The genome of Plasmodium berghei



Genome and genome sequence

See for detailed information on the genome and genome sequence of P. berghei (and other rodent malaria parasites) the following papers:


Table: Features of the genomes of rodent malaria parasites (and compared with the human parasite P. falciparum) (from Otto et al. (2014).  BMC Biol. 2014 Oct 30;12:86).

Genome features P. berghei ANKA P. c. chabaudi AS P. y. yoelii (YM) P. falciparuma 
Nuclear genome    
Genome size (Mb) 18.5 18.8 21.9 23.3
G+C content (%) 22.1 23.6 21.1 19.4
Chromosomes 14 14 14 14
Synteny breaksb - 1 0 ND
Contigs 220 40 195 14
Sequence coverage 237x 109x 627x -
Genesc 4,979 5,139 5,675 5,419
Genes with functional annotationd 2,781 (56%) 2,927 (57%) 3,485 (61%) 3,234 (60%)
Mitochondrial genome    
 Genome size (bp) 5,957 5,949 6,512 5,967
 G+C content (%) 30.9 30.9 30.7 31.6
 Number of genes 3 3 3 3
Apicoplast genome    
 Genome size (bp) 30,302 29,468 29,736 29,430
 G+C content (%) 13.5 13.7 14.1 13.1
 Number of genes 30 30 30 30
aGenome version: 1.5.2013; Apicoplast genome from accession numbers: X95275, X95276; bcompared to the PbA genome; cin new versions, this includes pseudogenes and partial genes, but does not include non-coding RNA genes; dfigures include all genes except those annotated as `hypothetical', `conserved Plasmodium protein, unknown function', `conserved protein, unknown function', `conserved rodent malaria protein, unknown function' or `Plasmodium exported protein, unknown function'.



The first draft genome of a rodent malaria parasite (RMP) was published in 2002 for P. yoelii yoelii 17XNL. This was followed by publication of draft genomes of P. berghei ANKA (PbA) and P. chabaudi chabaudi AS (PcAS) in 2005. Comparisons with the genome of the human parasite P. falciparum and other primate malaria species defined a large set of core genes that are shared between RMPs and primate malarias. Although availability of draft RMP genomes made a significant impact in applying post-genomic technologies for understanding malaria biology and were used in many follow-up functional genomics studies to analyse gene regulation and function, these RMP genomes were highly fragmented and were annotated with little or no manual curation. The fragmented nature of the genomes has hampered genome wide analysis of gene regulation and function, especially of the (subtelomeric) multigene families. To utilise RMP models to their full potential, high quality reference genomes were produced in 2014: for PbA and PcAS large-scale improvement of their existing genomes, with re-sequencing, re-analysis and manual re-annotation, and for P. y. yoelii a genome sequence was produced de novo from the virulent YM line using the latest sequencing technologies and computational algorithms. In addition, comprehensive RNA-seq data was produced, derived from a number of life-cycle stages to both improve gene model prediction and to provide genome-wide, quantitative data on gene expression. By sequencing additional isolates/lines of P. berghei, P. yoelii and P. chabaudi (including the subspecies P. c. adami)  genotypic diversity was documented that exists within different RMP species. The availability of RMP reference genomes in combination with the RNA-seq and genotypic diversity data serve as excellent resources for gene-function and post-genomic analyses and, therefore, better interrogation of Plasmodium biology and development of anti-malaria interventions (from: Otto et al. (2014). A comprehensive evaluation of rodent malaria parasite genomes and gene expression. BMC Biol. 2014 Oct 30;12:86).


Genome size, base composition, mitochondrial and plastid DNA, DNA replication

Genome  size

The genome is organised into 14 chromosomes and has a total size of 18.5 Mb. 

Base composition

Like P. falciparum, the nuclear DNA of P. berghei has an high overall A+T content of about 80%. This (A+T)-rich bias is unevenly distributed between protein coding and non-coding regions. All open reading frames are relatively (G+C)-rich (25-30%), while the (A+T) composition of the vast majority of the intergenic regions and intragenic introns can rise to more than 90%.

