The genome of P. berghei

• Genome and genome sequence
• Genome size, base composition, mitochondrial and plastid DNA, DNA replication
• Chromosomes, centromeres, telomeres, subtelomeric and core regions
• Chromosome size variation, DNA-rearrangements
• References

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Genome and genome sequence

Information will follow

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

Genome  size

By comparative and quantitative cytophotometric measurements of DNA of the haploid stages of P. berghei the genome size has been estimated at 2.5 x 107 bp (ref. #1). By comparison of the size of pulsed field gel electrophoretic separated chromosomes the genome size has been estimated at 2.3-2.4 x 107 bp (ref. #2). This genome size of P. berghei is comparable to the genome size of 22.8Mb of P. falciparum (ref. #56). The genome is organised into 14 chromosomes (see below).

Base composition

Like P. falciparum, the nuclear DNA of P. berghei has an extremely high overall A+T content of about 82% (ref. #3). 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 (organellar genomes). The circular plastid genome is 30.7 kb in size (35kb in P. falciparum) (ref. #4). Partial DNA sequence analysis revealed 69.9-95.5% homology to sequences of P. falciparum. Arrangement of the genes (rRNA, rpo-B and tRNA) on the P. berghei plastid genome is similar to that found in P. falciparum (ref. #4).
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 (ref. #5). The mt genome of P. berghei has not been sequenced completely. The mt DNA of the closely related rodent parasite P. yoelii shows a remarkably 90% conservation with the mt genome of P. falciparum, P. vivax and P. gallinaceum (ref. #6). Part of mt genome of P. berghei has been sequenced including the cytochrome b gene. Mutations in the cytochrome b gene of P. berghei were correlated with resistance to atovaquone (ref. #7).

DNA content; DNA synthesis; replication origins

Merozoites, gametes and sporozoites are haploid. The only diploid stage is the 'young' zygote, just after fertilisation (ref. #8,9). 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 (ref. #8) 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 (ref. #9). 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 (ref. #10). Replication of the (extra-nuclear) plastid genome of P. berghei occurs (just before and) during schizogony (ref. #11 ) as has been found in P. falciparum (ref. #12).
There is evidence that during gametocyte development of both P. falciparum and P. berghei limited synthesis of DNA occurs, up to 1.3-2 times the amount of haploid DNA (ref. #9). This has been demonstrated with cytophotometric methods (quantitative determination of amount of DNA after staining with DNA-specific fluorescent dyes) but has not yet been confirmed by independent methods for analysis of the nuclear DNA of gametocytes, own observations).
During male gametogenesis, three rounds of genome replication take place within 10 minutes after activation of the male gametocytes (ref. #8,9). 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. Assuming the rate of replication fork movement in Plasmodium to be equal to that in other eukaryotes (about 50 base pairs per second or 9.6 kb in 3.2 min), it follows from the estimation of the genome size of P. berghei of 25Mb that there must be at least 1300 (25000/(2x9.6)) origins of replication with a mean distance of at most 2x9.6kbp (ref. #9). Origins of replication have not been defined yet in Plasmodium at the structural or functional level.
In the diploid zygotes of P. berghei, DNA synthesis (up to the tetraploid value) coincides with meiotic division (ref. #8,13). This suggests that this DNA synthesis represents the genome replication during the first meiotic division like in other eukaryotes.
Different DNA polymerases have been partly characterised from P. berghei and polymerase-α is sensitive to the inhibitor aphidicolin, which is a specific inhibitor for eukaryotic polymerase-α. Aphidicolin has been used to specifically block DNA synthesis in blood stages and during male gametogenesis (ref. #9). Since aphidicolin is a reversible inhibitor, it has been used to synchronise asexual development in P. falciparum by blocking DNA synthesis (ref. #14)

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Chromosomes, centromeres, telomeres, subtelomeric and core regions

Chromosomes

See Figs. 1-5 for pictures of 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 0.61-3.8 Mb (ref. #2,14; see figure 1-5). The number and size range of the P. berghei chromosomes (see table 1) are comparable to P. falciparum, resulting in a comparable genome size of about 25Mbp (see above).

