DNA synthesis in Physarum polycephalum.
thesisposted on 19.11.2015, 09:08 by Martin William. Cunningham
This thesis describes investigations of DNA synthesis in the myxomycete, Physarum polycephalum, using an isotope dilution technique capable of measuring macromolecular synthesis directly, in contrast to methods which follow the incorporation of a radioactive precursor. Using petri dish and large surface cultures, the pattern of either nuclear or total DNA accumulation during the mitotic cycle was determined in three media with cycle lengths varying between 9 and 12 h. In common with previously published results, no G1 phase was detected. Statistical analysis suggested that, within the range examined, changes in cycle length were confined mainly, if not solely, to G2, S phase remaining constant at approximately 120 min. During G2 an amount of synthesis was detected which ranged from 10 to 27% of nuclear DNA, more than could be due to nucleolar DNA. In most cases, DNA content showed less than a doubling between divisions, and possible causes of this and of G2-synthesis are discussed. Examining DNA synthesis in the presence of cycloheximide which at 20 mug per ml could, within 15 min, inhibit by 95% the incorporation of carbon-14 labelled lysine, it was found that (i) inhibition of replication was detectable after 20 min exposure but, at shorter times, the method employed was insufficiently sensitive to detect inhibition consistently; (ii) residual synthesis of approximately 15 - 20% of total or nuclear DNA can occur during 3 h exposure to cycloheximide (20 mug per ml) and (iii) approximately 50% inhibition of protein synthesis by 0.25 mug per ml cycloheximide produced no detectable effect on the level of DNA synthesis. Other experiments described are: (i) preliminary attempts to construct an in vitro replicating system from plasmodial homogenates, assaying synthesis by isotope dilution; (ii) measurements in small plate cultures of macromolecular synthesis between inoculation and M1, and DNA synthesis within the inoculum region; (iii) determinations and statistical analyses of the growth rate of microplasmodia and surface plasmodia under various conditions.