Replicational organization of three weakly expressed loci in Physarum polycephalum

We previously mapped early-activated replication origins in the promoter regions of five abundantly transcribed genes in the slime mold Physarum polycephalum. This physical linkage between origins and genes is congruent with the preferential early replication of the active genes in mammalian cells. To determine how general this replicational organization is in the synchronous plasmodium of Physarum, we analyzed the replication of three weakly expressed genes. Bromodeoxyuridine (BrdUrd) density-shift and gene dosage experiments indicated that the redB (regulated in development) and redE genes replicate early, whereas redA replicates in mid-S phase. Bi-dimensional gel electrophoresis revealed that redA coincides with an origin that appears to be activated within a large temporal window in S phase so that the replication of the gene is not well defined temporally. The early replication of the redB and redE genes is due to the simultaneous activation of flanking origins at the onset of S phase. As a result, these two genes correspond to termination sites of DNA replication. Our data demonstrate that not all the Physarum promoters are preferred sites of initiation but, so far, all the expressed genes analyzed in detail either coincide with a replication origin or are embedded into a cluster of early firing replicons.     

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.     

Maternal inheritance of mitochondria: multipolarity, multiallelism and hierarchical transmission of mitochondrial DNA in the true slime mold Physarum polycephalum

Direct evidence of digestion of paternal mitochondrial DNA (mtDNA) has been found in the true slime mold Physarum polycephalum. This is the first report on the selective digestion of mtDNA inside the zygote, and is striking evidence for the mechanism of maternal inheritance of mitochondria. Moreover, two mitochondrial nuclease activities were detected in this organism as candidates for the nucleases responsible for selective digestion of mtDNA. In the true slime mold, there is an additional feature of the uniparental inheritance of mitochondria. Although mitochondria are believed to be inherited from the maternal lineage in nearly all eukaryotes, the mating types of the true slime mold P. polycephalum is not restricted to two: there are three mating loci—matA, matB, and matC—and these loci have 16, 15, and 3 alleles, respectively. Interestingly, the transmission patterns of mtDNA are determined by the matA locus, in a hierarchical fashion (matA hierarchy) as follows: matA7 > matA2 > matA11 > matA12 > matA15/matA16 > matA1 > matA6. The strain possessing the higher status of matA would be the mtDNA donor in crosses. Furthermore, we have found that some crosses showed biparental inheritance of mitochondria. This review describes the phenomenon of hierarchical transmission of mtDNA in true slime molds, and discusses the presumed molecular mechanism of maternal and biparental inheritance.     

Involvement of extracellular cAMP-specific phosphodiesterase in the control of motile activity of <Emphasis Type="Italic">Physarum polycephalum</Emphasis> plasmodium

Possible involvement of extracellular cAMP-specific phosphodiesterase in the control of cell motile behavior has been investigated in Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the autooscillatory mode of motility. It was found that the rate of the hydrolysis of 10 mM cAMP by a partially purified preparation of cAMP-specific phosphodiesterase secreted by the plasmodium in the course of migration decreases 20–30 times under the action of 1 mM dithiothreitol. In the presence of 1–5 mM of this strong reducing agent, the onset of the plasmodium spreading and the transition to the stage of migration were delayed in a concentration-dependent manner. In accordance with the morphological pattern of motile behavior, the duration of the maintenance of high frequency autooscillations, which normally precede the increase in the rate of the spreading and appear also in response to the application of attractants at spatially uniform concentrations, strongly increased by the action of dithiothreitol. The results obtained suggest that the autocrine production of cAMP and extracellular cAMP-specific phosphodiesterase is an important constituent of the mechanism controlling the motile behavior of the Physarum polycephalum plasmodium.     

A New Mating Compatibility Locus in PHYSARUM POLYCEPHALUM

The rate and extent of plasmodium formation were studied in mating tests involving pairs of largely isogenic amoebal strains compatible for mating-type (mt) alleles. A systematic variability was observed: plasmodia formed either rapidly and extensively or slowly and inefficiently. Plasmodium formation was found to be 10³- to 10⁴-fold more extensive in "rapid" crosses than in "slow" crosses. A genetic analysis revealed that the variability reflects the influence of a multiallelic compatibility locus that determines mating efficiency. This compatibility locus (designated matB), together with the original mating type locus, mt (in this work designated matA), constitute a tetrapolar mating specificity system in Physarum polycephalum.     

