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Effects of 8-Azaguanine Florian, Svatopluk Fred

Abstract

An investigation was made of the effects of the purine analogs, 8-azaguanine, 8-azaxanthine, and 6-mercaptopurine, and the purines adenine and guanine, on cell growth, mitosis, and desoxyribonucleic acid (DNA) synthesis in primary roots of Vicia faba seedlings grown in aerated solutions. Root elongation was used as a measure of cell elongation; mitotic frequency was determined in free cell suspensions prepared from 1 mm-long root tips; the relative content of DNA was determined microspectrophotometrically by the two-wavelength method. It was shown that the balance between root elongation and mitosis in the root tip could be influenced by the amount of aeration and by adenine. Increased aeration stimulated root elongation and depressed mitotic frequency; adenine stimulated mitosis, inhibiting, at the same time, root elongation. 8-azaguanine, in concentrations of 10 p.p.m. and higher, stopped mitosis within 24 hours and greatly reduced root elongation and the fresh and dry weights of roots within 72 hours. This inhibitory effect on both root elongation and mitosis was positively correlated with aeration. 8-azaguanine in a concentration of 1 p.p.m. significantly reduced mitotic frequency but slightly stimulated root elongation. The inhibition of root elongation could be best, though incompletely, reversed by 40 p.p.m. adenine or guanine. The mitotic inhibition could be partially reversed by 40 p.p.m. guanine; adenine, at a concentration of 80 p.p.m., not only completely relieved mitotic inhibition, but increased the mitotic frequency to a level higher than that of the water controls. Concentrations of 6-mercaptopurine and 8-azaxanthine comparable with those of 8-azaguanine had no inhibitory effects. Roots treated with 20 p.p.m. 8-azaguanine for 24 hours and then transferred into 80 p.p.m. adenine showed a higher mitotic frequency than the control within 24 hours after transfer. Roots transferred from 8-azaguanine into water showed some mitosis 48 hours after transfer; in this case mitosis was restricted to the partially differentiated and elongated cells of the provascular bundles. DNA content of interphase nuclei in the controls showed this distribution: a sharp 2C peak (about 65 per cent nuclei), a much lower 4C peak (about 20 per cent nuclei), and intermediates (about fifteen per cent). There were no polyploid nuclei in the apical meristem of the root. The DNA content of chromosomal groups in different mitotic stages demonstrated the accuracy of the two-wavelength method which was used. The DNA content of nuclei in roots treated with 20 p.p.m. 8-azaguanine was distributed in a 2C peak (about 80 per cent nuclei) and a 4C peak (about 20 per cent nuclei). There were no intermediates in treated roots and no nuclei contained a higher amount of DNA than 4C. The percentage of 4C nuclei did not increase with time. From the evidence that the mitotic inhibition induced by 8-azaguanine could be completely reversed within 24 hours by subsequent treatment with adenine, and from the findings concerning the distribution of DNA in inhibited nuclei, it may be concluded that 8-azaguanine was not incorporated into DNA. The possibility that 8-azaguanine exerts its inhibitory effects through interference with ATP metabolism is discussed.

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