Mitotic Cycle Of Physarum Polycephalum Research Articles

Translocation of dna polymerase-α during the mitotic cycle of Physarum polycephalum

The location of DNA polymerase-α within nuclei of a macroplasmodium of Physarum polycephalum was identified at successive stages of the highly synchronous mitotic cycle by indirect immunofluorescence. The distribution of α-tubulin, the nucleolar protein fibrillarin and DNA were also identified. In G2-phase the polymerase-α was found mainly in the region of the nucleolus. In prophase the polymerase became dispersed into the nucleus with the breakdown of the nucleolus. In metaphase, the polymerase-α was located in two zones separated by the metaphase plate. In S-phase, which follows immediately after mitosis, the distribution of the polymerase was initially punctate but later segments were noted during nucleolar reconstruction. We infer that DNA polymerase-α is stored mainly in the region of the nucleolus (or the reconstructing nucleolus) when it is not needed for DNA replication.

Synthesis of actinomycin-insensitive RNA during the first post-irradiation mitotic cycle, in the synchronously mitotic plasmodia of Physarum polycephalum

A sucrose density gradient analysis of3H-uridine pulse-labelled RNA from the first postirradiation mitotic cycle ofPhysarum polycephalum shows that all the density classes of RNA synthesized during this period are resistant to the peptide-antibiotic, actinomycin D. In fact, the synthesis is found to be greater in the presence of the drug. The heterogenously sedimenting synthetic activity here may represent a single species of RNA and its precursors or more than one kind of RNA. Further characterization of this RNA is meaningful in view of the actinomycin insensitivity of the postirradiation mitotic cycle itself to this antibiotic.

Biosynthesis and activity of DNA polymerase throughout the mitotic cycle of Physarum polycephalum

Measurements of DNA polymerase protein levels and polymerase activity through the naturally synchronous mitotic cycle of Physarum polycephalum show that active DNA polymerase-α is synthesized throughout the G 2 phase, in step with the profile of general protein biosynthesis. Three main components of P. polycephalum DNA polymerase of 200, 112 and 70 kDa were found to be immunologically related.

Replication timing of the H4 histone genes in Physarum polycephalum.

The time of replication of the two H4 histone genes (H41 and H42) was determined during the naturally synchronous mitotic cycle of Physarum polycephalum. 5-Bromo-2'-deoxyuridine labeling and density gradient centrifugation was used to isolate newly synthesized DNA from defined periods of S phase. The DNA was analyzed by Southern hybridization with a cloned probe containing one of the H4 histone genes of Physarum. The results indicate that the two H4 histone genes are replicated in the first 30 min of S phase but not exactly at the same time. H41 is replicated during the first 10 min of S phase, when only 15% of the genome is duplicated, whereas H42 replicates between 20 and 30 min after the onset of S phase. The possible relationship between the periodic expression of the genes and the timing of their replication is discussed.

High diadenosine tetraphosphate (Ap 4A) level at initiation of S phase in the naturally synchronous mitotic cycle of Physarum polycephalum

Levels of the diadenosine tetraphosphate Ap 4A are high during exponential growth of Physarum, decrease during encystment (spherulation) and increase again during excystment. Consistently, a rapid 8–30-fold increase in Ap 4A level occurs at entry into S phase of the mitotic cycle and is maintained during the first half of genome replication. The elevated Ap 4A level depends significantly on ongoing DNA replication and is completely sensitive to the protein synthesis inhibitor cycloheximide administered either before or after initiation of S phase.

Cell-cycle-dependent effects of sodium-n-butyrate in Physarum polycephalum.

Sodium-n-butyrate affects the length of the mitotic cycle of Physarum polycephalum. Application during S- or early G2-period results in a delay of the subsequent mitosis, whereas application later in the cycle has no delaying effect. Interestingly, the second mitotic cycle after application is considerably shortened when butyrate has been administered during S- or early G2-period of the preceding cycle. In comparison, other homologous short-chain fatty acids were tested; the retarding effect on mitosis increases with the number of carbon atoms, although only butyrate can shorten the second mitotic cycle. It is shown that butyrate causes an immediate depression of synthesis of DNA, RNA and protein. After a certain time-interval the plasmodium overcomes the butyrate block. DNA synthesis is fully recovered and the inhibition of RNA and protein synthesis is even overcompensated until the next mitosis, as reflected by elevated levels of RNA and protein.

