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[Immunochemical identification of kinesin in Physarum polycephalum].

Physarum polycephalum, a low eukaryote ameba provides an attractive system for studying contractile proteins. In this work, we have identified a kinesin-like protein in the plasmodium of Physarum polycephalum by western blotting, using monoclonal antibody against kinesin (bovine brain). The molecular weight of the polypeptide which immunologically cross-reacts with kinesin from bovine brain is about 137kd. It suggests that the 137kd polypeptide is the heavy chain of the kinesin in Physarum polycephalum.

Animals↗

Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum.

Physarum polycephalum, an acellular slime mould, serves as a model system to study cell-cycle-dependent events since nuclear division is naturally synchronous. This organism was shown to release isoxanthopterin which is structurally related to tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases and of nitric oxide synthases (NOSs) (EC 1.14.13.39). Here, we studied Physarum pteridine biosynthesis in more detail and found that high amounts of tetrahydrobiopterin are produced and NOS activity is expressed. Physarum pteridine biosynthesis is peculiar in as much as 7,8-dihydroneopterin aldolase (EC 4.1.2.25), an enzyme of folic acid biosynthesis usually not found in organisms producing tetrahydrobiopterin, is detected in parallel. NOS purified from Physarum depends on NADPH, tetrahydrobiopterin and flavins. Enzyme activity is independent of exogenous Ca2+ and is inhibited by arginine analogues. The purified enzyme (with a molecular mass of 130 kDa) contains tightly bound tetrahydrobiopterin and flavins. During the synchronous cell cycle of Physarum, pteridine biosynthesis increases during S-phase whereas NOS activity peaks during mitosis, drops at telophase and peaks again during early S-phase. Our results characterize Physarum pteridine biosynthesis and NOS and suggest a possible link between NOS activity and mitosis.

Amino Acid Oxidoreductases↗

Phosphorylation of ribosomal proteins during the cell cycle of Physarum polycephalum.

Physarum polycephalum has been used as a model system to study the phosphorylation of ribosomal proteins during the cell cycle. The results showed that the phosphate content of S3, the major ribosomal phosphoprotein in this organism, was constant during all phases of the cell cycle. No additional ribosomal phosphoproteins were observed. These results differ significantly from those reported earlier by Rupp, R.G., Humphrey, R.M. and Shaeffer, J.R. (Biochim. Biophys. Acta (1976) 418, 81-92) and suggest that the use of thymidine or hydroxyurea to synchronize cell population may affect the phosphorylation of ribosomal proteins. The results are discussed in relation to protein synthesis and cAMP level during the cell cycle.

Animals↗

Organisation of genes for ribosomal RNA in Physarum polycephalum.

Physarum polycephalum nucleolar satellite DNA has been analysed by restriction enzyme digests and hybridisation to ribosomal RNA. The nucleolar DNA is isolated as molecules of molecular weight 39 X 10(6). The positions of sites of endo R-EcoRI and endo R-HindIII digestion have been determined accurately in the whole molecule. The endo R-EcoRI sites and one endo R-HindIII site are within the26-S rRNA complementary sequence and the other endo R-HindIII site is within the 19-S rRNA complementary sequence. The sites are arranged symmetrically about the centre of the molecule as if it were a palindrome.

Cell Nucleolus↗

Structure and expression of an actin gene of Physarum polycephalum.

Physarum polycephalum (strain M3CVIII) contains four unlinked actin gene loci, each with two alleles (ardA1, ardA2, ardB1, ardB2, ardC1, ardC2, ardD1 and ardD2). The 4800 base HindIII fragment of the ardC2 allele was previously isolated as a recombinant phage lambda. We now report the structure of the actin gene sequences (C-actin gene). The gene, which contains four intervening sequences, codes for the principal actin isotype of plasmodia and it is expressed in both the haploid myxamoebal and diploid plasmodial phases of the life cycle. The C-actin isotype is closely related to actins of Dictyostelium, Acanthamoebae, Drosophila, sea urchin and mammalian cytoplasmic actin, and more distantly related to actins of yeast, Entamoebae and Tetrahymena. The ardC1 and ardC2 alleles differ by a 700(+/- 100) base-pair insertion/deletion in the vicinity of the 3' end of the transcribed region of the gene.

