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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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Structure and gene content of gch1 amplicons.The span of each amplicon was established by real-time PCR and breakpoint specific PCR assays. (a) Real-time PCR estimates of relative copy number plotted for 9 genes including gch1 on chr. 12. The error bars show 95% confidence intervals around the copy number estimate. The three plots show the profiles observed for the 2.3 kb (top), 7.3 kb (middle) and 8.7 kb (bottom) amplicons. The location of gch1 is marked by a red bar (b) Plot showing the span of the 5 amplicons types. The abundance of each of the amplicon types in the Thai sample is shown in white on the bars. The gene content in this region of the chr. 12 are shown beneath the graph: all breakpoints were between genes. The 5′ boundary for the >11 kb amplicon fell outside the range of our real-time PCR assays and was not defined: the bar shows the minimum size estimate for this amplicon.
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pgen-1000243-g002: Structure and gene content of gch1 amplicons.The span of each amplicon was established by real-time PCR and breakpoint specific PCR assays. (a) Real-time PCR estimates of relative copy number plotted for 9 genes including gch1 on chr. 12. The error bars show 95% confidence intervals around the copy number estimate. The three plots show the profiles observed for the 2.3 kb (top), 7.3 kb (middle) and 8.7 kb (bottom) amplicons. The location of gch1 is marked by a red bar (b) Plot showing the span of the 5 amplicons types. The abundance of each of the amplicon types in the Thai sample is shown in white on the bars. The gene content in this region of the chr. 12 are shown beneath the graph: all breakpoints were between genes. The 5′ boundary for the >11 kb amplicon fell outside the range of our real-time PCR assays and was not defined: the bar shows the minimum size estimate for this amplicon.

Mentions: Initially, we sampled P. falciparum from infected patients visiting a single clinic on the Thailand-Burma border. Following removal of multiple clone infections 140 samples were available. We found between 1 and 11 copies of gch1 by taqman PCR with 72% of parasites sampled carrying >1 copy. To determine the arrangement and size of these genome amplifications we measured copy number in genes surrounding gch1 (Figure 2a) and designed PCR assays to identify chromosome breakpoints [14] (Table S3, Figure S1). We found 5 different amplicon types containing one to >six genes (Figure 2b). Breakpoint specific PCR assays indicate that these were arranged in tandem. However we note that duplicative transposition of some amplicons would not be detectable using this assay. The most common of these amplicons contained only gch1 and accounted for 48% of all amplicon types, while the largest amplicon (>11 kb), was found in only one sample. We sequenced the chromosome breakpoints for 4 of the 5 amplicons. In each case breakpoints were found in between genes in microsatellite sequences or monomeric tracts (Figure S1). The amplicon size data provides a natural mapping experiment. Gch1 was the only gene observed in all amplicons. Hence, if CNP results from selection, then gch1 is clearly the gene that is targeted.


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)

Structure and gene content of gch1 amplicons.The span of each amplicon was established by real-time PCR and breakpoint specific PCR assays. (a) Real-time PCR estimates of relative copy number plotted for 9 genes including gch1 on chr. 12. The error bars show 95% confidence intervals around the copy number estimate. The three plots show the profiles observed for the 2.3 kb (top), 7.3 kb (middle) and 8.7 kb (bottom) amplicons. The location of gch1 is marked by a red bar (b) Plot showing the span of the 5 amplicons types. The abundance of each of the amplicon types in the Thai sample is shown in white on the bars. The gene content in this region of the chr. 12 are shown beneath the graph: all breakpoints were between genes. The 5′ boundary for the >11 kb amplicon fell outside the range of our real-time PCR assays and was not defined: the bar shows the minimum size estimate for this amplicon.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000243-g002: Structure and gene content of gch1 amplicons.The span of each amplicon was established by real-time PCR and breakpoint specific PCR assays. (a) Real-time PCR estimates of relative copy number plotted for 9 genes including gch1 on chr. 12. The error bars show 95% confidence intervals around the copy number estimate. The three plots show the profiles observed for the 2.3 kb (top), 7.3 kb (middle) and 8.7 kb (bottom) amplicons. The location of gch1 is marked by a red bar (b) Plot showing the span of the 5 amplicons types. The abundance of each of the amplicon types in the Thai sample is shown in white on the bars. The gene content in this region of the chr. 12 are shown beneath the graph: all breakpoints were between genes. The 5′ boundary for the >11 kb amplicon fell outside the range of our real-time PCR assays and was not defined: the bar shows the minimum size estimate for this amplicon.
Mentions: Initially, we sampled P. falciparum from infected patients visiting a single clinic on the Thailand-Burma border. Following removal of multiple clone infections 140 samples were available. We found between 1 and 11 copies of gch1 by taqman PCR with 72% of parasites sampled carrying >1 copy. To determine the arrangement and size of these genome amplifications we measured copy number in genes surrounding gch1 (Figure 2a) and designed PCR assays to identify chromosome breakpoints [14] (Table S3, Figure S1). We found 5 different amplicon types containing one to >six genes (Figure 2b). Breakpoint specific PCR assays indicate that these were arranged in tandem. However we note that duplicative transposition of some amplicons would not be detectable using this assay. The most common of these amplicons contained only gch1 and accounted for 48% of all amplicon types, while the largest amplicon (>11 kb), was found in only one sample. We sequenced the chromosome breakpoints for 4 of the 5 amplicons. In each case breakpoints were found in between genes in microsatellite sequences or monomeric tracts (Figure S1). The amplicon size data provides a natural mapping experiment. Gch1 was the only gene observed in all amplicons. Hence, if CNP results from selection, then gch1 is clearly the gene that is targeted.

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