Extra nuclear DNA: mitochondrial and plastid genome

P. berghei has two extra-nuclear DNA elements comparable to P. falciparum: the mitochondrial DNA and the plastid DNA (organelle genomes). All Plasmodium species analysed so far contain a ~6 kb tandemly repeated mitochondrial (mt) genome which codes for only three proteins (cytochrome b and two subunits of cytochrome oxidase) as well as two fragmented rRNA's. The circular plastid genome (apicoplast) is ~30 kb in size. The apicoplast is of similar secondary endosymbiotic origin to the chloroplast of green plants. Although the apicoplast is not photosynthetic, it nonetheless retains several plant-like metabolic pathways. A key process within the apicoplast is synthesis of the five-carbon isoprenoid precursor molecules. All isoprenoids are derived from these precursors and isoprenoid functions are required in all living cells. These molecules fulfill a variety of cellular roles, including participation in key processes such as N-glycosylation, electron transport (ubiquinone), and protein prenylation. Ioprenoid biosynthesis in apicomplexan parasites occurs via a metabolic pathway housed in the apicoplast, known as the methylerythritol phosphate (MEP) pathway. Because this organelle is cyanobacterial in origin, the MEP pathway is shared by the majority of eubacteria and other plastid-containing eukaryotes, such as plants and algae.

DNA synthesis and ploidy of life cycle stages

Merozoites, gametes and sporozoites are haploid. The only diploid stage is the 'young' zygote, just after fertilisation. The dividing stages, such as schizonts and oocysts are 'polyploid', because DNA replication and nuclear division is not immediately followed by cell division, resulting in a 'syncytial' cell with many nuclei. Only towards the end of schizogony/sporogony does the parasite start to divide its cytoplasm by budding of uninuclear haploid merozoites/sporozoites. The ookinete stage has a nucleus containing the 'tetraploid' amount of DNA resulting from fertilisation and meiosis without immediate nuclear division. Nuclear division only starts in the oocyst stage.
During asexual blood stage development DNA synthesis starts around 16 hour after invasion in the old trophozoite, just before the first nuclear division in the schizont. Throughout schizogony DNA replication and genome segregation are alternating events. The timing and rate of DNA synthesis in blood stages of P. berghei is comparable to that in P. falciparum blood stages, where DNA synthesis also starts in the old trophozoites and continues during schizogony. 

Replication of the (extra-nuclear) plastid genome of P. berghei occurs (just before and) during schizogony as has been found in P. falciparum.

During male gametogenesis, three rounds of genome replication take place within 10 minutes after activation of the male gametocytes. The content of resulting 'octoploid' nucleus is divided over the eight gametes, resulting in the haploid male gametes. It has been calculated that the entire haploid genome is replicated in, on average, 3.2 min. 

In the diploid zygotes of P. berghei, DNA synthesis (up to the tetraploid value) coincides with meiotic division. This suggests that this DNA synthesis represents the genome replication during the first meiotic division like in other eukaryotes.


Chromosomes, centromeres, telomeres, subtelomeric and core regions

Chromosomes

See Figs. 1-5 for pictures of the 14 chromosomes separated by pulsed field gel electrophoresis.
The chromosomes of Plasmodium species do not condense prior to mitosis like in most other eukaryotes. Due to the lack of condensation and the small size chromosomes cannot be visualised by light-microscopy or by standard electron microscopy. Pulsed field separation of chromosomes revealed that P. berghei has 14 chromosomes in the size range of 0.5-4 Mb (see figure 1-5). 

