Purification and characterization of a novel intracellular acid proteinase from the plasmodia of a true slime mold, Physarum polycephalum

An acid proteinase was purified to apparent homogeneity from the plasmodia of a slime mold, Physarum polycephalum, by a combination of detergent extraction, acid precipitation, and column chromatographies on DEAE-Sephadex, hydroxylapatite, CM-Sephadex, and Sephadex G-100. The enzyme was shown to be composed of two polypeptide chains (a 31-kDa heavy chain and a 23-kDa light chain) cross-linked by disulfide bond(s). The NH2-terminal amino acid sequence of the heavy chain was determined to be Ala-Gly-Val- Asp-Gly-Tyr-Ile-Val-Pro-Tyr-Val-Ile-Phe-Asp-Leu-Tyr-Gly-Ile-Pro-Tyr and that of the light chain to be Ala-Glu-Pro-Pro-Ile. The heavy chain contained carbohydrate moiety composed of mannose, glucosamine, fucose, and glucose. The enzyme was optimally active at pH 1.7 toward hemoglobin as a substrate. Among the proteinase inhibitors tested only diazoacetyl-D,L-norleucine methyl ester, a typical aspartic proteinase inhibitor, inhibited the acid proteinase in the presence of cupric ions. It was insensitive to the other typical aspartic proteinase inhibitors, pepstatin A and 1,2-epoxy-3-(p-nitrophenoxy)propane. The enzyme hydrolyzed Lys-Pro-Ile-Glu-Phe(4-NO2)-Arg-Leu at the Phe-Phe(4-NO2) bond, but could not hydrolyze another synthetic pepsin-substrate, N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. The enzyme showed a unique substrate specificity toward oxidized insulin B chain. The major cleavage sites were the bonds Gly8-Ser9, Leu11-Val12, Cya19-Gly20, and Phe24-Phe25, and the Gly8-Ser9 bond was most susceptible. These results indicate that the enzyme is a novel type of intracellular acid proteinase with a unique substrate specificity.     

Isolation, identification, and characterization of histones from plasmodia of the true slime mold Physarum polycephalum using extraction with guanidine hydrochloride

Histones from plasmodia of the true slime mold Physarum polycephalum have been prepared free of slime by an approach to histone isolation that uses extraction of nuclei with 40% guanidine hydrochloride and chromatography of the extract on Bio-Rex 70. This procedure followed by chromatography or electrophoresis has been used to obtain pure fractions of histones from Physarum microplasmodia. Physarum microplasmodia have five major histone fractions, and we show by amino acid analysis, apparent molecular weight on three gel systems containing sodium dodecyl sulfate, mobility on gels containing Triton X-100, and other characterizations that these fractions are analogous to mammalian histones H1, H2A, H2B, H3, and H4. Significant differences between Physarum and mammalian histones are noted, with histone H1 showing by far the greatest variation. Histones H1 and H4 from Physarum microplasmodia have similar, but not identical, products of partial chymotryptic digestion compared with those of calf thymus histones H1 and H4. Labeling experiments, in vivo, showed that histone H1 is the major phosphorylated histone and approximately 15 separate phosphopeptides are present in a tryptic digest of Physarum histone H1. The core histones from Physarum, histones H2A, H2B, H3, and H4, are rapidly acetylated; histone H4 shows five subfractions, analogous to the five subfractions of mammalian histone H4 (containing zero to four acetyllysine residues per molecule); histone H3 has a more complex pattern that we interpret as zero to four acetyllysine residues on each of two sequence variants of histone H3; histones H2A and H2B show less heterogeneity. Overall, the data show that Physarum microplasmodia have a set of histones that is closely analogous to mammalian histones.     

Unusual polyamines in slime molds Physarum polycephalum and Dictyostelium discoideum

We analyzed the cellular contents of not only major polyamines but also minor polyamines in slime molds Physarum polycephalum and Dictyostelium discoideum. The presence of putrescine and spermidine in either plasmodia or myxamoebae of these molds as major polyamines was confirmed. In addition to these polyamines, appreciable amounts of 1,3-diaminopropane were detected in P. polycephalum and D. discoideum. Cadaverine and sym-homospermidine were detected in P. polycephalum even when the slime mold was cultured in a chemically defined growth medium. Spermine was not detected when these molds were grown in synthetic media. Other "unusual" polyamines such as norspermidine, norspermine, thermospermine, aminopropylcadaverine, and canavalmine were not detected in either mold.     

