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.     

Expression of ras-family genes in the cell cycle and during differentiation of the lower eukaryote Physarum polycephalum.

Messenger RNA levels of three ras-family genes (Ppras1, Ppras2, and Pprap1) were measured in different life forms and throughout the cell cycle of the slime mold Physarum polycephalum. All three genes are expressed at constant rates in the uninucleate amoebae and flagellates, regardless of the culture conditions (solid or liquid medium, particulate or dissolved nutrients). In the multinucleate stages (micro- and macroplasmodia) Ppras1 and Pprap1 mRNAs are somewhat less abundant, while Ppras2 is not expressed at all. The early stages of the amoeba-plasmodium transition proceed without any drop in Ppras2 expression. During the synchronous cell cycle in macroplasmodia Ppras1 and Pprap1 are expressed at a constant level.     

Building a plasmodium: Development in the acellular slime mould Physarum polycephalum

The two vegetative cell types of the acellular slime mould Physarum polycephalum - amoebae and plasmodia - differ greatly in cellular organisation and behaviour as a result of differences in gene expression. The development of uninucleate amoebae into multinucleate, syncytial plasmodia is under the control of the mating-type locus matA, which is a complex, multi-functional locus. A key period during plasmodium development is the extended cell cycle, which occurs in the developing uninucleate cell. During this long cell cycle, many of the changes in cellular organisation that accompany development into the multinucleate stage are initiated including, for example, alterations in microtubule organisation. Genes have been identified that show cell-type specific expression in either amoebae or plasmodia and many of these genes alter their pattern of expression during the extended cell cycle. With the introduction of a DNA transformation system for P. polycephalum, it is now possible to investigate the functions of genes in the vegetative cell types and their roles in the cellular reorganisations accompanying development.     

A novel gene encoding a ras-like GTP-binding protein from Trypanosoma brucei: an evolutionary ancestor of the ras and rap genes of higher eukaryotes?

The ras superfamily of GTP binding proteins encompasses a wide range of family members, related by conserved amino-acid motifs, and act as molecular binary switches that play key roles in cellular processes. Gene duplication and divergence has been postulated as the mechanism by which such family members have evolved their specific functions. We have cloned and sequenced a ras-like gene, tbrlp, from the primitive eukaryote Trypanosoma brucei. The gene encodes a protein of 227 amino acids and contains the six conserved subdomains that designate it as a ras/rap subfamily member. However, the presence of key diagnostic residues characteristic of both the ras and rap families of GTP confuse the familial classification of this gene. Phylogenetic analysis of the GTP binding domain places its origins at the divergence point of the ras/rap families and suggests that tbrlp is an ancestral gene to the ras/rap genes of higher eukaryotes.     

Mating type and the differentiated state in Physarum polycephalum.

In the acellular slime mold, Physarum polycephalum, the differentiation of amoebae into plasmodia is controlled by a mating type locus, mt. Amoebae carrying heterothallic alleles usually do not differentiate within clones; plasmodia form when two amoebae carrying different alleles fuse and undergo karyogamy. In this paper, we show that amoebae heterozygous for heterothallic alleles can be isolated and maintained as amoebae; the amoebae form plasmodia in clones without a change in ploidy. Plasmodia were also found to be formed, infrequently, by heterothallic amoebae of a single mating type. The plasmodia are healthy and are also formed without a change in ploidy. Thus, the presence of two different heterothallic mating type genes in a single nucleus is compatible with the amoebal state and one heterothallic mating type gene is compatible with the plasmodial state, once established.     

Multiple Alleles and Other Factors Affecting Plasmodium Formation in the True Slime Mold Physarum polycephalum Schw*

SYNOPSIS. The life cycle of the true slime mold Physarum polycephalum includes 2 vegetative stages: the multinucleate coenocytic plasmodium and the uninucleate amoeba. A clone of amoebae established from a single spore does not normally yield plasmodia. Plasmodia are formed when amoebae from particular clones are mixed; thus plasmodium formation is said to be controlled by a ‘mating-type’ system. Previous work by the author with a sample of P. polycephalum derived from a single source revealed that 2 mating types were present and were determined by a pair of alleles at 1 locus. The present paper reveals the presence of 2 more mating types in a sample of P. polycephalum derived from a different source and provides evidence that these are determined by 2 alleles at the same locus as the other 2. Evidence for the presence of other inherited factors affecting plasmodium formation, the mode of action of these factors and possible explanations for the occurrence of plasmodia in single-spore cultures are also discussed.     