Actins↗

A method for separation of pigments from plasmodia of the true slime molds, Physarum polycephalum and Physarum nudum.

Plasmodia of the true slime molds Physarum polycephalum and Physarum nudum were grown on agar semidefined medium (J. W. Daniel and H. Baldwin (1964) in Methods in Cell Physiology (Precsott, D. M., Ed.), Vol. 1, pp. 9-44, Academic Press, New York.) and pigments were extracted from 8-day-old plasmodia with the same solvent mixture that was used later as a mobile phase in the HPLC analysis. The separation was carried out on a strong anion exchanger column in a methanol/borate buffer solution. Under these conditions eight pigments from P. polycephalum and eight from P. nudum were revealed. This method can be used for detailed studies of plasmodial pigments in these species.

Chromatography, High Pressure Liquid↗

Tubulin proteins and RNA during the myxamoeba-flagellate transformation of Physarum polycephalum.

Physarum myxamoebae can be reversibly induced to become flagellates. Physarum flagellates contain a new form of tubulin, alpha 3, that is not found in nonflagellated cells. Evidence is presented that suggests that alpha 3 tubulin arises through posttranslational modification of a preexisting alpha tubulin. Pulse-chase experiments showed that labeled alpha 3 tubulin could be detected when flagellates formed after a chase. RNA was isolated from myxamoebae at different times after induction of flagellum formation. When this RNA was translated in vitro, the resulting products contained no alpha 3 tubulin, also consistent with alpha 3 being made by posttranslational modification. Levels of alpha and beta tubulin RNA increased with the proportion of flagellates in the culture. These elevated tubulin RNA levels declined after the number of flagellates in the population achieved plateau values.

Actins↗

A novel 66-kDa stress protein, p66, associated with the process of cyst formation of Physarum polycephalum is a Physarum homologue of a yeast actin-interacting protein, AIP1.

When exposed to various stresses including heat shock, myxoamoebae, growing haploid cells of Physarum polycephalum, show marked morphological changes and consequently become disk-shaped microcysts. We have found that p66 is induced exclusively in the course of microcyst formation and has an actin-binding activity. In this study, we purified p66 to homogeneity and isolated a p66 cDNA. The deduced protein sequence contained 601 amino acids and showed 31% identity to a yeast actin-interacting protein, AIP1. Northern blot analysis revealed that the amount of p66 mRNA was significantly increased by heat shock in myxoamoebae but not in plasmodia. Thus, p66 seems to be a developmentally-expressed stress protein which regulates the rearrangement of actin organization during microcyst formation in P. polycephalum.

Actins↗

[Structure and histone composition of chromatin in Physarum polycephalum].

In Physarum polycephalum several degrees of organisation of deoxyribonucleoprotein fibres were found. The complexes of histones and the DNA duplex seem to "be packed" at first into a 100 A fibre and then into a 200 A fibre of DNP. In Ph. polycephalum the electrophoretic mobilities of histone fractions 4 and 6 are comparable to that of fractions f3/f2b and f2a1 of calf thymus, resp. Histone fractions 3 and 5 move a bit faster than fractions f1 and f2a2, resp. Thus, the myxomycete P. polycephalum is similar to higher eukaryotes as concerns the ultrastructure of chromatin and electrophoretic properties of histones.

Amino Acids↗

[Protein-bound mono (adenosine-diphosphate-ribose) levels during the cell cycle of the slime mold Physarum polycephalum].

In Physarum polycephalum two fractions of ADPR-protein conjugates could be differentiated on the basis of their susceptibility towards hydroxylamine. Quantitation during the cell cycle revealed independent synthesis of the two species, the NH2OH-resistant fraction being formed during S phase, while the NH2OH-sensitive conjugate increased sharply at the S/G2 boundary. These findings indicated that nuclear ADP-ribosylation reactions are more than one function.