Specific nuclear elimination in polyploid plasmodia of the slime mold Physarum polycephalum

In growing plasmodia of the myxomycete Physarum polycephalum (G2-phase), three distinct classes of nuclei with a relative DNA content of 1x, 2x, and 4x are observed in the presumed haploid strain CL. The 2x and 4x species comprise up to 35% and 5% of the nuclei. Quantitative cytofluorometric studies of nuclei isolated in either G2- or S-phase or after FUDR treatment (G1 arrest) show that the three nuclear populations undergo a synchronous mitotic cycle and that the relative DNA content of the nuclear fractions in G-2 phase reflects the 2c, 4c, and 8c state. The heterogeneity of the nuclear population does, however, seem to be restricted to the growth phase. During a starvation period of 4 days that always preceeds sporulation (and also meiosis), the 4c nuclear population is reduced to 7%, 8c nuclei are no longer detected. These results suggest that a mechanism exists in Physarum for the selective detection and elimination of polyploid nuclei.     

Cytoplasmic control of mitosis in true slime mold, Physarum polycephalum

To resolve the problem of whether mitosis is controlled by anuclear or cytoplasmic stimulus, plasmodia from various periodsof the mitotic cycle were fused with one another. Mitosis inthe fused plasmodiura occurred about midway between mitosesof the donors. Treatment with cyclohcximide during the G₂-period.delayed the next mitosis for a period equal to the time of treatment. (Received March 2, 1971; )     

A method for isolation of cytoplasmic RNA from a slime mold,Physarum polycephalum

A procedure for fast and simple preparation of cytoplasmic ribonucleic acid from Physarum polycephalum microplasmodia is described. Microplasmodia are homogenized in a high-magnesium-high-ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid buffer and nuclei are pelleted. The supernatant is extracted with sodium dodecyl sulfate-phenol-chloroform and crude RNA is precipitated. This is further purified by selective ethanol precipitation from 6 m guanidinum hydrochloride. This RNA preparation is suitable for direct use in hybridization studies.     

The life cycle of mitochondria in the true slime mould,Physarum polycephalum

The cytoplasmic aspects of mitochondrial biogenesis have been the focus of much recent attention and a review is presented here of studies on the life cycle of mitochom dria inPhysarum polycephalum. Such studies have focused predominantly on behavior of the mitochondrial genome throughout the mitochondrial life cycle and have been designed to reveal details about (1) the role of the DNA-membrane complex in the segregation of the mitoehondrial genome; (2) the regulation of mitochondrial activity associated with changes in ptoidy of the mitoehondrial nucleus; (3) the hierarchical pattern of transmission of the mitochondrial genome as it relates to the mating-type locus (matA) during the sexual development; and (4) the fusion of mitoehondria that is promoted by a mitochondrial plasmid. The results of such studies contribute significantly to efforts towards a better understanding not only of the mitochondrial life cycle inP. potycephalum but also of the biogenesis of mitoehondria and plastids in many other organisms. Recipient of the Botanical Society Award for Young Scientists, 1988.     

Identification and sequence analysis of a rap gene from the true slime mold Physarum polycephalum

A member of the ras gene superfamily, belonging to the rap family and designated Pprap1, was isolated from a cDNA library from the true slime mold Physarum polycephalum by plaque hybridization in combination with 5′-RACE. The assembled nucleotide sequence of Pprap1 (1062 bp) has an open reading frame coding for a protein of 188 amino acids of a calculated M_r of 21035. This protein exhibits: (i) a highly conserved GTP binding domain containing a putative effector domain, with the threonine-for-glutamine substitution characteristic of rap proteins, (ii) a hypervariable domain, and (iii) the CAAX motif. Analysis of the C-terminal amino acid sequence of Pprap1 shows that it presumably undergoes geranylgeranylation but is not palmitoylated; however, it contains a lysine-rich domain which might serve as the second membrane localization signal. Pprap1 exhibits significantly high amino acid homology within the GTP binding domain with its homologues: Ddrap1 from Dictyostelium discoideum (92%) and human Rap1A (83%), and relatively low homology (59%) with the Saccharomyces cerevisiae homologue, RSR1. It has also 59% and 61% homology with the P. polycephalum Ppras1 and Ppras2 proteins, respectively. This gene is the third member of the ras gene superfamily identified in P. polycephalum so far.     

A cytoplasmic inhibitor of DNA polymerase from the plasmodia of Physarum polycephalum.

