Size and structure of mitochondrial DNA from Physarum polycephalum

One band of DNA with a buoyant density of 1.688 g cm⁻³ is found when isolated mitochondria of the slime mold Physarum polycephalum are incubated with deoxyribonuclease prior to lysis. The DNA consisted mainly of linear molecules up to about 20 μm in length. As many as 10% of the molecules were, however, of open circular conformation with a circumference of 19.1±0.5 μm. Mild lysis conditions favoured the isolation of DNA/protein complexes with no visible free ends. The data suggest that the mitochondrial DNA (mtDNA) of Physarum is a circle and that circularity may be maintained by DNA/protein interaction.     

Stage-specific antigens of Physarum polycephalum

Antisera were prepared in isogenic F₁ hybrid rats against three amoebal strains and against two genetically related plasmodial strains of Physarum polycephalum. Differences in specificities between the antisera were studied using immunofluorescence tests and Ouchterlony double diffusion tests. There were no strain-specific differences between any of the three anti-amoebal sera, nor were any strain-specific differences found between the two anti-plasmodial sera. However, both ubiquitous and stage-specific antigens were detected.     

Cycloheximide-induced mitotic delay in Physarum polycephalum

Cycloheximide pulses applied to Physarum polycephalum surface plasmodia delay mitosis. Pulses applied in G2 cause a delay of mitosis which is linearly dependent on the phase in the cell cycle at which the pulse is applied. A 30 min pulse of 10 micrograms/ml cycloheximide starting in G2 at time t after mitosis induces an excess delay (delay in excess of pulse duration) of the next mitosis of (0.55) t-1.3 h. The excess delays induced by 7 h pulses during G2 are at most 1 h larger. Pulses applied less than 30 min before mitosis induce only small delays.     

Protoplasmic streaming during the cell cycle of Physarum polycephalum

It has been reported that protoplasmic streaming stops during the synchronous mitosis exhibited by growing plasmodia of P. polycephalum. Our data reveal that at no time during the mitotic cycle did streaming stop. However, during a 3–5 min period at anaphase the percent of each oscillation period accounted for by an outward flow was precisely equal in duration to the corresponding inward flow. At all other periods the duration of outward flow exceeded that of inward flow. Plasmodial migration or locomotion was briefly arrested at telophase, although shuttle streaming persisted.     

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.     

Characterization of thermosensitive mutants of Physarum polycephalum

Summary Amoebal thermosensitive mutants of Physarum polycephalum have been isolated after mutagenesis of the amoebal form by nitrosoguanidine treatment. About 70% of the independent thermosensitive amoebal mutants obtained were also thermosensitive in the plasmodial form. Two basic screening methods were applied at the same time to thermosensitive microplasmodia in order to detect strains defective in premitotic events, mitosis or chromosomal DNA synthesis. The first method consists in the determination of increase in protein. RNA and DNA with incubation time at the non-permissive temperature. It allowed the detection of four independent thermosensitive mutant strains, showing an early arrest in DNA synthesis. The second one is the quantification of the variations of the different nuclear types at the restrictive temperature. Two mutant strains presented very large nuclei, uni- or multinucleolate, very similar to those obtained after methyl benzimidazole carbamate treatment, suggesting a defect in one of the mitotic processes. One of these two mutant strains showed an early arrest in DNA synthesis at the restrictive temperature. These two screening procedures were completed by electron microscopic observation. This technique allowed the detection of intra-nuclear macrotubular crystal-like structures in a thermosensitive mutant showing a reduced DNA synthesis at the non-permissive temperature.     

Isolation of cell cycle mutants of Physarum polycephalum

Summary Asynchronous amoebal cultures of temperature-sensitive mutants of Physarum polycephalum were examined cytologically, and two cell cycle mutants were identified. Genetic analysis indicated that each mutant carried a single mutation that was expressed in both amoebal and plasmodial phases. Thus it is possible to isolate cell cycle mutations expressed in plasmodia by initial isolation and analysis of amoebal mutants, a quicker procedure than the alternative of isolating plasmodial mutants directly. The two mutants were studied further by measuring nuclear DNA contents and synthesis of macromolecules. Both mutants gave results consistent with a block in nuclear division.     