Adenosine Diphosphate Sugars↗

Nucleotide sequence of a ribosomal RNA gene intron from slime mold Physarum polycephalum.

The Physarum polycephalum 26S ribosomal RNA gene contains two intervening sequences (introns). The DNA sequence of one of these introns was analyzed together with that of its flanking regions (exons). In addition, the nucleotide sequence of the corresponding region of the reverse transcript of 26S rRNA was determined, and from comparisons of both sequences the precise location and size of the intron were determined. Our findings, when compared with those for Tetrahymena and Chlamydomonas rRNA gene introns, led to the conclusion that certain characteristics exist near the ends of these introns. (i) An exon ends in T at the exon/intron junction and an intron ends in G at the intron/exon junction in all cases. (ii) For each intron, direct repeats several nucleotides long are present 5 to approximately 30 nucleotides upstream from both the exon/intron and intron/exon junctions.

Base Sequence↗

Histone H4 mRNA is stored as a small cytoplasmic RNP during the G2 phase in Physarum polycephalum.

In Physarum polycephalum the triggering of histone H4 gene transcription occurs in G2 phase. The rate of synthesis of histone H4 mRNA was measured by in vivo pulse-labeling experiments. We show that it begins to increase in mid-G2. During the second part of G2 it increases approximately 20 fold over its minimum value and reaches a maximum at the end of G2. After entry of the cells in S, histone H4 gene transcription rate begins to decrease and reaches a minimum value in early G2. The histone H4 mRNA which accumulates in G2 is not translated immediately into proteins but is stored in an inactive form until the beginning of the next S phase. Immediately after its transcription the H4 mRNA is transported to the cytoplasm where it is stored and stabilized as an inactive mRNP complex. This was shown by fractionation of cytoplasmic RNP in sucrose gradients and blot hybridization of subcellular fractions.

Cytoplasm↗

Developmentally regulated late mRNAs in the encystment of Physarum polycephalum plasmodia.

Physarum polycephalum plasmodia survive adverse conditions by transforming into encysted cells called spherules. In this work we analysed the developmentally regulated mRNAs from the late stages of spherulation. A cDNA library was constructed and four abundant mRNAs were identified. One of the mRNAs was present in trace amounts in early spherules, while the other three were found only in late spherules. A cDNA clone for one of the late spherulation specific mRNAs was sequenced. It codes for a 332-amino-acid protein that did not show significant similarities with any known protein. Since the mRNA for this protein accumulates during spherulation, the protein was called spherulin 4. This protein has many features of a plasma membrane protein; it contains a signal peptide and a long hydrophobic region, which could serve as a transmembrane anchor. Another interesting feature is the presence of seven consecutive glycine residues in the N-terminal region. This is even more remarkable since the protein is not rich in glycine.

Amino Acid Sequence↗

Variance of ploidy in mitochondrial nucleus during spherulation in Physarum polycephalum.

In Physarum polycephalum, microplasmodia differentiated into spherules when cultures were aged for 8-10 days. Respiration rates of the microplasmodia decreased rapidly with ageing to a 90% decrease in oxygen consumption over 9 days. We studied this phenomena by isolating and characterizing mitochondria from microplasmodia and spherules at different stages of spherulation. Oxygen uptake by the isolated mitochondria decreased with spherulation. Morphological and biochemical analyses showed that mitochondrial differentiation to inactive state was characterized by a decrease not only in dimension but also of content (DNA, RNA and protein). Diminutive mitochondria contained small particle-shaped mitochondrial nuclei. The DNA content, measured by microscopic fluorometry, was about 1.15 and 0.58 X 10(-10) g, which corresponded to about 16 and 8 genome copies, respectively (e.g., 32 genome copies per mitochondrion at mitochondrial G1). Restriction endonuclease analysis showed that the physical structure and methylation pattern of the mtDNA had not changed although the DNA content per mitochondrion had decreased remarkably with spherulation. This showed that changes in the ploidy level of the mitochondrial nucleus during spherulation were due to reduction in the number of whole mitochondrial genomes.