A factor which inhibited DNA polymerase [EC 2.7.7.7] activity was isolated from the cytoplasm of plasmodia of true slime mold, Physarum polycephalum. This factor was purified by DEAE-Sephadex and CM-cellulose column chromatographies, heat treatment and gel filtration. This inhibitor was heat-stable, insensitive to trypsin [EC 3.4.21.4] and was not digested by RNase [EC 3.1.4.22] or DNase [EC 3.1.4.5]. The molecular weight was 16,000 as determined by gel filtration, and the isoelectric point was determined to be pH 10.1. In the presence of the inhibitor, Km for DNA in the DNA polymerizing reaction was markedly increased. The inhibitory effect was eliminated by addition of excess DNA, but the addition of excess enzyme or deoxyribonucleoside triphosphates had no effect on the inhibition.     

Identification of the telomeric sequence of the acellular slime molds Didymium iridis and Physarum polycephalum.

We have determined the telomeric DNA sequence of the acellular slime molds Didymium iridis and Physarum polycephalum. In both organisms the telomeres consist of tandem repeats of the hexamer 5'(TTAGGG)3'. This sequence was determined by cloning and sequencing the telomeric fragment of the linear extrachromosomal ribosomal DNA from Didymium, as well as direct end labeling and sequencing the rDNA from both organisms. Interestingly, this sequence is identical to the telomeric DNA sequence of the flagellated protozoan Trypanosoma brucei, and suggests that despite the diversity of telomeric sequences previously determined in lower eukaryotes, the necessity to create functional telomeres has led to constraints on these sequences.     

DNA synthesis in isolated nuclei from synchronized plasmodia of Physarum polycephalum

Nuclei were isolated from synchronized plasmodia of a true slime mold, Physarum polycephalum, in S-phase, and DNA synthesis in the nuclei was studied in vitro. The nuclei catalyzed DNA synthesis at the rate of 0.7 ng DNA/1.0 X 10(6) nuclei/30 min at 25 degrees C, which was 5 times higher than that catalyzed in G2-phase nuclei. The DNA synthesis required Mg2+, four kinds of deoxyribonucleoside 5'-triphosphates and ATP, suggesting that the mode of synthesis is a replicative-type, but not a repair-one. Sedimentation analysis of the DNA products revealed that the nuclei produced 2-4S DNA fragments mainly during a 30-sec pulse incubation, and 2-4S, 5-12S and longer fragments during a 15-min incubation. The pulse- and chase-labeling experiments showed that the 2-4S fragments shifted discontinuously to longer fragments. These results indicate that the nuclei catalyze the formation of 2-4S Okazaki fragments first and then their subsequent ligation. Eighty % and 96% of the DNA synthesis was inhibited by 200 micrograms/ml aphidicolin and 40 mM N-ethylmaleimide, respectively, but 80% of the activity was resistant to 100 microM 2',3'-dideoxythymidine 5'-triphosphate. These results suggest that the DNA synthesis is catalyzed by the alpha-type DNA polymerase of Physarum polycephalum.     

Isolation and partial characterization of haemagglutinins from plasmodia of Physarum polycephalum.

The soluble haemagglutinins produced by plasmodia of Physarum polycephalum were purified by chromatographic methods and resolved into haemagglutinins I and II. On SDS-PAGE, purified haemagglutinins I and II each gave a single band with an apparent molecular mass of 6 and 11 kDa, respectively. The results of gel-filtration chromatography suggested that both haemagglutinins were dimers of the respective subunits under non-denaturing conditions. Rabbit erythrocytes were preferentially agglutinated by both haemagglutinins. The human type A, B and O erythrocytes were agglutinated by haemagglutinin II to an equal degree but were not agglutinated by haemagglutinin I. Simple sugars failed to inhibit the activities of both haemagglutinins. The activities, however, were effectively inhibited by the addition of thyroglobulin. Other glycoproteins such as fetuin, orsomucoid and transferrin inhibited the activity of haemagglutinin I but not that of haemagglutinin II. These haemagglutinins were detected in a slime fraction obtained from the culture media of starved plasmodia, suggesting that they are released to the outside of the plasmalemma to become associated with the slime layer on the plasmodial surface.     

