The Molecular Biology of Physarum polycephalum
During sporulation, the protoplasmic mass develops into multiple fruiting bodies.
Each sporangium contains hundreds of haploid n , mononucleate spores that have been formed through meiosis. Apogamic cycle: A haploid, mononucleate amoeba that carries a gadAh mutant allele may develop into a multinucleate, haploid plasmodium without mating. Upon sporulation, the low number of diploid nuclei that have been formed within the plasmodium gives rise to viable spores. Apogamic development can be suppressed experimentally by elevated temperature, which allows propagation of amoebal clones or the formation of a diploid plasmodium by mating of two amoebae of different mating type.
As mating of amoebal cells and plasmodium formation are controlled by different mating type genes, sophisticated approaches based on Mendelian genetics are possible Dee B Phylogenetic subtree of some Amoebozoa species with completed genomes. The tree is based on 30 highly conserved genes and rooted with other eukaryotes. The complete tree including representatives from plants, animals, and fungi is shown in supplementary fig S The maximum likelihood method with the JTT matrix was used.
Entamoeba histolytica genes were omitted for calculating the tree, because endoparasites are known to evolve at different rates as compared with free-living organisms.
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For details, see Supplemental Methods, Results , and Discussion. From an evolutionary biology point of view, P. The model organism most closely related to P. The two model species differ in several important aspects.
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A comparison of P. The established lab strains of D. The classical genetics are well-established in P. Amoebal strains derived from different natural plasmodial isolates, such as Wis-1 and Wis-2 see Appendix A of Supplemental Methods, Results , and Discussion , allow forward genetic approaches by mapping genes identified in phenotypic screens. Mendelian analysis based on the co-segregation of phenotype and single nucleotide polymorphisms Schedl and Dove can be performed by sequencing pools of cDNAs of phenotype-carrying segregants Barrantes and Marwan, unpublished results.
These high-throughput methods combined with Mendelian genetics mitigate current difficulties of reproducibly making transgenic lines. Methods for knocking down gene expression have recently been developed for P. Here, we present an overview of the whole genome of P. Combined sequence reads from different libraries including 3- and 8-kb mate pairs were assembled using the Newbler software version 2.
The The assembled coverage is We removed from the assembly all contaminating sequences, trimmed vectors X , and ambiguous bases N. The draft genome sequence of P. This initial analysis yielded , potential transcripts. After clustering with usearch Edgar , we obtained 31, clusters, each represented by the largest sequence in a cluster. The mean length of transcripts in the thus constructed reference transcriptome is 1, bases. These transcripts were mapped onto the genome using blat Kent Only transcripts could not be mapped to the genome sequence, of which only 90 have a blast score of or higher in a search against the reference sequence database NCBI July Fifteen of these 90 appear to be of P.
The Molecular Biology of Physarum Polycephalum - samsumonsheman.gq
Thus, only a minor fraction of transcripts is not represented by the assembled genome. Inability to assemble genome junctions at these positions is likely caused by the low complexity of intron sequences and the small size of the missing exons. As contiguity of a sequence is required for accurate gene prediction, we relied heavily on the transcript data for annotation and analysis.
For an ab initio gene prediction, we used Augustus Stanke et al. The transcriptome data were used to train this program according to the manual, and to provide splice information for the algorithm. The program itself was run with default values. This way we predicted 47, protein coding genes. We noticed, however, that the fragmented nature of the genome sequence and missing contiguity within scaffolds led to an overprediction. To get a better estimate of the number of protein coding genes in the P. To this end, we mapped the reference transcriptome data to the genome.
In this way, we defined 34, gene loci, of which 17, are not supported by transcript data table 1. As this specific experiment was performed on mitochondrial preparations, we only expect the more abundant nuclear small RNAs to be observed. However, the absence of poly-A selection and the use of a small RNA kit in library preparation promised a view orthogonal to traditional RNA-Seq approaches. For detailed information on strains used, preparation of nucleic acids, and for bioinformatic methods, see supplementary file 1.
The P. Construction of gene models therefore heavily relied on accompanying transcriptome data see Materials and Methods. The genetic material used for sequencing the P. However, assembling the genome has been challenging owing to extremely long stretches of di-, tri-, and tetranucleotide repeats and large homopolymeric tracts, which likely lead to polymerase slippage and premature polymerase termination in vitro.
The unresolvable sequence patches cause innumerable gaps in the final sequence, resulting in a mean scaffold size of only slightly larger than 2 kb. PacBio sequencing yielded only minimal improvement. We therefore tested whether our assembly captures most of the coding information of the genome by investigating the primary metabolism capacity encoded by the assembly. We found that all expected pathways were entirely present and, therefore, conclude that the current assembly is complete enough for further analysis. To further evaluate gene content and completeness, we generated transcriptome data sets from multiple life-cycle stages and constructed a reference transcriptome see Materials and Methods.
We next trained the gene prediction program Augustus Stanke et al.
Gene predictions using the splicing information from all transcripts initially yielded more than 47, gene models table 1. However, because in the current assembly some shorter introns and exons are missing in scaffolds owing to simple repeat sequences at both ends, we used the reference transcripts together with the gene predictions to define gene loci. Transcripts and predicted genes overlapping each other or being in close vicinity on the assembly in the range of an intron length were fused to a common gene locus.
A total of 34, gene loci were defined in this way table 1 , half of which are represented by transcripts 17, The gene loci with corresponding transcripts have a mean length of bases, whereas those without transcript support have a mean length of only bases. This suggests that the majority of unsupported gene loci may represent false-positive predictions, which is fairly typical of highly fragmented genome assemblies. There are scores of complex repeats in the genome, which contains a minimum of integrase domains PF and 1, reverse transcriptase domains PF and PF This greatly exceeds the number present in other Amoebozoa species such as A.
In total, P. Most P.
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The median size of the , introns confirmed by transcript data is bases, and the total number of predicted introns is , Thus, the mean number of confirmed introns per gene is approximately 5. If only the genes with transcript data are counted, the number of introns per gene increases to well above 9.
This intron frequency is higher than the estimated number of introns in the last common ancestor LCA of eukaryotes Zhou et al. Thus, P. Using iprscan Zdobnov and Apweiler , we defined the domain content for each gene locus.
Decomposition by Physarum polycephalum
A comparison with A. In terms of distinct domains, A. Further analysis of domains revealed that domains were present at least once in the genomes of P.
Among these are a considerable number of domains associated with signaling functions. Domains enriched compared with those of A.