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.     

Encapsulation of 8-azaguanine in single multiple compartment liposomes

Incorporation of [2-¹⁴C]-8-azaguanine into positively charged single and multiple component dipalmitoyl-DL-α-phosphatidylcholine and egg yolk phosphatidylcholine liposomes has been established. The extent of encapsulation in single compartment liposomes is 0.70–1.80% and it depends on the concentration of the added 8-azaguanine and the sonication time. Utilizing dialysis, the leakage of the drug from the single compartment liposomes after 20 hours was determined to be 4–34%. At high concentration of the added drug it is possible to encapsulate 18×10^?9 pmole of 8-azaguanine per liposome. Percentages of uptake into multicompartment liposomes are 3.6–5.4%. A preliminary study has been carried out on the effects of free and single and multiple compartment encapsulated 8-azaguanine on the survival and weight gains of leukemia L-1210 bearing mice.     

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.