguanine. Three clones (AGRO, AGR3 and AGR6) were found to be resistant to 50 μg/ml of 8-azaguanine. They had only 10–15% as much hypoxanthineguanine phosphoribosyltransferase activity as wild type cells and a low plating efficiency of 1–3 × 10⁻⁶ in the selective medium, HAT. Potential uses of these variants for studying EBV-lymphoblastoid cell interactions as well as human genetics by cell hybridization are discussed.     

8-azaguanine and flavinogenesis in Eremothecium ashbyii.

8-Azaguanine (10- minus 4 M) supplementation in synthetic medium inhibited flavinogenesis in Eremothecium ashbyii to far greater extent (68per cent) than the growth (25 per cent). That enzymes comprising the biosynthetic pathway of riboflavin are synthesized during early growth phase of the organism is supported by the data presented. 8-Azaguanine mediated inhibition in flavinogenesis was closely related with decreased levels of ribose-5'-phosphatase, ribose reductase and ribitol kinase, the enzymes involved in supplying ribitol for flavinogenesis. Addition of guanine and not ribitol during early growth phase to 8-azaguanine-added cultures released the inhibition of riboflavin synthesis and restored the enzyme levels in the presence of the antimetabolite.     

alpha-Amanitin resistant RNA polymerase II from Aedes albopictus cell mutants resistant to alpha-amanitin

Spontaneous and EMS-induced alpha-amanitin-resistant Aedes albopictus cells have been isolated and characterized. Two mutant sublines, one of intermediate resistance (alpha A2) and the other highly resistant (Ama18) contained RNA polymerase II activity, the resistance of which in vitro to alpha-amanitin correlated well with the resistance of these cells in vivo. The resistance of these cells to alpha-amanitin can likely be attributed to the presence of an altered RNA polymerase II.