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.     

Studies on microplasmodia of Physarum polycephalum

Summary Fluorescently labeled actin (TRITC-G-actin) and heavy meromyosin (TRITC-HMM) derived from skeletal muscle and injected into microplasmodia of the acellular slime mold Physarum polycephalum were used to analyze the function of a cortical and fibrillar actin system in living specimens. The plasma membrane-attached cortical system can be labeled with TRITC-G-actin as well as with TRITC-HMM and visualized as a continuous sheath along the entire cell surface. Long-term experiments over time periods of several hours in conjunction with digital grey-value evaluations revealed that changes in the intensity of the fluorescent signal, as caused by alternative contraction and relaxation cycles of the cortical system, are distinctly correlated with periodic changes in the volume and shuttle streaming activity of the microplasmodia. The fibrillar actin system extending through the cytoplasmic matrix can be labeled only with TRITC-HMM. Formation and disappearance of fibrils were found to take place during relaxation and contraction of the cortical system, respectively. Results of the present paper indicate that the cortical actin system is mainly involved in motive force generation for alterations in cell surface morphology and locomotion activity, whereas the fibrillar actin system rather appears to maintain the mechanical stability of microplasmodia.Abbreviations ATP adenosine-5'-triphosphate - BSA bovine serum 'albumin - DTE 1,4-dithioerythrit - EGTA ethyleneglycol-bis-(-amino-ethylether)-N,N,N,N,-tetraacetic acid - HMM heavy meromyosin - PIPES l,4-piperazine-N,N-bis-(2-ethanesulfonic acid) - Rh rhodamine - TRIS Tris-(hydroxylmethyl)-aminomethane - TRITC tetramethyl rhodamine isothiocyanate     

Membrane histochemistry of Physarum polycephalum myxamoebae

Myxamoebae of Physarum polycephalum are the uninucleate, haploid stage of the organism. Histochemical studies were undertaken to characterize intracellular and plasma membranes, and to provide a basis for assaying subcellular fractions for enrichment in plasma membranes. Lead salts deposition techniques were employed for hydrolytic enzymes. Alcian blue-ruthenium red, osmium tetroxide-potassium ferrocyanide, and phosphotungstic acid-chromic acid stains were evaluated for specificity for plasma membranes. Glucose 6-phosphatase was localized in endoplasmic reticulum, Golgi apparatus, and perinuclear space. 5'-Nucleotidase was localized in food vacuoles, chromatin, and plasmalemma. Acid phosphatase was in food vacuoles and Golgi apparatus. Alkaline phosphatase was in food vacuoles and endoplasmic reticulum. We conclude that none of the above enzymes is suitable as a cytochemical marker for plasma membranes of Physarum myxamoebae, but recommend instead staining ultrathin sections of membrane pellets with phosphotungstic acid-chromic acid, which stains plasma membranes selectively.     

Studies on microplasmodia of Physarum polycephalum

Summary Fragments excised from front regions of thinspread Physarum plasmodia were used to examine a possible correlation between the periodical dynamic activity of such specimens and the spatial organization of actin fibrils. Under isotonic conditions, symmetrical contractions and relaxations of the entire fragment alternate with a period of 1–4 min, whereas under isometric conditions local contractions and relaxations occur simultaneously in different regions of the same specimen. Rapid fixation and phalloidin-staining at distinct stages of the contraction-relaxation cycle demonstrates the permanent existence of cytoplasmic actin fibrils under both isometric and isotonic conditions. During the transition from relaxation to contraction the fibrils shorten in length from 25.5 m to 21.0 m and increase in density from 1.2 fibrils/1000 m² to 2.3 fibrils/1000 m². The present results demonstrate that actin fibrils in Physarum plasmodia are not completely decomposed and reformed every contraction-relaxation cycle.Series Studies on microplasmodia of Physarum polycephalum VIII     

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.     

Nuclear matrix proteins of Physarum polycephalum

The proteins of nuclear matrix preparations from Physarum polycephalum were compared with analogous mammalian fractions by gel electrophoresis, DNA-binding studies and immunological tests. Polypeptides of 28 and 36 K dalton, which dominate in Physarum preparations, differed from calf thymus matrix proteins in that they were basic and showed low affinity to DNA. These polypeptides were present at about 1.2 mg per mg of nuclear DNA. Polypeptides of higher molecular weight occurred in the preparation at about 0.5 mg per mg of nuclear DNA. At least some of the latter proteins showed high affinity to DNA and cross-reacted with the antiserum against calf thymus matrix proteins.     

