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Adaptive copy number evolution in malaria parasites.

Nair S, Miller B, Barends M, Jaidee A, Patel J, Mayxay M, Newton P, Nosten F, Ferdig MT, Anderson TJ - PLoS Genet. (2008)

Bottom Line: The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP.These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function.More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.

View Article: PubMed Central - PubMed

Affiliation: Southwest Foundation for Biomedical Research (SFBR), San Antonio, TX, USA.

ABSTRACT
Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2-11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.

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Correlation between gch1 CNP and expression for 6 parasite isolates.We measured gene expression and copy number at 4 different time points in the asexual cycle. Error bars indicate 95% CI for estimates of both CNP and expression. Details of the real-time PCR assay are shown in Table S2. Linear regression summary statistics (slope, r2, F, p-value) for the four time points were as follows: 0–6 hrs: 0.63, 0.71, 10.36, 0.032, 8–14 hrs: 0.649, 0.87, 25.89, 0.007, 24–30 hrs: 0.419, 0.621, 6.54, 0.063, 32–40 hrs: 0.46, 0.85, 22.01, 0.009).
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pgen-1000243-g007: Correlation between gch1 CNP and expression for 6 parasite isolates.We measured gene expression and copy number at 4 different time points in the asexual cycle. Error bars indicate 95% CI for estimates of both CNP and expression. Details of the real-time PCR assay are shown in Table S2. Linear regression summary statistics (slope, r2, F, p-value) for the four time points were as follows: 0–6 hrs: 0.63, 0.71, 10.36, 0.032, 8–14 hrs: 0.649, 0.87, 25.89, 0.007, 24–30 hrs: 0.419, 0.621, 6.54, 0.063, 32–40 hrs: 0.46, 0.85, 22.01, 0.009).

Mentions: The data presented provides strong evidence that gch1 CNP is adaptive and associated with antifolate drug selection. An important assumption made is that gene dosage is reflected in increased transcription of amplified genes. To test this assumption directly, we grew six parasites in the laboratory that vary in gch1 copy number, and harvested mRNA at 4 different time periods in the asexual cycle. We observe strong correlations between copy number and expression in all stages of the cycle validating this assumption (Figure 7). Hence, increased expression of gch1 has the potential to increase flux in the folate pathway. However, detailed dissection of protein flux through the pathway will be required to fully understand the impact of copy number of the dynamics of folate biosynthesis in Plasmodium.


Adaptive copy number evolution in malaria parasites.

Nair S, Miller B, Barends M, Jaidee A, Patel J, Mayxay M, Newton P, Nosten F, Ferdig MT, Anderson TJ - PLoS Genet. (2008)

Correlation between gch1 CNP and expression for 6 parasite isolates.We measured gene expression and copy number at 4 different time points in the asexual cycle. Error bars indicate 95% CI for estimates of both CNP and expression. Details of the real-time PCR assay are shown in Table S2. Linear regression summary statistics (slope, r2, F, p-value) for the four time points were as follows: 0–6 hrs: 0.63, 0.71, 10.36, 0.032, 8–14 hrs: 0.649, 0.87, 25.89, 0.007, 24–30 hrs: 0.419, 0.621, 6.54, 0.063, 32–40 hrs: 0.46, 0.85, 22.01, 0.009).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2570623&req=5

pgen-1000243-g007: Correlation between gch1 CNP and expression for 6 parasite isolates.We measured gene expression and copy number at 4 different time points in the asexual cycle. Error bars indicate 95% CI for estimates of both CNP and expression. Details of the real-time PCR assay are shown in Table S2. Linear regression summary statistics (slope, r2, F, p-value) for the four time points were as follows: 0–6 hrs: 0.63, 0.71, 10.36, 0.032, 8–14 hrs: 0.649, 0.87, 25.89, 0.007, 24–30 hrs: 0.419, 0.621, 6.54, 0.063, 32–40 hrs: 0.46, 0.85, 22.01, 0.009).
Mentions: The data presented provides strong evidence that gch1 CNP is adaptive and associated with antifolate drug selection. An important assumption made is that gene dosage is reflected in increased transcription of amplified genes. To test this assumption directly, we grew six parasites in the laboratory that vary in gch1 copy number, and harvested mRNA at 4 different time periods in the asexual cycle. We observe strong correlations between copy number and expression in all stages of the cycle validating this assumption (Figure 7). Hence, increased expression of gch1 has the potential to increase flux in the folate pathway. However, detailed dissection of protein flux through the pathway will be required to fully understand the impact of copy number of the dynamics of folate biosynthesis in Plasmodium.

Bottom Line: The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP.These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function.More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.

View Article: PubMed Central - PubMed

Affiliation: Southwest Foundation for Biomedical Research (SFBR), San Antonio, TX, USA.

ABSTRACT
Copy number polymorphism (CNP) is ubiquitous in eukaryotic genomes, but the degree to which this reflects the action of positive selection is poorly understood. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gch1), shows extensive CNP. We provide compelling evidence that gch1 CNP is an adaptive consequence of selection by antifolate drugs, which target enzymes downstream in this pathway. (1) We compared gch1 CNP in parasites from Thailand (strong historical antifolate selection) with those from neighboring Laos (weak antifolate selection). Two percent of chromosomes had amplified copy number in Laos, while 72% carried multiple (2-11) copies in Thailand, and differentiation exceeded that observed at 73 synonymous SNPs. (2) We found five amplicon types containing one to greater than six genes and spanning 1 to >11 kb, consistent with parallel evolution and strong selection for this gene amplification. gch1 was the only gene occurring in all amplicons suggesting that this locus is the target of selection. (3) We observed reduced microsatellite variation and increased linkage disequilibrium (LD) in a 900-kb region flanking gch1 in parasites from Thailand, consistent with rapid recent spread of chromosomes carrying multiple copies of gch1. (4) We found that parasites bearing dhfr-164L, which causes high-level resistance to antifolate drugs, carry significantly (p = 0.00003) higher copy numbers of gch1 than parasites bearing 164I, indicating functional association between genes located on different chromosomes but linked in the same biochemical pathway. These results demonstrate that CNP at gch1 is adaptive and the associations with dhfr-164L strongly suggest a compensatory function. More generally, these data demonstrate how selection affects multiple enzymes in a single biochemical pathway, and suggest that investigation of structural variation may provide a fast-track to locating genes underlying adaptation.

Show MeSH
Related in: MedlinePlus