Physarum polycephalum is a member of the class/superclass Myxogastridae (or myxomycetes) commonly referred to as plasmodial or true slime molds. Although historically classified as fungi, molecular data now clearly show that they are most closely related to the cellular slime molds (Dictyostelidae). Together they form the supergroup Amoebozoa, which also includes amoebae with lobose (broad) pseudopodia (e.g. Acanthamoeba), archamoebae (e.g. Entamoeba), and pelobionts (flagellated amoebae without mitochondria). The vegetative stage (macroplasmodium) is a large, single cell containing multiple dipoid nuclei that divide precisely at the same time. Macroplasmodia migrate by protoplasmic streaming which reverses every 30-60 s. Plasmodia engulf bacteria, myxomycete amoebae and other microbes. They also secrete enzymes for digesting the engulfed material. Under adverse conditions, plasmodia can reversibly transformed into a dormant hardened mass (sclerotium) that can survive for long periods. Alternatively, starvation of the macroplasmodium in the presence of light induces differentiation into specialized sacs (sporangia), a highly regulated process, which includes the complete conversion of the macroplasmodium into "fruiting bodies", or fructifications. Haploid spores are produced inside the fructification by meiosis (chromosome reduction by 1/2 of that of the diploid cell). The germinated spore can transform into either an amoeba-like myxamoeba cell or a flagellated swarm cell (mxyoflagellate). Myxamoebae can reversibly transform into mxyoflagellates in liquid environments, or form cysts upon drying; cysts can transform into flagellates if the cell wall has not fully developed prior to re-exposure to liquids. Two myxamoeba cells of different mating types may join together in a cellular fusion (mating, plasmogamy) followed by nuclear fusion (karyogamy). This mixing of cellular contents (protoplasts) represents a very primitive form of sexual reproduction, providing a source of genetic variability. The fusion of two haploid cells results in a diploid zygote, which transforms into a developing plasmodium by nuclear division in the absence of cellular division (cytokinesis). In the laboratory, apogamic strains of Physarum have been derived in which haploid amoebae are able to grow into haploid plasmodia without forming a zygote. In these mutant strains, apogamic development is suppressed at temperatures above 30C, but formation of a diploid plasmodium through mating is still possible. The switch between haploid and diploid stages is an important tool for genetic analysis.
Slime molds are important heterotrophs (cannot synthesize their own food) in the decomposition of organic matter in temperate and tropical forests. Physarum and other acellular slime molds are composed of a syncytial mass of protoplasm (called a plasmodium) with no cell walls in their main vegetative state, although they can take on a variety of different microscopic and macroscopic forms (see Biology section below). Depending on the species, the plasmodium may range from only a few millimeters in diameter to well over 12 inches (30 cm) across. The plasmodium moves like a giant amoeba, flowing over the surface as it ingests dead leaves and wood. The bright yellow Physarum polycephalum commonly studied in general botany courses and research laboratories is typically found in damp, shady areas of temperate forests, although they may move to bright areas to "fruit." Some tropical slime molds are bioluminescent and glow in the dark.
The Physarum polycephalum genome has been targeted for a high quality draft and assembly by the NHGRI. An examination of available data available from the community and our preliminary results indicate that the ~300 Mb genome is about 40% repetitive, including about 100 families of 2000 members that are relics of transposition events. There are many repetitive elements in the intergenic regions, and there are homopolymer sequences in the introns of many genes. They are composed of poly A or poly C stretches of 10-25 nt on the same strand. From analysis of the genome survey sequence, it has been determined that there are two transoposon types, an 8.3 kb LTR-retrotransposon-like sequence (TP1) that has inserted into itself, generating 20-50-kb islands of repeated, hypermethylated DNA, and another LTR-retrotransposon-like sequence of 1.68 kb (TP2). These repeats may confound the assembly process, but we feel that an initial attempt at 6X coverage, along with EST data, will give us ample information on which to formulate alternatives, if necessary.
Gernot Gloeckner and Wofgang Marwan, Physarum community researchers, plan to produce a map with markers spaced every 100 kb. The map is to be produced in parallel to the sequencing effort, and is to be integrated with the sequence. In addition, the community has cDNA resources available that can be used for the sequencing effort. The community has already determined that the available Dictyostelium genome sequence will be of great use in annotating the Physarum sequence (Gerard Pierrion, personal communication).
This initial plan was developed in consultation with and using data input from members of the community as Jonatha Gott, Gerard Pierrion, Gernot Gloeckner, Wofgang Marwan and others. The goal is to provide resources that will be useful to the community, taking into consideration the anticipated difficulties of the genome and relative cost effectiveness of the project.
DNA is in house and was prepared from haploid amoebe provided by the laboratory of Gerard Pierrion. We propose to perform a 6X WGS with plasmid (5.7X) and fosmid end sequences (0.3X). We also propose to perform cDNA sequencing on the 454 FLX Sequencer, obtaining as much full length sequence as possible, and to use community cDNA library resources for EST production. The whole genome data will be assembled, and we will evaluate the genome, perhaps returning at a later date with additional requests, armed with additional data. If it appears that a standard 6X WGS will not assemble well, options might include:
The National Human Genome Research Institute (NHGRI), National Institutes of Health (NIH) have provided funding for the sequence characterization of the Physarum polycephalum genome.