Identification of three genes expressed primarily during development in Physarum polycephalum

During the life cycle of Physarum polycephalum, uninucleate amoebae develop into multinucleate syncytial plasmodia. These two cell types differ greatly in cellular organisation, behaviour and gene expression. Classical genetic analysis has identified the mating-type gene, matA, as the key gene controlling the initiation of plasmodium development, but nothing is known about the molecular events controlled by matA. In order to identify genes involved in regulating plasmodium formation, we constructed a subtracted cDNA library from cells undergoing development. Three genes that have their highest levels of expression during plasmodium development were identified: redA, redB (regulated in development) and mynD (myosin). Both redA and redB are single-copy genes and are not members of gene families. Although redA has no significant sequence similarities to known genes, redB has sequence similarity to invertebrate sarcoplasmic calcium-binding proteins. The mynD gene is closely related to type II myosin heavy-chain genes from many organisms and is one of a family of type II myosin genes in P. polycephalum. Our results indicate that many more red genes remain to be identified, some of which may play key roles in controlling plasmodium formation. Received: 21 June 1999 / Accepted: 17 August 1999     

A gene,imz, affecting the pH sensitivity of zygote formation inPhysarum polycephalum

Mating inPhysarum polycephalum involves the fusion of two haploid amoebae and the differentiation of the resulting diploid zygote into a multinucleate plasmodium. Mating proceeds optimally with amoebae growing on an agar medium at pH 5.0. At pH 6.2, the amoebae still grow normally, but mating is completely blocked. The barrier at pH 6.2 is not in the differentiation step, since preformed diploids readily convert to plasmodia at this pH. The barrier can be overcome by raising the ionic strength of the agar medium; the effect, moreover, is not ion-specific. We have discovered a genetic locus,imz (ionicmodulation of zygote formation), that affects the upper pH limit for mating; the respective limits associated with the two known alleles,imz-1 andimz-2, are pH 5.6 and pH 6.0 at low ionic strength. Animz-1×imz-2 mating displays the pH 6.0 limit;imz-2 is therefore “dominant”. We suggest that this new gene affects a cell component that is exposed to the exterior of the amoeba and is involved in the fusion step of mating.     

Isolation of Physarum polycephalum plasmodial mutants altered in sporulation by chemical mutagenesis of flagellates

Plasmodia are giant, multinucleate single cells which develop from mononucleate amoebae during the developmental cycle of Physarum polycephalum. In visible light, starving plasmodia lose their unlimited replicative potential and terminally differentiate into fruiting bodies (sporulation). Aiming at genetic dissection of the circuits controlling commitment and differentiation, we worked out a standardized procedure for the generation and screening of plasmodial mutants altered in sporulation by mutagenesis with ethylnitrosourea. To obtain a homogeneous population of cells of those strains which cannot grow axenically, we describe a protocol for preparing a suspension of flagellates to be used as starting material for mutagenesis. Flagellates can transform into plasmodia via the amoebal stage. Pilot phenotypic screening yielded plasmodial mutants altered in the photocontrol of sporulation or with disturbed developmental program. The existence of mutants with a disturbed developmental program indicates that the sequence and synchrony of morphogenetic steps of fruiting body formation can be uncoupled through mutation. Complementation testing by plasmodial fusion identified three complementation groups of non-sporulating mutants. The work described provides an experimental basis for performing mass screens for Physarum mutants altered in sporulation.     

Apogamic development of plasmodia in the myxomycetePhysarum polycephalum: a cinematographic analysis

Summary Strain CL ofPhysarum polycephalum forms multinucleate plasmodia within clones of uninucleate amoebae. The plasmodia have the same nuclear DNA content as the amoebae. Analysis of plasmodial development, using time-lapse cinematography, showed that binucleate cells were formed as a result of nuclear division in uninucleate cells. Binucleate cells developed into plasmodia by further nuclear divisions and cell fusions. No fusions involving uninucleate cells were observed. A temporary increase in cell and nuclear size occurred at the time of binucleate cell formation.     

A relaxed mutant with an altered ribosomal protein L11

Summary A replica plating method for isolating ts amoebal mutants of Physarum polycephalum has been devised. Temperature-sensitive mutations occur at a frequency after nitrosoguanidine mutagenesis of 10^-3 per survivor, are stable but are not usually expressed in the plasmodia formed from these amoebae in clones. Some of these mutants appear to be cell-cycle stage specific.     

