Mutant/mutation

The mutants contain circular or linear 'Plasmodium artificial chromosomes' (C-PAC; L-PAC).

The C-PAC is a circular construct containing a Plasmodium centromere. The L-PAC is a linear construct containing a Plasmodium centromere and Plasmodium telomeric sequences

See the following papers for more information:

Iwanaga S, Khan SM, Kaneko I, Christodoulou Z, Newbold C, Yuda M, Janse CJ, Waters AP. Functional identification of the Plasmodium centromere and generation of a Plasmodium artificial chromosome. Cell Host Microbe. 2010 Mar 18;7(3):245-55.
PMID: 20227667

To generate a Plasmodium artificial chromosome (PAC), we had to first functionally identify and characterize the parasite's centromere. A putative centromere (pbcen5) was cloned from chromosome 5 of the rodent parasite P. berghei based on a Plasmodium gene-synteny map. Plasmids containing pbcen5 were stably maintained in parasites during a blood-stage infection with high segregation efficiency, without drug pressure. pbcen5-containing plasmids were also stably maintained during parasite meiosis and mitosis in the mosquito. A linear PAC (L-PAC) was generated by integrating pbcen5 and telomere into a plasmid. The L-PAC segregated with a high efficiency and was stably maintained throughout the parasite's life cycle, as either one or two copies. These results suggest that L-PAC behaves like a Plasmodium chromosome, which can be exploited as an experimental research tool.

Construction of Plasmids Containing PCEN (C-PAC)
The various (complete and partial) PCEN-containing constructs were cloned into the plasmid vector pbGFPcon, which contains a selectable marker cassette encoding the Toxoplasma gondii pyrimethamine-resistant dhfr-ts gene (Tgdhfr-ts) as well as a gfp expression cassette under the control of the P. berghei eef1αa promoter. pbGFPcon was digested with EcoRI, thereby removing its d-ssu-rrna sequence. Subsequently, the PCEN-containing DNA fragments were digested with EcoRI and cloned into the EcoRI-digested pbGFPcon.

Construction of the Plasmodium Artificial Chromosome (L-PAC)
The DNA fragment containing the two telomeric sequences, oriented head to head with an ∼500 bp spacer region, was digested with HindIII and then cloned into the HindIII-digested pbCEN5A/T plasmid. This resulted in a circular plasmid, termed C-PAC. When we digested the C-PAC with PmeI, removing the spacer region between the two telomeres, the remaining linearized fragment was designated L-PAC.

Iwanaga S, Kaneko I, Yuda M.A high-coverage artificial chromosome library for the genome-wide screening of drug-resistance genes in malaria parasites. Genome Res. 2012 May;22(5):985-92.
PMID: 22426943

Here, we report a robust method for identifying resistance genes from parasites by using a Plasmodium artificial chromosome (PAC). Large genomic DNA fragments (10-50 kb) from the drug-resistant rodent malaria parasite Plasmodium berghei were ligated into the PAC and directly introduced into the drug-sensitive (i.e., wild-type) parasite by electroporation, resulting in a PAC library that encompassed the whole genomic sequence of the parasite. Subsequently, the transformed parasites that acquired resistance were selected by screening with the drug, and the resistance gene in the PAC was successfully identified. Furthermore, the drug-resistance gene was identified from a PAC library that was made from the pyrimethamine-resistant parasite Plasmodium chabaudi, further demonstrating the utility of our method.