Adenylate cyclase and cyclic AMP phosphodiesterase activity during the mitotic cycle of Physarum polycephalum

The activity of adenylate cyclase (EC 4.6.1.1.) and cyclic AMP phosphodiesterase (EC 3.1.4.17) were measured in three subcellular fractions of the acellular slime mould Physarum polycephalum . Activity of adenylate cyclase was measured using the ATP analogue β-γ imido (8- 3 H) adenosine 5′ triphosphate. Cyclic AMP phosphodiesterase was measured using (8- 3 H) cyclic AMP as substrate in the presence of endogenous 5′nucleotidase (EC 3.2.2.4.). A particulate fraction showed a peak of adenylate cyclase activity late in G2 whereas other fractions showed no change at any time during the mitotic cycle. No changes in cyclic AMP phosphodiesterase activity were detected at any time in any fraction.

The relation of cycling of intracellular pH to mitosis in the acellular slime mould Physarum polycephalum.

The relation between intracellular pH and the mitotic cycle of Physarum polycephalum was studied by two-independent techniques. Both techniques revealed a long term cycling of intracellular pH which has the same period as the mitotic cycle, Qualitative detection of the changes in intracellular pH was made by measuring the changes in fluorescence of 4-methylesculetin which had been absorbed by the plasmodium. Quantitative measurements of intracellular pH were made throughout the mitotic cycle with antimony micro pH electrodes. The cycle of intracellular pH is sinusoidal in appearance. The maximum intracellular pH (pH 6.6) occurred at, or very near to, mitosis, and was approximately 0.6 pH units higher than the minimum pH, which occurred near the middle of the mitotic cycle.

Fluctuations in cyclic adenosine 3′:5′-monophosphate and cyclic guanosine 3′:5′-monophosphate during the mitotic cycle of the acellular slime mould Physarum polycephalum

Cyclic adenosine 3′:5′-monophosphate (cyclic AMP) and cyclic guanosine 3′:5′-monophosphate (cyclic GMP) have been determined at half-hourly intervals throughout the mitotic cycle of Physarum polycephalum. Cyclic AMP was constant at 1pmole/mg protein throughout except for a transient peak of 17pmoles/mg protein in the last quarter of G2. Cyclic GMP was more variable (2–4pmole/mg protein) rising to 9.5pmole/mg protein during the 3 hour S period and to 7pmole/mg protein during the last hour of G2. The significance of these changes is discussed.

Levels of RNA polymerases during the mitotic cycle of Physarum polycephalum

Two RNA polymerase activities were quantitatively solubilized in plasmodial homogenates from Physarum polycephalum by sonication at 0.5 M ammonium chloride concentration. The proportions of RNA polymerases A and B were determined by four different methods. Equal activity levels of both enzyme A and enzyme B were detected throughout the synchronous mitotic cycle of Physarum.

Fluctuations in deoxyribo- and ribonucleoside triphosphate pools during the mitotic cycle of Physarum polycephalum

Fluctuations in the pools of deoxyribo- and ribonucleoside triphosphates have been measured during the synchronous mitotic cycle of the slime mould Physarum polycephalum. The most pronounced fluctuation of the deoxyribonucleoside triphosphates was seen shortly before and after initiation of DNA synthesis. The pools of dTTP, dATP, dCTP and dGTP expanded before initiation of DNA synthesis and decreased again in early S phase. The pools of ribonucleoside triphosphates increased during mitosis and again 1 h after mitosis and 5 h after mitosis.

Differential RNA synthesis in the mitotic cycle of Physarum polycephalum

Differential RNA synthesis in the mitotic cycle of Physarum polycephalum

DNA-membrane association during the mitotic cycle of Physarum polycephalum

Association of DNA with the nuclear membrane was studied in the naturally synchronous slime mold Physarum polycephalum. Sucrose gradient analyses showed that DNA is bound to the membrane during all stages of interphase. The association is labile, but can be stabilized by spermine, which presumably acts on the membrane. Experiments with pulse-labeled DNA suggest that replication does not occur at a membranal site.