Morphogenesis and alpha-tubulin gene of plasmodium in Didymium megalosporum

The morphogenesis of plasmodium in Didymium megalosporum was observed for the first time by hanging drop culture and 3 % oat-agar culture under controlled conditions. The development of plasmodium was characteristic formation process of phaneroplasmodium. The mature plasmodium was white yellow or yellow green in color, and had an extending fan-like sheet at the front, followed by a network of veins. It could be easy to fuse into a bigger plasmodium during the formation and die at high temperature or starvation. In view of the important role of alpha-tubulin during the morphogenesis of plasmodium, we sequenced partial sequence of alpha-tubulin gene, a total length of 1,159 bp, in this plasmodium. It had an intron area from 177 to 235 bp, and the exon area had a similarity of 91 % relative to altA locus of alpha-tubulin gene in Physarum polycephalum, the translated amino acid sequence was identical (100 % match) between the two.     

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.     

Revertants of selfing (gad) mutants in Physarum polycephalum

The conversion of the uninucleate amoebal form of Physarum polycephalum to the multi-nucleate plasmodial form is under the control of a genetic region which contains matA (or mt), a determinant of mating specificity. The region is the site of most gad mutations, which give amoebae the ability to produce plasmodia in clones without mating (ie, to self). In the present study, nonselfing revertants were isolated from two matA2-derived gad mutants and two matA3-derived gad mutants. Some revertants were found to have regained exactly, or nearly, the same phenotype as the original matA2 or matA3 strain. Others expressed new mating types, having gained the ability to mate with strains of the parental matA type. The results are compatible with a model in which new mating types arise from forward mutations (gad) and back mutations (npf or no plasmodium formation) occurring successively in a single gene, matA.     

Novel Sexual-Cycle-Specific Gene Silencing in Aspergillus nidulans

We report a novel sexual-cycle-specific gene-silencing system in the genetic model Aspergillus nidulans. Duplication of the mating type matA^HMG gene in this haploid organism triggers Mat-induced silencing (MatIS) of both endogenous and transgenic matA genes, eliminates function of the encoded SRY structural ortholog, and results in formation of barren fruiting bodies. MatIS is spatiotemporally restricted to the prezygotic stage of the sexual cycle and does not interfere with vegetative growth, asexual reproduction, differentiation of early sexual tissues, or fruiting body development. MatIS is reversible upon deletion of the matA transgene. In contrast to other sex-specific silencing phenomena, MatIS silencing has nearly 100% efficiency and appears to be independent of homologous duplicated DNA segments. Remarkably, transgene-derived matA RNA might be sufficient to induce MatIS. A unique feature of MatIS is that RNA-mediated silencing is RNA interference/Argonaute-independent and is restricted to the nucleus having the duplicated gene. The silencing phenomenon is recessive and does not spread between nuclei within the common cytoplasm of a multinucleate heterokaryon. Gene silencing induced by matA gene duplication emerges as a specific feature associated with matA^HMG regulation during sexual development.     

A genetic map of the Nicotiana alata S locus that includes three pollen-expressed genes

The S locus of solanaceous plants includes separate genes that control the self-incompatibility phenotype of the pistil and of the pollen. The gene controlling the self-incompatibility phenotype of the pistil encodes an extracellular ribonuclease, the S-RNase. The gene(s) controlling the self-incompatibility phenotype of pollen (the pollen-S gene) has yet to be identified. As part of a long-term strategy to clone the pollen-S gene by chromosome walking, a detailed map of the region near the S locus of Nicotiana alata was generated using a total of 251 F₂ plants. The map spans an interval of approximately 2.6 cM and contains five markers as well as the S-RNase gene. Two markers were detected with heterologous probes that also detect sequences linked to the S locus of Solanum tuberosum and the homologous region of the Lycopersicon genome. Three markers were identified by differential display using N. alata pollen RNA as a template. One of these markers is a pollen-expressed sequence, 48A, which detects a polymorphic marker no more than 0.5 cM from the S locus. RNA blot analysis indicates that the 48A gene is expressed primarily during pollen development after the completion of meiosis and is therefore a candidate for the pollen-S gene. The utility of these markers and the possible involvement of 48A in the molecular mechanism of self- incompatibility are discussed. Received: 28 June 1999 / Accepted: 24 September 1999     

Evidence for an additional cattle DQB locus

 Studying the genetic polymorphism of the major histocompatibility complex class II genes in cattle, we identified a sequence (KUH1) which resembles those encoding class II β chains. The gene was shown to be transcribed in peripheral blood leukocytes. Sequence comparisons, Southern blot, and phylogenetic analyses indicate that (1) KUH1 represents a distinct DQB locus, which we propose to designate BoLA-DQB5, (2) DQB5 constitutes an ancient DQB locus which diverged from a common ancestor gene prior to the duplication resulting in DQB1 and DQB2, (3) DQB5 is associated with haplotypes which contain DQA5 and a duplicated DQ region. Received: 26 January 1999 / Revised: 20 April 1999     

