E.N. Brewer

DNA replication in Physarum polycephalum

Abstract The DNA of Physarum polycephalum, a eukaryote whose nuclei divide synchronously, was uniformly-labeled with [14C]thymidine or pulse-labeled for 10 minutes with [3H]thymidine. Nuclei isolated from combined 14C- and 3H-labeled cultures were gently lysed on top of neutral and alkaline sucrose density gradients. In neutral gradients, uniformly-labeled DNA from G2-phase cultures sedimented at a rate corresponding to an average (peak) molecular weight of 2.3 × 108 daltons, while in alkaline gradients the average molecular weight was 4 × 107 daltons. Pulse-labeled nuclear DNA sedimented in alkaline sucrose according to an average molecular weight of 1.5 × 107 daltons. Upon continued incubation in the absence of [3H]thymidme, the molecular weight of these “precursor” pieces increased, reaching that of the non-replicating (G2) DNA after a 90-minute chase period. On the other hand, pulse-labeled double-strand DNA species were initially heavier (average molecular weight 3.6 × 108 daltons) and had a broader size distribution than did non-replicating DNA. These species do not contain protein and may, therefore, consist predominantly of intact replicating (forked) molecules. During a subsequent chase period the heavy intermediates were converted into molecules equal in size to the parental DNA. This transition was first observed after a 30-minute chase and was completed within 60 minutes. The presence of cycloheximide during the 10-minute pulse-labeling period had little effect on the profiles obtained for either neutral or alkaline sucrose gradients, but if the inhibitor was present during the chase period, the size increase of the pulselabeled single-strand units and the completion of synthesis of mature double-strand molecules were prevented. From these data, a working model for DNA replication in Physarum polycephalum is proposed. Replication is postulated to occur discontinuously, the ends of alternate single-strand “subunits” of the native molecule serving as initiation and termination sites.

A new mitotic cycle marker

Abstract The entry into metaphase of Physarum nuclei becomes insensitive to cycloheximide 10 min earlier than to γ-radiation, i.e., the “CH-marker” occurs earlier in prophase than the “γ-marker”. Gamma-irradiation of plasmodia wich have passed the CH marker and even the γ-marker leads to further sensitivity to CH. Thus, the data reveal a “γ-CH marker” in the mitotic cycle of this organism.

Studies on the mechanism of DNA replication in Physarum polycephalum

Abstract The synthesis of single-stranded DNA subunits (4 × 107 daltons) in Physarum polycephalum was studied by alkaline sucrose density gradient centrifugation. The results were compared with the synthesis of the double-stranded DNA molecules (2.3 × 108 daltons) which they comprise, as determined from neutral sucrose density gradient centrifugation patterns. Although the initiation of synthesis of most double-stranded DNA molecules takes place relatively early in the S period, synthesis of the subunits within them is initiated throughout at least the first two hours of this period. Similarly, replicating (presumably forked) DNA molecules appear to split into daughter DNA molecules prior to the completion of synthesis of the subunits therein. The average rate of DNA chain elongation within subunits is 0.3 × 106 daltons/minute. It is suggested that alkaline sucrose density gradient centrifugation may be a more sensitive method for determining the time required for the completion of replication than other methods based solely on the incorporation of radioactive DNA precursors into an acid-insoluble product.

Effects of cycloheximide on thymidine metabolism and on DNA strand elongation in Physarum polycephalum

Abstract Treatment of Physarum polycephalum with cycloheximide during the DNA synthesis period resulted in a reduction in the incorporation of [ 3 H]thymidine into DNA. This effect was caused by both a reduction in the specific activity of TTP and by an inhibition of progeny strand elongation within replication units. No effect of the drug on the initiation of synthesis of replication units or on the ligation of DNA fragments was detected.

Absence of a correlation between cyclic nucleotide fluctuations and cell cycle progression

Abstract The levels of cAMP and cGMP were determined throughout the mitotic cycle of two independent strains of the naturally synchronous slime mold, Physarum polycephalum. The normal range of values was approx. 0.5–2.5 pmoles cAMP/ mg protein and 0.01–0.08 pmoles cGMP/mg protein. In our standard laboratory strain, there was no systematic pattern to the variations in the values within the normal range and no unique time in the cycle when values significantly above normal were noted. In the other strain recently cultured from spherules, elevated levels of cGMP, but not cAMP, were observed in late G2 and in the S phase. The data suggest that elevated levels of these cyclic nucleotides are not required for normal progression of the Physarum mitotic cycle which is unperturbed by artificial synchronizing procedures.

Inhibition of alkaline ribonuclease activity by polyamines

Abstract The polyamines spermine and spermidine inhibit the activity of alkaline ribonuclease in cell-free preparations from rat liver. By inhibiting the activity of alkaline ribonuclease in vivo, these compounds may play a role in the regulation of RNA breakdown in mammalian tissues.

DNA synthesis in a sub-nuclear preparation isolated from Physarumpolycephalum

Abstract A sub-nuclear preparation capable of substantial levels of DNA synthesis in vitro has been obtained from isolated S-phase nuclei of Physarum polycephalum . Nuclei were disrupted by gentle resuspension in a dextran-free medium followed by immediate addition of dextran to stabilize the liberated replication complex. Synthesis continues for at least 120 min, and appears to occur by a semi-discontinuous mechanism. Little DNA synthesis occurs in preparations obtained from G2-phase nuclei.