Cycloheximide resistance of Physarum polycephalum.

In the presence of cycloheximide, wild-type plasmodia of Physarum polycephalum exhibit an immediate decrease in deoxyribonucleic acid synthesis, a reduction in the incorporation of [3H]thymidine into thymidine triphosphate, and an increase in the level of thymidine triphosphate, as well as a decrease in protein synthesis. In this study, we have utilized a cycloheximide-resistant (Cycr) amoebic strain selected from a population of cells mutagenized with nitrosoguanidine. Segregation data indicate that the resistance is due to a single mutation. We have used this Cycr mutant to construct Cycr plasmodial strains. Ribosomes isolated from such Cycr plasmodia showed resistance to cycloheximide in vitro, in contrast to ribosomes isolated from wild-type plasmodia. The Cycr plasmodia showed none of the cycloheximide-induced biochemical effects. Plasmodia heterozygous for the resistance marker were sensitive to cycloheximide with regard to growth but showed an intermediate response in the biochemical parameters. Heterokaryons formed by fusion of various proportions of the sensitive and resistant plasmodia showed a resistance with regard to both growth and biochemical parameters which was directly related to the fraction of Cycr plasmodia present in the heterokaryons. The data are consistent with the hypothesis that the effects of cycloheximide on deoxyribonucleic acid synthesis and nucleoside metabolism are secondary to the effect of the drug on protein synthesis in this organism.     

Polyadenylated RNA in the lower eukaryote Physarum polycephalum.

Utilizing a method which quantitatively extracts high molecular weight RNA, including intact precursor as well as mature ribosomal RNAs, adenylated molecules have been isolated from nuclear- and cytoplasmic-enriched fractions of Physarum microplasmodia labeled with [3H]-uridine. Electrophoretic analysis of denatured adenylated RNA from the nuclear-enriched fraction indicated the presence of a population of large molecules not found in the cytoplasmic-enriched fraction.     

Inheritance of extrachromosomal rDNA in Physarum polycephalum.

In the acellular slime mold Physarum polycephalum, the several hundred genes coding for rRNA are located on linear extrachromosomal DNA molecules of a discrete size, 60 kilobases. Each molecule contains two genes that are arranged in a palindromic fashion and separated by a central spacer region. We investigated how rDNA is inherited after meiosis. Two Physarum amoebal strains, each with an rDNA recognizable by its restriction endonuclease cleavage pattern, were mated, the resulting diploid plasmodium was induced to sporulate, and haploid progeny clones were isolated from the germinated spores. The type of rDNA in each was analyzed by blotting hybridization, with cloned rDNA sequences used as probes. This analysis showed that rDNA was inherited in an all-or-nothing fashion; that is, progeny clones contained one or the other parental rDNA type, but not both. However, the rDNA did not segregate in a simple Mendelian way; one rDNA type was inherited more frequently than the other. The same rDNA type was also in excess in the diploid plasmodium before meiosis, and the relative proportions of the two rDNAs changed after continued plasmodial growth. The proportion of the two rDNA types in the population of progeny clones reflected the proportion in the parent plasmodium before meoisis. The rDNAs in many of the progeny clones contained specific deletions of some of the inverted repeat sequences at the central palindromic symmetry axis. To explain the pattern of inheritance of Physarum rDNA, we postulate that a single copy of rDNA is inserted into each spore or is selectively replicated after meiosis.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.     

Methylation of nuclear DNA in Physarum polycephalum.

The restriction endonucleases HpaII and HhaI, whose action is inhibited by the presence of methylated base analogues at the recognition sequences in the DNA substrate, were used to investigate the distribution of 5-methylcytosine in nuclear DNA from Physarum polycephalum. Physarum DNA is digested into two fractions by these enzymes: a low-molecular-weight (M--) compartment comprising 80% of the DNA, and a high-molecular-weight (M+) compartment containing 20% of the DNA. The DNA fraction showing resistance to digestion by restriction endonuclease HpaII is cleaved by its isoschizomer MspI, indicating that methylated endonuclease-HpaII-specific sites are present in M + DNA. Additional properties of sequences in the M+ compartment were investigated.     

ADP-ribosylation in isolated nuclei of Physarum polycephalum.