Exon duplication from a fork head to a homeodomain protein

The evolution of complex organisms such as animals requires a large expansion of the number of genes controlling developmental events. In addition, it is thought that domains are shuffled between genes to further increase the complexity and generate new types of genes and functions. Working with the Caenorhabditis elegans homeobox gene ceh-43, the orthologue of fly Distal-less (Dll), we observed sequence similarity to the C. elegans gene fkh-1. Now, with the complete genomic sequence available, we examined this similarity in detail. The region of similarity is confined essentially to one exon in the carboxy terminus of the two genes. Based on the gene structure, we think that an exon of fkh-1 was duplicated to the carboxy terminus of ceh-43, where it was incorporated as the last exon. This duplication event seems to have happened recently since the similarity on the nucleotide level is higher than the sequence similarity between fkh-1 of C. elegans and C. briggsae. Potentially the duplication event was mediated via a short region of sequence similarity between the two open reading frames of the genes. This duplication event clear shows that a part of a gene can successfully be juxtaposed to another gene. These events may perhaps not be rare. Received: 28 March 1999 / Accepted: 8 June 1999     

Dynamic complexity inPhysarum polycephalum shuttle streaming

Summary The nature of the oscillator controlling shuttle streaming inPhysarum polycephalum is not well understood. To examine the possibility of complex behavior in shuttle streaming, the time between reversal of streaming direction was measured over several hours in an intact plasmodium to produce a time series. Time series data were then used to analyze shuttle streaming dynamics. Complexity in shuttle streaming is revealed by an inverse frequency (1/f) power spectrum where the amplitude of reversals is plotted against their frequency. The complex dynamics of shuttle streaming is also shown by a trajectory in phase space typical of a strange attractor. Finally, shuttle streaming time series data have a dominant Lyapunov exponent of approximately zero. Dynamic systems with a Lyapunov exponent of zero exist in a state at the edge of chaos. Systems at the edge exhibit self-organized criticality, which produces complex behavior in many physical and biological systems. We propose that complex dynamics inPhysarum shuttle streaming is an example of self-organized criticality in the cytoplasm. The complex behavior ofPhysarum is an emergent phenomenon that probably results from the interaction of actin filaments, myosin, ATP, and other components involved in cell motility.     

Allelic diversity at class II DRB1 and DQB loci of the pig MHC (SLA)

 The loci encoding the β chain of the pig major histocompatibility complex (MHC) class II antigens, SLA-DR and -DQ, have been known to exhibit a remarkable degree of allelic polymorphism. Here, to understand the generation of SLA class II polymorphism, 25 SLA-DRB1 and 24 SLA-DQB genes including newly identified 12 SLA-DRB1 and 7 SLA-DQB genes obtained from miniature pigs were analyzed based on the nucleotide and deduced amino acid sequences. Most of the allelic diversity was attributed to the variable sequences which encode a β₁ domain consisting of a β-pleated sheet followed by an α helix. In the β₁ domain coding region, there were four GC-rich sequences, which have been considered to involve the intra-exon sequence exchange also in other gene evolutions. The first and second GC-rich sequences were χ-like sequences, which have been shown to be a putative recombination signal, and were stably conserved among SLA-DRB1 and DQB genes. These χ-like sequences identified in SLA-DRB1 and SLA-DQB were found to encode the first turning point of the β-pleated sheet and the boundary between the β-pleated sheet and the α helix. Analysis of clustered sequence variation also suggested intra-exon gene conversions in which the χ-like sequences act as putative breakpoints. In addition to point mutations and selection mechanism, intra-exon gene conversions must be an important mechanism in the generation of allelic polymorphism at the SLA-DRB1 and SLA-DQB. Received: 3 December 1998 / Revised: 29 June 1999     

A carotenogenic gene cluster from Brevibacterium linens with novel lycopene cyclase genes involved in the synthesis of aromatic carotenoids