Table: The size of the 14 chromosomes of P. berghei (ANKA strain, clone 8417)

Chromosome Number 

Estimated size (Mb)Presence of 2.3 kb repeats1Remarks
13/143.8 yes 13 and 14 co-migrate in most lines; in several clones size differences exist which allows separation by pulsed field gel electrophoresis 
121.9 yes P. chabaudi clones exist in which chr. 9, 10 and 11 can be separated, allowing determination of chromosome location of genes 
9/10/111.8 yes 
81.6 no?  
71.5 yes Size is highly variable as a result of loss and acquisition of 2.3kb repeats. Chr. 7 may have the same size or can be smaller then chr. 5/6 (see Fig. 1)
61.3 yes Size is also variable (comparable to chr. 7); therefore not always separated from chr. 5 or 7 (see Fig. 1) 
51.2 no 
40.8 no 
30.75 no 
20.7 no 
10.65 yes 

1Presence of subtelomeric 2.3kb repeat units as shown by hybridisation experiments 

Figures 1 to 5: Images and descriptions of chromosomes, chromosome size variation, karyotypes and presence of 2.3 kb subtelomeric repeats. (Click to see larger images and descriptions in a separate window.)

chromosomes Plasmodium berghei
figure 1

chromosomes Plasmodium berghei
figure 2
chromosomes rodent malaria parasites
figure3
chromosomes rodent malaria parasites
figure 4
2.3kb subtelomeric repeats in chromosomes of P. berghei
figure 5

Centromeres and telomeres

Centromeres and telomeres have been (functionally) characterised in P. berghei. Telomeres with the repeat sequence of CCCTA(G)AA have been characterised and this sequence is similar in all Plasmodium species analysed so far. The total length of a telomere is about 1-1.2kb.

Subtelomeric regions

Like the subtelomeric regions of P. falciparum chromosomes, these regions of P. berghei chromosomes contain many (different), non-coding subtelomeric repeat sequences that are species specific. A widely distributed subtelomeric repeat  sequence in P. berghei is the 2.3 kb repeat unit that are directly joined to the telomeric repeats of several chromosomes (see table 1). These repeats and variation in copy number have been characterised in detail.

The genomes of RMP contain a number of multigene families located in the subtelomeric chromosomal regions. These include a large family of so-called `Plasmodium interspersed repeat genes' (pir), that are present also in other human/primate Plasmodium species, such as the human parasite P.vivax . Most of these gene families are expressed in blood stages and these proteins show features that have been reported to contribute to immune evasion through antigenic variation and may play a role in the sequestration of infected red blood cells and virulence. 

Table: Different (subtelomeric) multigene families in the genomes of rodent malaria parasite (RMP) 

 Gene family (new name)Other (previous) namesNumber of genes 
       
   PbA  PcAS  PyYM 
  CGPSG FG CG PSG FG CG 

PSG

FG 
pir pir, bir, cir, yir 100 88 12 194  3 4  583 40 172 
RMP-fam-aPb-fam-1;Pc-fam-1;fam-a;PYSTA 23 16 3 132  2 0 94 811
RMP-fam-bPb-fam-3;PYSTB 34 1 5  26 0 0 48 24
RMP-fam-cPYSTC 60 - 10 0 - 22  0-
RMP-fam-dPc-fam 1 0 - 17 4- 5  0-
Early transcribed membrane protein etramp 7 - - 13 - - 12 - -
Reticulocyte binding protein, putativeP235;235kDA protein rhoptry protein, putative  6 - 8 8 - 0 11 - 3
Lysophospholipase  4 1 - 28  0  - 11 1
RMP-erythrocyte membrane antigen (RMP-EMA1)pcema1 1 0 0 13 1 1 1 0 0
haloacid dehalogenase-like hydrolase, putative  1 - - 9 - -  1 - -
'Other subtelomeric genes'  46 - - 67 - - 46 - -

(from Otto et al. (2014).  BMC Biol. 2014 Oct 30;12:86). PbA = P. berghei ANKA, PcAS = P. c. chabaudi AS, PyYM = P. y. yoelii YM. CG: complete gene; FG: fragment; PSG: pseudogene

Core regions of chromosomes

In comparison with the highly variable subtelomeric regions of Plasmodium chromosomes, the central, core regions are much more stable and conserved between different rodent malaria parasite (RMP) species. A high level of conservation of gene linkage groups (synteny: gene location and order on chromosomes) exists between the four RMP species rodent parasites, emphasizing a low frequency of  large-scale rearrangements in the core regions of their chromosomes. Although the level of synteny of genes is lower when the genomes of rodent and human species of Plasmodium are compared, significant conservation of genome organization has been observed.