ADP-ribosylation of histones and non-histone nuclear proteins was studied in isolated nuclei during the naturally synchronous cell cycle of Physarum polycephalum. Aside from ADP-ribosyltransferase (ADPRT) itself, histones and high mobility group-like proteins are the main acceptors for ADP-ribose. The majority of these ADP-ribose residues is NH2OH-labile. ADP-ribosylation of the nuclear proteins is periodic during the cell cycle with maximum incorporation in early to mid G2-phase. In activity gels two enzyme forms with Mr of 115,000 and 75,000 can be identified. Both enzyme forms are present at a constant ratio of 3:1 during the cell cycle. The higher molecular mass form cannot be converted in vitro to the low molecular mass form, excluding an artificial degradation during isolation of nuclei. The ADPRT forms were purified and separated by h.p.l.c. The low molecular mass form is inhibited by different ADPRT inhibitors to a stronger extent and is the main acceptor for auto-ADP-ribosylation. The high molecular mass form is only moderately auto-ADP-ribosylated.     

A calcium-sensitive preparation from Physarum polycephalum.

Differential ultracentrifugation of an extract of the plasmodium of Physarum polycephalum yields a high-speed fraction which exhibits calcium-sensitive adenosine triphosphate activity at low ionic strength. The rate of inorganic phosphate production increased from 2- to 25-fold in different preparations when the calcium concentration was increased from about 10(-8) to 10(-5) M. Complement fixation using specific antibody to Physarum myosin showed the fraction to contain 3% myosin. By electron microscopy, actin-like microfilaments 50--150 nm long were present. Addition of pure rabbit F-actin or myosin to this fraction activated the ATPase measured in EGTA and so partially reversed the calcium sensitivity. If muscle myosin was added to the supernatant from which the fraction was centrifuged, a "hybrid complex" was obtained which included actin and additional protein from the plasmodium, and this hybrid was also calcium sensitive. Over 85% of the calcium-sensitive, magnesium-activated ATPase could be precipitated by sequential "hybrid" formation. The calcium sensitivity of the hybrid was maximal when formed at the lowest ratios of added myosin to Physarum proteins. It is concluded that the results do not allow a simple interpretation along the lines of either actin-linked or myosin-linked sensitivity. Evidence consistent with both a form of actin-linked and myosin-linked sensitivity is present in our results.     

Cytoplasmic DNA-binding phosphoproteins of Physarum polycephalum.

The cytoplasmic DNA-binding proteins of Physarum polycephalum were recovered by chromatography of cytosol extracts on sequential columns of native and denatured calf thymus DNA-cellulose. 5.4% of the total cytosol protein was bound to native DNA-cellulose, while 4.4% was bound to denatured DNA-cellulose. Stepwise salt gradient elution of the columns separated the DNA-binding proteins into 9 fractions which were analysed by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Several hundred discrete polypeptide bands were identified, with many more high molecular weight polypeptides (greater than 100 000 D) binding to native than to denatured DNA. Continuous in vivo labelling of microplasmodia in KH₂[³²P]O₄ and [³H]leucine was used to determine which of the DNA-binding proteins were phosphorylated, and to approximate their phosphorus content. About 30–40 phosphoproteins were resolved among the DNA-binding proteins. Most phosphoproteins contained less than 3 phosphates per polypeptide, but a small number of low molecular weight phosphoproteins (less than 50 000 D) contained from 5 to 10 phosphates per polypeptide. The majority of high molecular weight DNA-binding phosphoproteins bound to native DNA and were eluted with 0.25 M NaCl. As a group, the DNA-binding proteins were enriched in protein-bound phosphorus when compared with the cytosol proteins which did not bind to DNA. The phosphorus content of the cytoplasmic DNA-binding proteins was similar to that of the acidic nuclear proteins.     

Translational control and the cytoskeleton in Physarum polycephalum

Translationally active plasmodia of the syncytial slime mold Physarum polycephalum develop into translationally dormant sclerotia during starvation. Although functional mRNA and ribosomes exist in sclerotia, protein synthesis is suppressed at the level of initiation. To test the possibility that alterations in the cytoskeleton may limit protein synthesis, we have examined the distribution of polysomes and actin mRNA in the cytoskeletal (CSK) and soluble (SOL) fractions of Triton X-100-extracted plasmodia and sclerotia. Most of the polysomes and actin mRNA were located in the CSK of plasmodia, while most of the ribosomes and actin mRNA were located in the SOL of sclerotia. The results suggest that ribosomes and mRNA shift from the CSK to the SOL as protein synthesis is suppressed during starvation. Plasmodia and sclerotia can be induced to accumulate excess polysomes by treatment with low levels of the elongation inhibitor cycloheximide. Treatment of plasmodia with cycloheximide caused excess polysomes to accumulate in the SOL, suggesting that the CSK contains a limited capacity for binding translational components and that the association of polysomes with the cytoskeleton is not required for protein synthesis. Treatment of sclerotia with cycloheximide, however, caused polysomes and actin mRNA to accumulate in the CSK, suggesting that the sclerotial cytoskeleton, although depleted in ribosomes and mRNA, is capable of binding translational components. It is concluded that alterations in the sclerotial cytoskeleton are not involved in translational control.     