Table 1: The size of the 14 chromosomes of P. berghei (ANKA strain, clone 8417)
Chromosome Number	Estimated size (Mb)	Presence of 2.3 kb repeats1	Remarks
13/14	3.8	yes	13 and 14 co-migrate in most lines; in several clones size differences exist which allows separation by pulsed field gel electrophoresis
12	1.9	yes	P. chabaudi clones exist in which chr. 9, 10 and 11 can be separated, allowing determination of chromosome location of genes (ref. #14)
9/10/11	1.8	yes
8	1.6	no?
7	1.5	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)
6	1.3	yes	Size is also variable (comparable to chr. 7); therefore not always separated from chr. 5 or 7 (see Fig. 1)
5	1.2	no
4	0.8	no
3	0.75	no
2	0.7	no
1	0.65	yes
1: Presence of subtelomeric 2.3kb repeat units as shown by hybridisation experiments (ref. #2,15)

 

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.)
figure 1	figure 2	figure3
figure 4	figure 5

Centromeres, replication origins, telomeres

No functional and structural information is yet available about centromeres and replication origins in P. berghei. Telomeres with the repeat sequence of CCCTA(G)AA have been characterised in P. berghei (ref. #16) and this sequence is similar in all Plasmodium species analysed so far. The total length of a telomere is about 1-1.2kb (ref. #17,18).

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 (refs. #2,15).
Characterisation of the subtelomeric regions of chromosome 5 of P. berghei demonstrated a symmetrical organisation of the two subtelomeric regions of a single chromosome (ref. #20). Symmetry of these regions has also been observed in P. falciparum chromosomes.
As in P. falciparum, the subtelomeric regions of P. berghei chromosomes contain (rapidly evolving) multigene families. An example is the family of subtelomeric variant genes (pir genes; Plasmodium interspersed repeat), that is also present in the other rodent parasites and the human parasite P.vivax (refs #58-60). In the genome of P. berghei 180 copies of pir genes are present (ref. #61). The proteins encoded by certain members of the pir gene family have been localised to the surface of erthrocytes, suggesting a role in antigenic variation  and immune evasion. Homologues of the major subtelomeric multigene families associated with antigenic variation in P. falciparum, such as var, stevor and rifin, have not been found yet in P. berghei. However, several smaller gene-families in the subtelomeric regions are shared between rodent parasites and P. falciparum (refs. 57, 62-64). Comparable to P. yoelli,  the subtelomeric regions contain members of a gene family encoding for 235-kDA surface proteins of merozoites (ref. #21). In P. yoelii it has been shown that a unique member of this family is expressed in each merozoite in a form of programmed antigenic variation (ref. #22).

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 species (refs. #27, 32, 57, 59, 61).  A high level of conservation of gene linkage groups (synteny: gene location and order on chromosomes) exists between the four species of rodent parasites (ref. #14, 57), 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 (refs. #27, 57, 59).

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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 (ref. #2,23). Size differences result from (large-scale) chromosomal rearrangements, mainly affecting the subtelomeric rgions. 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 ( table 2). The mechanisms underlying chromosome size polymorphisms in P. berghei are comparable to those of P. falciparum (table 2). 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 (ref. #55) and P. berghei (ref. #26) 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 (ref. #24, 25). These kinds of recombination events are significant, since they can result in genetic exchange, changing gene location and the clustering of genes.

Table 3: Chromosomal rearrangements shown to occur in P. berghei and P. falciparum
(large scale) rearrangements	P. berghei	reference	P. falciparum
Deletion of subtelomeric repeat sequences	yes	2	yes
Deletion of subtelomeric located genes	yes	20	yes
Increase in number of subtelomeric repeats	yes	2, 24
Acquisition of subtelomeric repeats by recombination between non-homologous chromosomes	yes	24	yes?
Chromosome translocation	yes	25	yes
Gene duplication	yes	26	yes
Chromosome breakage followed by healing through addition of telomeric sequences	yes	25	yes
Chromosome duplication	yes	26	no
Programmed rearrangements resulting in changes in gene expression	no		no