Cell Fractionation↗

Accessibility of the ribosomal genes to micrococcal nuclease in Physarum polycephalum.

In Physarum polycephalum most genes coding for ribosomal RNA are not integrated in chromosomes, but are located in many copies in the nucleolus as plasmid-like palindromic DNA molecules. To find out whether coding sequences of rDNA are organized in a chromatin-like structure similar to that of bulk chromatin, nuclei were treated with micrococcal nuclease and DNA fragments were isolated. From bulk chromatin multimers of a basic unit of 170-180 base pairs were obtained. Nuclease fragmented DNA hybridized with labelled 19-S + 26-S rRNA was found to give the same saturation value as did unfragmented control DNA. No preferential degradation of ribosomal genes to acid soluble products was observed. A more detailed analysis of the nuclease degradation products was carried out with fragments separated by preparative gel electrophoresis. DNA eluted from the gels was hybridized in solution with labelled 19-S + 26-S rRNA. The coding sequences of rRNA were found to be degraded to approximately nucleosome size slightly more quickly than was the DNA of bulk chromatin. However, the distribution of the rDNA fragments on the gels did not coincide with the distribution of the fragments derived from bulk chromatin nucleosomes and their oligomers. The amount of rDNA in the interband regions was about intermediate between that found in the two adjacent bands. These results lead to the conclusion that the ribosomal genes, most of which are presumably active during rapid growth, are protected by proteins, probably histones. However, the ribosomal genes are present in a structure differing in some way from that of bulk chromatin.

DNA↗

A developmental mutation (npfL1) resulting in cell death in Physarum polycephalum.

In Physarum, microscopic uninucleate amoebae develop into macroscopic multinucleate plasmodia. In the mutant strain, RA614, plasmodium development is blocked. RA614 carries a recessive mutation (npfL1) in a gene that functions in sexual as well as apogamic development. In npfL+ apogamic development, binucleate cells arise from uninucleate cells by mitosis without cytokinesis at the end of an extended cell cycle. In npfL1 cultures, apogamic development became abnormal at the end of the extended cell cycle. The cells developed a characteristic rounded, vacuolated appearance, nuclear fusion and vigorous cytoplasmic motion occurred, and the cells eventually died. Nuclei were not visible by phase-contrast microscopy in most of the abnormally developing cells, but fluorescence microscopy after DAPI staining revealed intensely staining, condensed nuclei without nucleoli. Studies of tubulin organization during npfL1 development indicated a high frequency of abnormal mitotic spindles and, in some interphase cells, abnormally thick microtubules. Some of these features were observed at low frequency in the parental npfL+ strain and may represent a pathway of cell death, resembling apoptosis, that may be triggered in more than one way. Nuclear fusion occurred during interphase and mitosis in npfL1 cells, and multipolar spindles were also observed. None of these features were observed in npfL+ cells, suggesting that a specific effect of the npfL1 mutation may be an incomplete alteration of nuclear structure from the amoebal to the plasmodial state.

Animals↗

Calcium regulation of the actin-myosin interaction of Physarum polycephalum.

Plasmodia of Physarum polycephalum show vigorous cytoplasmic streaming, the motive force of which is supported by the actin-myosin interaction. Calcium is not required for the interaction but inhibits it. This calcium inhibition, a regulatory mode first discovered in Physarum, is the overwhelming mode of regulation of cytoplasmic streaming of plant cells and lower eukaryotes, and it is diametrically opposite to calcium activation of the interaction found in muscle and nonmuscle cells of the animal kingdom. Myosin, myosin II in myosin superfamily, is the most important protein for Ca2+ action. Its essential light chain, called calcium-binding light chain, is the sole protein that binds Ca2+. Although phosphorylation and dephosphorylation of myosin modify its properties, regulation of physiological significance is shown to be Ca-binding to myosin. The actin-binding protein of Physarum amplifies calcium inhibition when Ca2+ binds to calmodulin and other calcium-binding proteins. This review also includes characterization of this and other calcium-binding proteins of Physarum.

Actins↗