Cell cycle-related transfer of proteins between the nucleus and cytoplasm of Physarum polycephalum

The proteins of wild-type and polyploid plasmodia of P. polycephalum were prelabelled with [³H]leucine and [¹⁴C]leucine. The two types of plasmodia were then fused for 2 h. Following fusion the nuclei were isolated and the smaller wild-type cell nuclei separated from the larger polyploid cell nuclei. The proteins were isolated from the recipient cell nuclei and the recipient nuclear proteins extracted. Ratios of ³H/¹⁴C in the various nuclear protein fractions show that during fusion differential transfer of labelled preformed proteins from the donor cell into the recipient cell nucleus occurs. The quantity of proteins transferred varies among the different fractions and with the phase of the cell cycle. Isotopic dilution experiments indicate that these differences in protein transfer are, in part, due to a high rate of synthesis and turnover of the nuclear proteins.     

Annular structures in isolated nuclei of Physarum polycephalum

Whole-mount preparations of isolated interphase nuclei of Physarum polycephalum show annular structures. The outer and inner diameters are about 1 080 and 580 Å respectively. Ultrastructural features of the annuli are described; an association of the rings with fibers of various diameters, probably of chromatin nature, is also reported.     

Temperature-sensitive mutants of the slime mould Physarum polycephalum

Summary Methods are described for the isolation and testing of temperature-sensitive plasmodial strains of Physarum polycephalum. Nineteen temperature-sensitive strains were found by screening plasmodia derived from mutagenised amoebae and the properties of these are described. A scheme is outlined for the detection of specific mitotic cycle lesions amongst temperature-sensitive strains, and the properties of a presumptive mitotic cycle mutant are described.     

Coordinate replication of dispersed repetitive sequences in Physarum polycephalum

The synchronous macroplasmodial growth phase of the slime mould Physarum polycephalum was used to study the in vivo replication of large chromosomal DNA segments. Newly replicated DNA was isolated at various points in S-phase by its preferential association with the nuclear matrix. This DNA was then used to probe cosmid clones of the Physarum genome. The results indicate that certain dispersed repetitive sequences in the genome are coordinately replicated. The observed pattern of replication may be due either to the presence of a replication origin within each repetitive sequence or to the systematic arrangement of these sequences around a replication origin. The latter appears more likely since the repetitive sequences are probably not randomly scattered within the genome.     

Chemotaxis toward carbohydrates and amino acids in Physarum polycephalum

A method is described for assaying chemotaxis in the acellular slime mold Physarum polycephalum. It consists of measuring the amount of plasmodium that moves on a strip of nitrocellulose membrane filter Millipore in response to a gradient of an attractant. Time course of chemotactic response of the slime mold is described. Different factors that affect chemotaxis in the slime mold such as: culture care and stage of growth of microplasmodia, substratum used for cell movement, nature of the gradient, effect of salts, pH and temperature are described. From concentration-response curves for different attractants several parameters of the chemotactic effect, such as threshold concentration, half maximal concentration, and maximal effective concentration can be determined. As a group, sugars are more effective chemotactic agents than amino acids. Glucose and galactose, which support the growth of the slime mold, are shown to have high positive chemotactic effect. 3-O-Methyl- -glucose and 2-deoxy- -glucose are two sugars that do not support growth but are very effective attractants. Conversely, fructose which supports slime mold growth is at best a weak attractant. The results support the view that the chemotactic effects of different sugars are not dependent on their growth-supporting value.     

Mutants with decreased differentiation to plasmodia in Physarum polycephalum

Summary Mutant (APT) amoebae that display reduced ability to form plasmodia asexually were isolated by the use of an enrichment procedure. The results of reconstruction experiments show that the procedure enriches only for mutants blocked early in the pathway from amoeba to plasmodium. Mutants were isolated from four parents, two of which produce plasmodia asexually because they carry the allele mth of the mating type locus, and two because they carry gad (greater asexual differentiation) mutations. The APT mutants varied widely in the frequency of residual plasmodium formation, which occurred, in some cases, by reversion. The mutants, called apt (amoeba to plasmodium transition), were recessive in diploids and linked to the mating type (mt) locus. Mutants derived from the gad parents, unlike the parents themselves, crossed readily with heterothallic amoebae. Progeny analysis from such crosses indicates that both gad mutations are linked to mt. The mutants derived from one of the mth parents fell into two groups on the basis of their ability to cross with the mutants derived from the mt2 gad-8 parent. The result suggests that the mth-derived mutants represent two or more complementation groups. Mutants derived from the mt2 gad-8 parent cross with mt2 amoebae and hence display an altered mating specificity.     

