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Centromere plasmid: a new genetic tool for the study of Plasmodium falciparum.

Iwanaga S, Kato T, Kaneko I, Yuda M - PLoS ONE (2012)

Bottom Line: This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum.In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence.Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions.

View Article: PubMed Central - PubMed

Affiliation: Mie University, School of Medicine, Tsu, Japan. iwanaga@doc.medic.mie-u.ac.jp

ABSTRACT
The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.

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Sequence Properties of the Centromere from Chromosome 5 of P. falciparum and the Plasmid Map of pFCEN.(A) The sequence analysis of the centromere of chromosome 5 of P. falciparum was performed using Artemis 11 with regard to its length and A/T content. The line at the bottom indicates the centromere, and its length and A/T content are 2170 bp and 97.5%, respectively. The genomic sequence data of chromosome 5 were obtained from PlasmoDB (http://plasmodb.org/). (B) The repetitive region in the centromere of chromosome 5 was identified by the dot matrix analysis using the Dotlet program. The inset box indicates the repetitive region in this centromere. (C) The centromere of chromosome 5 is schematically shown and named pfcen5 in this Figure. The line on the top indicates the repetitive and non-repetitive regions. The triangle indicates the 19 bp of the repeat sequence motif, and the number in parentheses is the number of repeats within the repetitive region. The schematic drawing of pfcen5-1.5 also is shown. The numbers at the bottom are based on the sequence number of pfcen5 and correspond to the beginning and the end of pfcen5-1.5. (D) The pFCEN plasmid is 8018 bp, and pfcen5-1.5 is placed downstream of the 3′ UTR of the dhfr-ts gene of P. berghei.
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pone-0033326-g002: Sequence Properties of the Centromere from Chromosome 5 of P. falciparum and the Plasmid Map of pFCEN.(A) The sequence analysis of the centromere of chromosome 5 of P. falciparum was performed using Artemis 11 with regard to its length and A/T content. The line at the bottom indicates the centromere, and its length and A/T content are 2170 bp and 97.5%, respectively. The genomic sequence data of chromosome 5 were obtained from PlasmoDB (http://plasmodb.org/). (B) The repetitive region in the centromere of chromosome 5 was identified by the dot matrix analysis using the Dotlet program. The inset box indicates the repetitive region in this centromere. (C) The centromere of chromosome 5 is schematically shown and named pfcen5 in this Figure. The line on the top indicates the repetitive and non-repetitive regions. The triangle indicates the 19 bp of the repeat sequence motif, and the number in parentheses is the number of repeats within the repetitive region. The schematic drawing of pfcen5-1.5 also is shown. The numbers at the bottom are based on the sequence number of pfcen5 and correspond to the beginning and the end of pfcen5-1.5. (D) The pFCEN plasmid is 8018 bp, and pfcen5-1.5 is placed downstream of the 3′ UTR of the dhfr-ts gene of P. berghei.

Mentions: Based on the sequence analyses of the centromeres of Plasmodium spp., we focused on the centromere from chromosome 5 of P. falciparum (Figure 2A and B) and then designed a set of primers to truncate the non-repetitive regions on both ends, as shown in Figure 2C. The amplified small centromere (1467 bp), pfcen5-1.5, consisted of the original repetitive region and a truncated non-repetitive region (Figure 2C); the repetitive region was placed at one end in pfcen5-1.5. The amplified pfcen5-1.5 sequence was cloned into a plasmid containing the human dihydrofolate reductase (hdhfr) gene and the green fluorescent protein (gfp) gene, which served as a drug-selectable marker and a fluorescence marker, respectively (Figure 2D). The transcription of the hdhfr and gfp genes was controlled under the dual-oriented promoter of elongation factor α of P. berghei. The resulting centromere plasmid harbouring pfcen5-1.5, named pFCEN, was stably maintained in E. coli cells, as expected. Furthermore, the sequence analysis of the recovered plasmid showed that there were no mutations or deletions over its entire sequence (data not shown).