The carotenogenic (crt) gene cluster from Brevibacterium linens, a member of the commercially important group of coryneform bacteria, was cloned and identified. An expression library of B. linens genes was constructed and a fragment of the crt cluster was obtained by functional complementation of a colourless B. flavum mutant, screening transformed cells for production of a yellow pigment. Subsequent screening of a cosmid library resulted in the cloning of the wholecrt cluster from B. linens. All genes necessary for the synthesis of the aromatic carotenoid isorenieratene were identified on the basis of sequence homologies. In addition a novel type of lycopene cyclase was identified by complementation of a lycopene-accumulating B. flavum mutant. Two genes, named crtYc and crtYd, which code for polypeptides of 125 and 107 amino acids, respectively, are necessary to convert lycopene to β-carotene. The amino acid sequences of these polypeptides show no similarity to any of the known lycopene cyclases. This is the first example of a carotenoid biosynthetic conversion in which two different gene products are involved, probably forming a heterodimer. Received: 17 July 1999 / Accepted: 7 December 1999     

The cell adhesion-associated protein Git2 regulates morphogenetic movements during zebrafish embryonic development

Signaling through cell adhesion complexes plays a critical role in coordinating cytoskeletal remodeling necessary for efficient cell migration. During embryonic development, normal morphogenesis depends on a series of concerted cell movements; but the roles of cell adhesion signaling during these movements are poorly understood. The transparent zebrafish embryo provides an excellent system to study cell migration during development. Here, we have identified zebrafish git2a and git2b, two new members of the GIT family of genes that encode ArfGAP proteins associated with cell adhesions. Loss-of-function studies revealed an essential role for Git2a in zebrafish cell movements during gastrulation. Time-lapse microscopy analysis demonstrated that antisense depletion of Git2a greatly reduced or arrested cell migration towards the vegetal pole of the embryo. These defects were rescued by expression of chicken GIT2, indicating a specific and conserved role for Git2 in controlling embryonic cell movements. Git2a knockdown embryos showed defects in cell morphology that were associated with reduced cell contractility. We show that Git2a is required for phosphorylation of myosin light chain (MLC), which regulates myosin II-mediated cell contractility. Consistent with this, embryos treated with Blebbistatin-a small molecule inhibitor for myosin II activity-exhibited cell movement defects similar to git2a knockdown embryos. These observations provide in vivo evidence of a physiologic role for Git2a in regulating cell morphogenesis and directed cell migration via myosin II activation during zebrafish embryonic development.     

THE LABORATORY CULTIVATION OF PHYSARUM FLAVICOMUM

Gray , William D. (Ohio State U., Columbus.) The laboratory cultivation of Physarum flavicomum. Amer. Jour. Bot. 48(3): 242–243. 1961.—The laboratory cultivation of P. flavicomum on artificial medium is reported here for the first time. The plasmodium of this species can be easily cultivated on dry rolled oats in a moist chamber in the same manner as plasmodia of P. polycephalum, or on 3% rolled-oats agar. Plasmodia form sclerotia from which new cultures can later be started or they can be obtained by sowing spores on rolled oats agar. Like P. polycephalum, the plasmodia of P. flavicomum must be illuminated with visible light in order to induce the onset of the sporulating stage. Sporangia which develop in culture vary most noticeably with respect to the type of peridium. The nature of the peridium is largely determined by whether the sporangium develops under moist or dry conditions; dry conditions favor the formation of a lime-encrusted peridium, while humid conditions favor the development of a thin, limeless peridium which has a metallic luster.     

Electron microscopy of the slime moldPhysarum polycephalum

Summary Electron micrographs of fixed sections of the plasmodium of the classic protoplasmic material,Physarum polycephalum, are presented. Numerous round nuclei having well-defined membranes and containing one to three dense nucleoli were especially prominent in the plasmodium. In the surrounding cytoplasm, many irregular membrane-limited bodies were evident, some containing tiny rod-like elements and others with inner structures resembling mitochondria. In addition, there can be seen many small dense granules plus various vacuoles and other inclusions.     

The role of phosphoinositide-3-kinase in the control of shape and directional movement of the Physarum polycephalum plasmodium

The effects of wortmannin and LY294002, specific inhibitors of phosphoinositide-3-kinase, on the shape, locomotive behavior, and glucose chemotaxis were studied using the Physarum polycephalum plasmodium, a multinuclear amoeboid cell with the self-oscillatory mode of locomotive behavior. Both inhibitors were shown to cause a reduction in the plasmodium frontal edge and a decrease in the efficiency of mass transfer during migration. They also suppressed the chemotaxis towards glucose and eliminated the characteristic changes in self-oscillatory behavior normally observed in response to the treatment of the whole plasmodium with glucose. The manifestation of these effects depended on the inhibitor concentration, treatment duration, and size of plasmodium. The involvement of phosphoinositide-3-kinase in formation of the frontal edge and control of P. polycephalum plasmodium chemotaxis suggests that the correlation of polar shape and directional movement of amoeboid cells with the distribution of phosphoinositides in the plasma membrane has a universal nature.