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.     

Kin Discrimination in Dictyostelium Social Amoebae

Evolved cooperation is stable only when the benefactor is compensated, either directly or through its relatives. Social amoebae cooperate by forming a mobile multicellular body in which, about 20% of participants ultimately die to form a stalk. This benefits the remaining individuals that become hardy spores at the top of the stalk, together making up the fruiting body. In studied species with stalked migration, P. violaceum, D. purpureum, and D. giganteum, sorting based on clone identity occurs in laboratory mixes, maintaining high relatedness within the fruiting bodies. D. discoideum has unstalked migration, where cell fate is not fixed until the slug forms a fruiting body. Laboratory mixes show some degree of both spatial and genotype‐based sorting, yet most laboratory fruiting bodies remain chimeric. However, wild fruiting bodies are made up mostly of clonemates. A genetic mechanism for sorting is likely to be cell adhesion genes tgrB1 and tgrC1, which bind to each other. They are highly variable, as expected for a kin discrimination gene. It is a puzzle that these genes do not cause stronger discrimination between mixed wild clones, but laboratory conditions or strong sorting early in the social stage diminished by later slug fusion could be explanations.     

Biological Activity of the Alternative Promoters of the Dictyostelium discoideum Adenylyl Cyclase A Gene

Amoebae of the Dictyostelium discoideum species form multicellular fruiting bodies upon starvation. Cyclic adenosine monophosphate (cAMP) is used as intercellular signalling molecule in cell-aggregation, cell differentiation and morphogenesis. This molecule is synthesized by three adenylyl cyclases, one of which, ACA, is required for cell aggregation. The gene coding for ACA (acaA) is transcribed from three different promoters that are active at different developmental stages. Promoter 1 is active during cell-aggregation, promoters 2 and 3 are active in prespore and prestalk tip cells at subsequent developmental stages. The biological relevance of acaA expression from each of the promoters has been studied in this article. The acaA gene was expressed in acaA-mutant cells, that do not aggregate, under control of each of the three acaA promoters. acaA expression under promoter 1 control induced cell aggregation although subsequent development was delayed, very small fruiting bodies were formed and cell differentiation genes were expressed at very low levels. Promoter 2-driven acaA expression induced the formation of small aggregates and small fruiting bodies were formed at the same time as in wild-type strains and differentiation genes were also expressed at lower levels. Expression of acaA from promoter 3 induced aggregates and fruiting bodies formation and their size and the expression of differentiation genes were more similar to that of wild-type cells. Expression of acaA from promoters 1 and 2 in AX4 cells also produced smaller structures. In conclusion, the expression of acaA under control of the aggregation-specific Promoter 1 is able to induce cell aggregation in acaA-mutant strains. Expression from promoters 2 and 3 also recovered aggregation and development although promoter 3 induced a more complete recovery of fruiting body formation.     

Regulation of proteolytic enzymes in axenically grown myxamoebae of Physarum polycephalum

The cultivation of Physarum polycephalum amoebae in two media with different protein contents revealed a regulation of aminopeptidases and proteases depending on the albumin content of the medium: in growing amoebae and plasmodia the aminopeptidases have similar isoenzyme patterns and relative activities against nitroanilides. One alanine and four leucine aminopeptidase isoenzymes were found within the slightly acid pH range. During growth amoebae secrete—different from plasmodia—leucine aminopeptidase into the medium with low protein content. In an albumin-rich medium additional alanine aminopeptidase activity was found. Out of nine plasmodial proteases four were found in amoebae too. Only one band (pI 3.6) was present in the protein-poor medium. No protease activity could be detected in the proteinrich medium.Abbreviations BSA bovine serum albumin - SD semi-defined (medium with low protein content; Table 1) The investigation formed a part of the Ph.D. thesis of A. Haars, Göttingen, 1976     

Events in the amoebal-plasmodial transition ofPhysarum polycephalum studied by enrichment for committed cells