Nucleoprotein chromatin subunit from Physarum polycephalum

The nucleoproteins resulting from digestion of the nuclei of the true slime mold Pysarum polycephalum with micrococcal nuclease have been resolved according to the size classes in linear sucrose gradients containg 0.5 M NaCl, and analysed for DNA, RNA and protein content. The basic nucleoprotein subunit has been found to contain a DNA fragment of about 150--170 base pairs complexed with an approximately equal amount, on a weight basis, of basic proteins and a relatively small amount of non-histone proteins (about 35% of the amount of DNA). Higher nucleoprotein oligomers were shown to contain spacer DNA fragments between adjacent subunits and a considerably higher ratio of non-histone proteins to DNA than the basic subunit. Both the basic subunit and higher nucleoprotein oligomers of Physarum chromatin contain some amount of tightly bound RNA. However, in contrast to the distribution of the non-histone proteins, the ratio of RNA to RNA is similar in both fractions.     

多头绒泡菌微原质团瞬时表达系统的构建

真核生物多头绒泡菌的原质团是研究细胞周期的好材料。但尚无合适的表达体系可供选择。本研究用多头绒泡菌ardC actin基因启动子和终止子分别替换哺乳动物细胞表达质粒pDsRed1-N1的CMVIE和SV40 polyA片段,构建了多头绒泡菌红色荧光蛋白(RFP)表达质粒pXM1;用PardC-MCS-DsRed1-TardC替换pTB38表达盒PardC-hph-TardC,构建了多头绒泡菌RFP表达质粒pXM2。将多头绒泡菌转录延伸因子类似蛋白(PELF1)基因与质粒pXM2重组,构建了PELF1红色荧光融合蛋白(PELF1-RFP)表达质粒pXM2-pelf1。通过荧光显微镜和激光扫描共聚焦显微镜观察RFP表达发现,电转参数为4kV/cm(电场)、1A(电流)、70μs(电击时间)时,质粒pXM1和pXM2电转多头绒泡菌微原质团(≤500μm)后24～48h内,RFP荧光最显著;而PELF1-RFP则主要聚集在多头绒泡菌细胞核,说明本试验建立的表达系统可以用于研究特定蛋白在多头绒泡菌内的瞬时表达。     

Multiple forms of actin in Physarum polycephalum

Actin in the acellular slime mold Physarum polycephalum consists of three major forms closely spaced at isoelectric point (IP) 4.7 and a minor form at IP 5.1. Amino acid analysis has shown the IP 5.1 actin to be nearly identical to the 4.7 actins. In actin purified from acetone powder, both actin forms were present. Both forms bound to DNase I and have the same molecular weight of about 43 000 on sodium dodecyl sulfate (SDS) polyacrylamide gels. On 2-D gels of nuclear proteins, both forms of actin were present. The IP 4.7 actins account for 8.6% of total plasmodial protein, and the IP 5.1 form for about 0.7%. In the nucleus the IP 4.7 actins comprise 2.7% of total nuclear protein, and the 5.1 actin about 0.4%. No cell cycle-associated change in the concentration of actins was observed in either total plasmodial extracts or in isolated nuclei. Pulse-labelling experiments have shown that in total plasmodia actin synthesis occurs throughout the cell cycle, with no relative changes in the rate of synthesis. In isolated nuclei labelled during mitosis and early S-phase, there is about twice as much labelled actin as in nuclei labelled prior to mitosis. This result may indicate an increase in the transport of actin into the nucleus.     

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.     

Activity of some enzymes in Physarum polycephalum

Summary The activity levels of seven enzymes were studied in growing plasmodia of Physarum polycephalum. The enzymes were isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, phosphodiesterase, -glucosidase, and histidase. Six of the enzymes showed a continuous increase in activity during the mitotic cycle; glutamate dehydrogenase exhibited a stepwise increase about 5 h after mitosis. Cycloheximide immediately inhibited the activity of all enzymes. Actinomycin D was ineffective in inhibiting enzyme activity until after one mitotic cycle had been completed; this indicates that mRNA was stable for all of these enzymes during the G2 period. Attempts to induce enzyme activity were unsuccessful.

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Zusammenfassung Der Aktivitätsverlauf von sieben Enzymen wurde in wachsenden Plasmodien von Physarum polycephalum untersucht. Es handelte sich um die Enzyme: Isocitrat-Dehydrogenase, Glucose-6-Phosphat-Dehydrogenase, Glutamat-Dehydrogenase, saure Phosphatase, Phosphodiesterase, -Glucosidase und Histidase. Sechs dieser Enzyme wiesen einen kontinuierlichen Aktivitätsanstieg während des Mitosecyclus auf; Glutamat-Dehydrogenase zeigte einen stufenförmigen Anstieg etwa 5 Std nach der Kernteilung. Cycloheximid hemmte sofort die Aktivität der Enzyme, während Actinomycin D erst nach Ablauf eines halben Teilungscyclus inhibierend wirkte. Dies deutet auf eine relativ stabile mRNA hin. Versuche, die Aktivität zu induzieren, schlugen fehl.