Centromere plasmid: a new genetic tool for the study of Plasmodium falciparum.

Iwanaga S, Kato T, Kaneko I, Yuda M - PLoS ONE (2012)

Sequence Properties of the Centromere from Chromosome 5 of P. falciparum and the Plasmid Map of pFCEN.(A) The sequence analysis of the centromere of chromosome 5 of P. falciparum was performed using Artemis 11 with regard to its length and A/T content. The line at the bottom indicates the centromere, and its length and A/T content are 2170 bp and 97.5%, respectively. The genomic sequence data of chromosome 5 were obtained from PlasmoDB (http://plasmodb.org/). (B) The repetitive region in the centromere of chromosome 5 was identified by the dot matrix analysis using the Dotlet program. The inset box indicates the repetitive region in this centromere. (C) The centromere of chromosome 5 is schematically shown and named pfcen5 in this Figure. The line on the top indicates the repetitive and non-repetitive regions. The triangle indicates the 19 bp of the repeat sequence motif, and the number in parentheses is the number of repeats within the repetitive region. The schematic drawing of pfcen5-1.5 also is shown. The numbers at the bottom are based on the sequence number of pfcen5 and correspond to the beginning and the end of pfcen5-1.5. (D) The pFCEN plasmid is 8018 bp, and pfcen5-1.5 is placed downstream of the 3′ UTR of the dhfr-ts gene of P. berghei.
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pone-0033326-g002: Sequence Properties of the Centromere from Chromosome 5 of P. falciparum and the Plasmid Map of pFCEN.(A) The sequence analysis of the centromere of chromosome 5 of P. falciparum was performed using Artemis 11 with regard to its length and A/T content. The line at the bottom indicates the centromere, and its length and A/T content are 2170 bp and 97.5%, respectively. The genomic sequence data of chromosome 5 were obtained from PlasmoDB (http://plasmodb.org/). (B) The repetitive region in the centromere of chromosome 5 was identified by the dot matrix analysis using the Dotlet program. The inset box indicates the repetitive region in this centromere. (C) The centromere of chromosome 5 is schematically shown and named pfcen5 in this Figure. The line on the top indicates the repetitive and non-repetitive regions. The triangle indicates the 19 bp of the repeat sequence motif, and the number in parentheses is the number of repeats within the repetitive region. The schematic drawing of pfcen5-1.5 also is shown. The numbers at the bottom are based on the sequence number of pfcen5 and correspond to the beginning and the end of pfcen5-1.5. (D) The pFCEN plasmid is 8018 bp, and pfcen5-1.5 is placed downstream of the 3′ UTR of the dhfr-ts gene of P. berghei.
Mentions: Based on the sequence analyses of the centromeres of Plasmodium spp., we focused on the centromere from chromosome 5 of P. falciparum (Figure 2A and B) and then designed a set of primers to truncate the non-repetitive regions on both ends, as shown in Figure 2C. The amplified small centromere (1467 bp), pfcen5-1.5, consisted of the original repetitive region and a truncated non-repetitive region (Figure 2C); the repetitive region was placed at one end in pfcen5-1.5. The amplified pfcen5-1.5 sequence was cloned into a plasmid containing the human dihydrofolate reductase (hdhfr) gene and the green fluorescent protein (gfp) gene, which served as a drug-selectable marker and a fluorescence marker, respectively (Figure 2D). The transcription of the hdhfr and gfp genes was controlled under the dual-oriented promoter of elongation factor α of P. berghei. The resulting centromere plasmid harbouring pfcen5-1.5, named pFCEN, was stably maintained in E. coli cells, as expected. Furthermore, the sequence analysis of the recovered plasmid showed that there were no mutations or deletions over its entire sequence (data not shown).

Bottom Line: This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum.In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence.Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions.

View Article: PubMed Central - PubMed

Affiliation: Mie University, School of Medicine, Tsu, Japan. iwanaga@doc.medic.mie-u.ac.jp

ABSTRACT
The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.

Show MeSH
Related in: MedlinePlus