Summary Amoebae of strain CLof Physarum polycephalum undergo apogamic development to form multinucleate plasmodia. During the amoebalplasmodial transition, large uninucleate cells become irreversibly committed to plasmodium development. In developing cultures, amoebae lose the ability to flagellate before they become committed. Enriched suspensions of committed cells can be obtained by inducing asynchronous differentiating cultures to flagellate and passing the cells through a glass bead column. Committed cells can be cultured to form plasmodia on bacterial lawns or in axenic liquid medium but cannot be cultured on axenic agar medium. Uninucleate committed cells express tubulin isotypes characteristic of amoebae, but after culture in axenic liquid medium, the cells express plasmodial specific tubulin isotypes.Abbrevations SDM Semi-defined medium - DSDM Dilute semidefined medium - LIA Liver infusion agar - SBS Standard bacterial suspension - IEF Isoelectric focussing - SDS Sodium dodecyl sulphate - PAUF Precommitted amoebae unable to flagellate (for the explanation of these cells see text).     

Rap1 Overexpression Reveals That Activated RasD Induces Separable Defects duringDictyosteliumDevelopment

One of theDictyostelium rasgenes,rasD,is expressed preferentially in prestalk cells at the slug stage of development and overexpression of this gene containing a G12T activating mutation causes the formation of aberrant multitipped aggregates that are blocked from further development (Reymondet al.,1986,Nature,323, 340–343). The ability of theDictyostelium rap1gene to suppress this abnormal developmental phenotype was investigated. Therap1gene and G12V activated and G10V negative mutant forms of therap1gene were independently linked to therasDpromoter and each construct used to transform M1, aDictyosteliumcell line expressing RasD[G12T]. Transformants of M1 that expressed Rap1 or Rap1[G12V] protein still formed multitipped aggregates, but most tips were able to complete development and form fruiting bodies. Cell lines showing this modified phenotype were designated ME (multitipped escape). Therap1[G10V] construct did not modify the M1 phenotype. These data suggest that overexpression of RasD[G12T] has two effects, the formation of a multitipped aggregate and a block in subsequent differentiation and that the expression of Rap1 or Rap1[G12V] reverses only the latter. Differentiation of ME cells in low density monolayers showed the identical low level of stalk and spore cell formation seen for M1 cells under the same conditions. Thus the cell autonomous defect in monolayer differentiation induced in the M1 strain was not corrected in the ME strain. Cell type-specific gene expression during the development of M1 cells is dramatically altered: prestalk cell-specific gene expression is greatly enhanced, whereas prespore-specific gene expression is almost suppressed (Louiset al.,1997,Mol. Biol. Cell,8, 303–312). During the development of ME cells,ecmA mRNA levels were restored to those seen for Ax3, andtagB mRNA levels were also markedly reduced, although not to Ax3 levels.cotCexpression in ME cells was enhanced severalfold relative to M1, although levels were still lower than those observed during the development of Ax3. The low expression ofcar1mRNA during early development of the M1 strain remained low during the development of ME cells. These data are consistent with the idea that the expression of RasD[G12T] affects two independent and temporally separated events and that only the later defect is reversed byrap1.     

Biochemical characterization of cultivated Cordyceps bassiana mycelia and fruiting bodies by 1H nuclear magnetic resonance spectroscopy

In this study, nuclear magnetic resonance techniques coupled with multivariate data analysis were used for the metabolic profiling of mycelia and fruiting bodies of the entomopathogenic fungi, Cordyceps bassiana according to developmental stages. A direct extraction method using two deuterated solvents of D₂O and CDCl₃ was used to investigate the relative levels of identified metabolites in each extraction condition in the mycelium and fruiting body formation stages. There was a clear separation among mycelia and fruiting bodies with various developmental stages in partial least-squares discriminant analysis (PLS-DA) derived score plots. During the transition from mycelia to fruiting bodies, the major metabolic change observed was the conversion of glucose to mannitol, and beauvericin to phenylalanine and 1-hydroxyisovaleric acid. In the developmental stages of fruiting bodies studied, there was a clear separation between stage 3 and the other stages in PLS-DA derived score plots. Nineteen compounds including 13 amino acids, 2 nucleosides, 3 organic acids, and glucose showed the highest levels in stage 3 fruiting bodies. The flavonoid content in the fruiting bodies showed similar levels during stages 1, 2, and 3, whereas the level at stage 4 was significantly decreased compared to the other stages. Results suggest that the fruiting body of C. bassiana is richer in natural resources at stage 3 compared to the other fruiting body stages due to its high abundance of compounds including total flavonoids. The metabolome information acquired in this study can be useful criteria for the quality control of commercial use of C. bassiana.