Thymidine kinase-deficient mutants of Physarum polycephalum : Biochemical characterization

Thymidine kinase (TK) and deoxycytidine kinase (dCK) activity levels, [³H]thymidine (TdR) and 5-bromo-2′-deoxyuridine (BUdR) incorporation and 5-fluoro-2′-deoxyuridine (FUdR) sensitivity have been compared in TK-deficient (TU63 and TU84) and normal (TU291 and M₃b) strains of the myxomycete, Physarum polycephalum. The mutants had about 2% of the TK and 100% of the dCK activity of wild-type (wt) strains. They incorporated some TdR into both nuclear (nDNA) and mitochondrial DNA (mtDNA) but incorporated too little BUdR to give a buoyant density shift in nuclear DNA. They grew in the presence of levels of FUdR which completely blocked DNA synthesis in TU291. The FUdR sensitivity of strain M₃b could be increased by supplementing growth medium with folic acid.     

Mitotic Segregation of the Nucleolar Ribosomal RNA in Physarum polycephalum

In the naturally synchronous mitosis of the syncytial plasmodium of Physarum polycephalum , the nucleolus disintegrates in prophase, releasing a large amount of ribosomal RNA. Using biotinylated rDNA probes, we studied by high-resolution in situ hybridization the behavior of this nucleolar RNA throughout mitosis. Our results demonstrate that this rRNA is stable and maintained within the mitotic nucleus mainly, but not exclusively, associated with fibrillar nucleolar remnants. The distribution of these rRNA molecules on both sides of the cleavage plane in telophase is indicative of a precise mechanism of mitotic partition of the nucleolar components, supporting our recent findings concerning the rDNA minichromosomes (Puvion-Dutilleul and Pierron, 1992, Exp. Cell Res. 203, 354-364). Taking advantage of the stability of this RNA component in mitosis, we unambiguously demonstrate that the nucleolar remnants are the precursors of the prenucleolar bodies appearing in the newly divided nuclei which, by fusion, reconstitute a single nucleolus. Our data exemplify the persistence of the nucleolar rRNA in mitosis and demonstrate that in Physarum, following its disintegration, the nucleolus is segregated and inherited.     

Acceleration of mitosis induced by mitotic stimulators of Physarum polycephalum

Extracts of the myxomycete Physarum polycephalum exhibit an accelerating effect on nuclear division which fluctuates during the synchronous nuclear division cycle. Extracts from late G2 phase plasmodia can advance mitosis in recipient test plasmodia by up to 30% of the length of the control cycle. The advancing capacity of extracts is heat- and ammonium sulphate-precipitable, non-dialysable and destroyed by pronase, suggesting that the active substance is a protein. The advance of mitosis is in strong correlation with the applied dose of stimulatory material.     

Response of the dTMP-synthesizing enzymes to differentiation processes in Physarum polycephalum

Synthesis of deoxythymidylate (dTMP) is a rate-limiting step in DNA synthesis; there are two main enzymes which are responsible for dTMP production, thymidylate synthetase (ts) and thymidine kinase (tk). Both enzymes were studied during several differentiation processes of the myxomycete Physarum polycephalum. In all stages of proliferation (microplasmodia, macroplasmodia, germinating microsclerotia and germinating spores) tk is the dominant enzyme in terms of activity, whereas ts is the predominant enzyme in quiescent stages (microsclerotia, sporangia, respectively spores); this is expressed by calculating the tk/ts ratio. This ratio is greater than 1 during proliferation and much less than 1 during quiescence. Our results clearly show that ts is the basic enzyme for dTMP production during all differentiation stages, whereas tk, if required, is shut on and represents an additional potential for dTMP synthesis during rapid proliferation.     

Induction of polyploid nuclei in Physarum polycephalum by cycloheximide treatment in prophase

Macroplasmodia of the acellular slime mold Physarum polycephalum were treated with pulses of cycloheximide (10 micrograms/ml medium, for 3 h), initiated 10-20 min before metaphase in the synchronous nuclear division cycle. This treatment interfered with normal division of the nuclei, but permitted DNA synthesis in the next S phase. This interpretation is supported by measurements of the DNA content per nucleus in cycloheximide-treated cultures as compared to control cultures, which show that some nuclei after cycloheximide treatment are polyploid. By this method we can produce polyploid strains of Physarum, but the elevated nuclear DNA content is not stable, and after several months the strains have reverted to the normal diploid DNA content.     

Thymidine-dependent growth of Physarum polycephalum amoebae treated with methotrexate in axenic culture

Low levels of methotrexate block the growth of Physarum amoebae in axenic culture. The block is readily reversed by adding thymidine to the medium.