A DNA polymerase with unusual properties from the slime mold Physarum polycephalum

Two forms of a DNA polymerase have been purified from microplasmodia of Physarum polycephalum by poly(ethyleneimine) precipitation and chromatography on DEAE-Sephacel, phosphocellulose, heparin Sepharose, hydroxyapatite, DNA-agarose, blue-Sepharose. They were separated from DNA polymerase alpha on phosphocellulose and from each other on heparin-Sepharose. Form HS1 enzyme was 30-40% pure and form HS2 enzyme 60% with regard to protein contents of the preparations. Form HS2 enzyme was generated from form HS1 enzyme on prolonged standing of enzyme preparations. The DNA polymerases were obtained as complexes of a 60-kDa protein associated with either a 135-kDa (HS1) or a 110-kDa (HS2) DNA-polymerizing polypeptide in a 1:1 molar stoichiometry. The biochemical function of the 60-kDa protein remained unknown. The complexes tended to dissociate during gradient centrifugation and during partition chromatography as well as during polyacrylamide gradient gel electrophoresis under nondenaturing conditions at high dilutions of samples. Both forms existed in plasmodia extracts, their proportions depending on several factors including those which promoted proteolysis. The DNA polymerases resembled eucaryotic DNA polymerase beta by several criteria and were functionally indistinguishable from each other. It is suggested that lower eucaryotes contain repair DNA polymerases, which are similar to those of eubacteria on a molecular mass basis.     

Immunocytochemistry of the acellular slime mold Physarum polycephalum

Abstract. The polygonal arrangement of actomyosin fibrils in different stages of the acellular slime mold Physarum polycephalum is correlated with morphogenetic processes at the cell surface. Light and electron microscopic investigations on both endoplasmic drops and thin-spread small plasmodia demonstrate that the differentiation of a polygonal pattern depends on a transient deficiency of plasma membrane invaginations.
Glycerol-extracted specimens show condensation and drastic spatial changes in the organization of the polygonal net after addition of ATP, thus indicating contractile properties of this system. Observations with the polarizing microscope reveal rhythmic changes in fibrillar birefringence intensity corresponding to the protoplasmic streaming activity, i.e., birefringence increases during contraction and decreases during relaxation. Cell fusion experiments, local irradiation with blue light (450 nm), and chemical treatment by impeding the mitochondria1 function with DNP (2,4-di-nitrophenol) demonstrate morphological as well as physiological interdependences of the actomyosin system, the motive force generation, and the expression of a locomotor polarity in plasmodia of Physarum polycephalum.     

Environment-dependent morphology in plasmodium of true slime mold Physarum polycephalum and a network growth model

Branching network growth patterns, depending on environmental conditions, in plasmodium of true slime mold Physarum polycephalum were investigated. Surprisingly, the patterns resemble those in bacterial colonies even though the biological mechanisms differ greatly. Bacterial colonies are collectives of microorganisms in which individual organisms have motility and interact through nutritious and chemical fields. In contrast, the plasmodium is a giant amoeba-like multinucleated unicellular organism that forms a network of tubular structures through which protoplasm streams. The cell motility of the plasmodium is generated by oscillation phenomena observed in the partial bodies, which interact through the tubular structures. First, we analyze characteristics of the morphology quantitatively, then we abstract local rules governing the growing process to construct a simple network growth model. This model is independent of specific systems, in which only two rules are applied. Finally, we discuss the mechanism of commonly observed biological pattern formations through comparison with the system of bacterial colonies.     

DNA synthesis by the isolated nuclear matrix from synchronized plasmodia of Physarum polycephalum

Nuclear matrices were isolated from plasmodia of a true slime mold, Physarum polycephalum, and the DNA synthetic activity in vitro was examined. These matrices isolated in S-phase catalyzed DNA synthesis requiring Mg2+, deoxyribonucleoside 5'-triphosphates and ATP, without exogenous templates. The activity changed during S-phase with the rate of in vivo DNA replication. Product analysis by gel electrophoresis revealed that the matrices produced Okazaki fragments. These results suggest that DNA synthesis partially reflects in vivo DNA replication. DNA synthesis was sensitive to aphidicolin, heparin and N-ethylmaleimide, indicating involvement of the alpha-like DNA polymerase of Physarum. Exogenous addition of activated DNA stimulated DNA synthesis 4-10-fold and suggested that only some of the existing enzymes are involved in endogenous DNA synthesis. Matrices isolated in G2-phase were also associated with a similar DNA synthetic activity, but they did not produce Okazaki fragments in vitro. It is, therefore, concluded that nuclear matrices are associated with alpha-like DNA polymerase throughout the cell cycle, and that some of the enzymes participate in in vivo DNA replication in S-phase; thus, DNA replication is possibly controlled by this process. The relationship between DNA synthetic activities by the isolated nuclei and matrices was also discussed.