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References

1. Cornelissen et al. (1984) Parasitology 88, 531-553
2. Ponzi et al. (1990) Mol. Biochem. Parasitol. 41, 73-82
3. McCutchan et al (1984) Science 225, 808-811
4. Yap et al. (1997) Gene 200, 91-98
5. Wilson and Williamson (1997) Microbiol. Mol. Biol. Rev. 61, 1-16
6. McIntosh et al. (1998). Mol. Biochem. Parasitol. 95, 69-80.
7. Syafruddin et al. (1999) Mol. Biochem. Parasitol. 104, 185-194
8. Janse et al. (1986). Trans. Roy. Soc.Trop. Med. Hyg. 80, 154-157
9. Janse et al. (1986) Mol. Biochem. Parasitol. 20, 173-182
10. Inselburg & Banyal (1984) Mol. Biochem. Parasitol. 10, 79-87
11. van Dijk et al. (1997) Mol. Biochem. Parasitol. 86, 155-162
12. Smeijsters et al. (1994) Mol. Biochem. Parasitol. 67, 115-124
13. Sinden et al. (1985) Parasitology 91, 227-244
14. Janse et al. (1994) Mol. Biochem. Parasitol. 68, 285-296
15. Dore et al. (1990) Mol. Cel. Biol. 10, 2423-2427
16. Ponzi et al. (1985) EMBO J 4, 2991-2995
17. Dore et al. (1994) Mol. Biol. Rep. 20, 27-33
18. Pace et al. (2000) Genome Research 10, 1414-1420
19. van Dijk et al (1996) Science 271, 662-665
20. van Lin et al. (1997) Mol. Biochem. Parasitol. 86, 111-115
21. Owen et al. (1999) Mol. Biochem. Parasitol. 99, 183-92
22. Preiser et al. (1999) Nature 398, 618-22
23. Janse et al. (1989) Exp. Parasitol. 68, 274-282
24. Pace et al. (1990) Mol. Cell. Biol. 10, 6759-6764
25. Janse et al. (1992) Nucleic Acids Research 20, 581-586
26. van Dijk et al. (1994) Mol. Biochem. Parasitol. 68, 167-171
27. Carlton et al. (1998) Mol. Biochem. Parasitol. 93, 285-294
28. Vinkenoog et al. (1998) Mol. Biochem. Parasitol. 94, 1-12
29. van Lin et al. (2001) Nucleic Acids Research 29, 2059-2068
30. Thompson et al. (2001) Mol. Biochem. Parasitol. 20, 3975-83
31. van Dijk et al (2001) Cell 104, 153-164
32. van Lin et al. (2000) Int. J. Parasitol. 30, 357-370
33. Birago et al. (1996) Mol. Biochem. Parasitol. 80, 193-202
34. Kappe and Adams (1996) Mol. Biochem. Parasitol. 78, 279-283
35. Jennings et al. (1998) Mol. Biochem. Parasitol. 93, 43-55
36. Kedzierski et al. (2000). Mol. Biochem. Parasitol. 105, 137-47
37. Kappe et al. (1998) Proc. Natl. Acad. Sci. USA 95, 1230-5
38. Sam-Yellowe et al. (1998) Exp. Parasitol. 89, 271-84
39. Rayener at al. (2000) Proc. Natl. Acad. Sci. USA 97, 9648-53
40. Taylor et al. (2001) Infect. Immun 69, 3635-45
41. Eichinger et al. (1986) Mol Cell Biol 6, 3965-72
42. Menard et al. (1997) Nature 385, 336-40
43. Robson et al (1997) Mol. Biochem. Parasitol. 84, 1-12
44. Sultan, et al. (1997) Cell 90, 511-22
45. Spaccapelo et al. (1997) Lancet 350, 335
46. Tsuboi et al. (1997) Mol. Biochem. Parasitol. 85, 131-4
47. Tsuboi et al. (1997) Infect. Immun 65, 2260-4
48. Siden-Kiamos et al. (2000) J. Cell Sci 113, 3419-26
49. Thomas et al. (2001) EMBO J (in press)
50. Paton et al (1993) Mol. Biochem. Parasitol. 59, 263-75
51. Yuda et al. (1999) J. Exp. Med. 189, 1947-52
52. Yuda et al. (1999) J. Exp. Med. 190, 1711-6
53. Dessens et al. (1999) EMBO J. 18, 6221-7
54. Yuda et al. (2001) Mol. Biochem. Parasitol. 116, 65-72
55. Cowman and Lew (1999) Mol. Biochem. Parasitol. 42, 21-9
56. Gardner M.J. et al (2002). Nature 419, 498-511
57. Kooij et al. (2004) Submitted for publication
58. Janssen et al. (2002) Proc R Soc Lond B Biol Sci. 269: 431-6;
59. Carlton J.M. et al. (2002). Nature 419: 512-9.
60. del Portillo H.A. et al. (2001). Nature 410, 839
61. Dale et al (2004) Submitted for publication
62. Fischer K. et al. (2003) Mol Microbiol. 48, 1209-23
63. Birago C et al. (2003) Mol. Biochem. Parasitol. 126 (209-18)
64. Sam-Yellowe T.Y (2004) Genome Res. 14, 1052-9

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