Aminoacylation of transfer ribonucleic acid in vitro during the mitotic cycle of Physarum polycephalum.

Aminoacylation of transfer ribonucleic acid in vitro during the mitotic cycle of Physarum polycephalum.

Methylation of the Deoxyribonucleic Acid of Physarum polycephalum at Various Periods during the Mitotic Cycle

The methylation of major nuclear DNA-cytosine was found to occur throughout the mitotic cycle of Physarum polycephalum, during the period of DNA synthesis (S), as well as during the remainder of interphase (G2) when essentially no synthesis de novo of the major nuclear DNA takes place. After incubation of the mold with methionine methyl-3H, 5-methylcytosine was the only radioactive methylated minor base detected in major nuclear DNA, although 3H was also found to be incorporated during the S period into DNA-adenine, -guanine, and -thymine, presumably by conversion of the methionine methyl group to 1-carbon intermediates incorporated into DNA nucleotides during synthesis de novo. During the G2 period, methylation of major nuclear DNA occurred at a relatively constant rate which was approximately 50% of that observed during the S period. Incorporation of 3H from methionine methyl-3H into 5-methylcytosine of both mitochondrial DNA and the nuclear heavy satellite DNA was also observed. It was estimated that the three DNA fractions of Physarum contain 1 methylcytosine residue for every 12 to 24 cytosine residues.

Polysome patterns and protein synthesis during the mitotic cycle of Physarum polycephalum.

Polysome patterns and protein synthesis during the mitotic cycle of Physarum polycephalum.

Ribonucleic acid synthesis in vivo in the synchronously dividing Physarum polycephalum studied by cell fractionation

The incorporation in vivo of tritiated uridine into different cell fractions was studied in the mitotic cycle of Physarum polycephalum, a myxomycete with naturally synchronous nuclear divisions. In 10-min pulses approx. 70 % of the recovered radioactivity was in the nuclei at all times of interphase, whereas this figure was lower at mitosis. The biphasic pattern of incorporation previously observed in this organism was also seen in nuclei after isolation.

Sedimentation patterns of pulse-labeled RNA in the mitotic cycle of Physarum polycephalum

Plasmodia of the synchronously dividing myxomycete Physarum polycephalum have been exposed to short pulses of [ 3H]uridine at different stages of the mitotic cycle. RNA was extracted by a modified phenol procedure and separated on sucrose density gradients. At all times of the cycle investigated, ribosomal and soluble RNA were made. After a 3-min pulse followed by a 60-min chase, the absorbance and activity patterns coincided at all times of the cycle. No significant differences were seen in the sedimentation pattern of 10-min pulse-labeled RNA made at different times of the mitotic cycle. Peaks with sedimentation values of about 33 S and 19 S were always observed; these may be precursors of ribosomal RNA. With 3-min pulse-labeling polydisperse RNA was obtained. Sedimentation values ranged from about 4 S to more than 50 S with two peaks at about 6 S and 13 S. The patterns were very similar at different times of the mitotic cycle; however, 1 h before mitosis the 13-S peak seemed particularly high, and in mitosis there was partial shift of the labeling pattern to the lighter side.

RNA synthesis in the mitotic cycle of physarum polycephalum

The rate of uptake of [ 3H]uridine into RNA has been studied in synchronous cultures of Physarum polycephalum. RNA synthesis was reduced at mitosis and again in mid-interphase, and sharply increased following each of these periods. The maximum increase early in interphase coincided with the time of nucleolar reconstruction and DNA synthesis, while the second maximum occured at the time of nucleolar swelling. Such swelling is the first cytological indication of the approaching mitosis. Pretreatment with actinomycin D reduced the postmitotic synthesis one half and completely eliminated premitotic synthesis. These results suggest a different physical or chemical nature of some of the RNA's made during the two periods of synthesis.