Identification of mRNA in the slime mold Physarum polycephalum.

Using a differential extraction procedure which had previously been shown to yield one nucleic acid fraction enriched in cytoplasmic RNA and another enriched in nuclear RNA, we have been able to isolate two polyadenylated RNA populations from microplasmodia of Physarum polycephalum. The poly(A)-containing RNA from the cytoplasmic-enriched fraction accounts for approximately 1.2% of the cytoplasmic nucleic acid, has a number-average nucleotide size of 1339+/- 39 nucleotides, and has been shown, in a protein-synthesizing system in vitro, to be capable of directing the synthesis of peptides which have also been shown to be synthesized in vivo by microplasmodia. The poly(A)-containing RNA from the nuclear-enriched fraction has a number-average nucleotide size of 1533 +/- 104 nucleotides and represents a mixture of cytoplasmic and nuclear adenylated RNA molecules. Based upon these observations, we have identified the polyadenylated RNA isolated from the fraction enriched in cytoplasmic nuclei acid as Physarum poly(A)-containing messenger RNA.     

Glycosidases from the culture medium of Physarum polycephalum.

Eight exo-glycosidase activities were detected in the axenic culture medium of the myxomycete, Physarum polycephalum. The secretion of each enzyme examined followed the growth curve and continued during the stationary phase after the cessation of growth. Two or more forms of each enzyme were detected after electrophoretic separation. The beta-N-acetyl-D-hexosaminidase activity was readily separated into its two electrophoretic forms, X and Y, which were purified 145- and 306-fold respectively. These beta-N-acetyl-D-hexosaminidases had several similar characteristics. Evidence is presented that the major electrophoretic form of alpha-D-galactosidase is heterogeneous. The possible functions of extracellular glycosidases in teir occurrence and properties.     

Control of chemotaxis in Physarum polycephalum

Plasmodia migrate towards those situations which increase the frequency of their alternations in streaming, and away from those which decrease the frequency. Therefore peristalsis-like waves in Physarum move in the direction opposite from the net movement of the organism. The mechanism is fundamentally related to other known types of chemotaxis.     

Mutagenesis and mutant selection in Physarum polycephalum

Summary Physarum polycephalum myxamoebae were exposed to ultraviolet irradiation and plated in the absence and presence of caffeine. Caffeine reduces the shoulder on the UV¹ dose-survival curve, thereby increasing the UV-sensitivity for survival. Caffeine alone is a moderate mutagen. Used in conjunction with UV a strong mutagenic action is observed. Active growth is required for both of these mutagenic actions.Populations of Physarum myxamoebae mutagenized with NMG or EMS could be enriched for two classes of mutants by incubating at high temperature (30°C) with 5-bromodeoxyuridine-substituted bacteria followed by irradiation with long wave UV light and recovery at low temperature (23°C). One class of mutants was obtained in high yields after repeated cycles of light inactivation. These are not heat sensitive. Rather they are defective in utilization of DNA precursors provided by the bacteria. The other mutant class, obtained in low yields after limited selection, are heat sensitive. Three independent mutants of this kind, all eaky, were obtained. Reconstruction experiments show that all are selectants.     

A calcium ion-dependent atp pyrophosphohydrolase in Physarum polycephalum.

An activity of ATP Pyrophosphohydrolase (EC 3.6.1.8) was found in the soluble fraction of the plasmodium of Physarum polycephalum. The products of the enzyme reaction were inorganic pyrophosphate and 5'-AMP in equimolar quantities. The enzyme had a pronounced requirement for Ca2+ with high specificity. Mg-2+ was not an essential cofactor but stimulated the enzyme activity about 2.5-fold of the control. The enzyme hydrolyzed ITP, GTP and beta,gamma-methylene ATP at a limited rate. Among inhibitors tested, 3 mM caffeine reduced the activity to about 75% of the control. The enzyme had a broad pH optimum around pH=7.0 and the Km for ATP was 2.0 mM. An Arrhenius plot showed a break at about 18 degrees C and the calculated activation energies were 6.7 and 11.4 kcal/mol above and below the transition temperature, respectively. Disc electrophoresis in dodecyl sulfate and gel filtration on Sephadex -g-200 gave apparent molecular weights of 56 000 and 240 000, respectively, suggesting that the native enzyme was built up from 4 polypeptide chains. The possible role of the enzyme in vivo was discussed.