A comparison was made of all predicted RMP protein-coding genes with those of three primate malaria species, P. falciparum, P. knowlesi and P. vivax using OrthoMCL where the predicted RMP proteome was divided into three different categories: (1) RMP proteins with orthologs in any of the primate malarias; (2) RMP-specific proteins with no orthologs in primate malarias; and (3) primate malaria-specific proteins with no orthologs in any of the RMP. Between the predicted RMP proteomes (15,793 proteins in total) and primate malaria proteomes (15,853 proteins in total), approximately 87% of the RMP proteins had detectable orthologs in at least one of the primate malarias and only 2,104 proteins (13.3%) were predicted to be RMP-specific. Of those 2,104 proteins, 1,854 (88.1%) are from multigene families. For 2,306 primate malaria proteins (14.6%) no orthologs have been detected in the RMP. Of these primate malaria specific genes, approximately 1,635 (70.9%) are subtelomeric genes or members of subtelomeric gene families (from Otto et al. (2014).  BMC Biol. 2014 Oct 30;12:86).


Chromosome size variation,  DNA-rearrangements

(see figures 1 to 5)

Size differences between homologous chromosomes of up to 0.5Mb have been detected in parasites from different strains or clones of P. berghei . Size differences result from (large-scale) chromosomal rearrangements, mainly affecting the subtelomeric regions. At this moment no direct evidence exists for the presence of developmentally regulated or programmed DNA rearrangements in Plasmodium by which parasites increase antigenic variation or regulate gene transcription. Most size polymorphisms of chromosomes result from 'aberrant' chromosomal rearrangements. These DNA rearrangements occur frequently in the subtelomeric regions, while the internal parts of the chromosomes (core regions) appear to be more conserved. Since many multi-gene families that are associated with antigenic variation are located in the subtelomeric regions, these rearrangements may constitute a significant form of genetic variation. A number of chromosomal rearrangements leading to significant changes in the size of chromosomes have been characterised in P. berghei, including chromosome breakage and healing, loss and acquisition of subtelomeric repeats and chromosome translocation. The mechanisms underlying chromosome size polymorphisms in P. berghei are comparable to those of P. falciparum. In P. berghei chromosome size polymorphisms are most frequently the result of loss and acquisition of subtelomeric repeat sequences. In rodent parasites kept under laboratory conditions, large-scale rearrangements have even been shown to affect chromosome number. In response to drug pressure using the antifolate drug pyrimethamine, part of chromosome 7 containing the drug-sensitive dihydrofolate reductase-thymidylate synthase gene was duplicated to form a small 'mini-chromosome' of 400-500kb. This (partial) chromosome duplication, found both in P. chabaudi and P. berghei resulted in a population of parasites with 15 instead of 14 chromosomes.
Research on the mechanisms of chromosome size variation in P. berghei has provided evidence that recombination can occur between non-homologous chromosomes. These kinds of recombination events are significant, since they can result in genetic exchange, changing gene location and the clustering of genes.

Table: Chromosomal rearrangements shown to occur in P. berghei and P. falciparum

(large scale) rearrangementsP. berghei 
P. falciparum
Deletion of subtelomeric repeat sequencesyes
yes
Deletion of subtelomeric located genesyes
yes 
Increase in number of subtelomeric repeatsyes
 
Acquisition of subtelomeric repeats by recombination between non-homologous chromosomesyes
yes?
Chromosome translocationyesyes
Gene duplicationyesyes
Chromosome breakage followed by healing through addition of telomeric sequencesyesyes
Chromosome duplicationyesno
Programmed rearrangements resulting in changes in gene expressionno no

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