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25C-055 (7) Letter to the Editor 573 from sequential single-gene duplications. Multiple mRNA(M) kl ♦ M ♦ rounds of genome duplication associated with preferen- kf tial retention of dosage-sensitive interacting partners might explain the existence of paralogous networks and nM 4 Mn also the tendency toward nonrandom gene association ilcr in the eukaryotic genome. The perspective outlined b here is compatible with, and does not diminish the dM _kl-k2M-dMn _0 evolutionary importance of,segmental duplications.In- dt dt deed, TEICHMANN and VEITIA (2004) have shown the dMn n M existence of an excess of linked gene pairs encoding dt -krn=0 subunits of stable protein complexes in yeast.We specu- 14f n n kl lated that these pairs may be modules (perhaps gener- M�-—M - .M ; M=ki ated by the nonrandom retention of dosage-sensitive kr k2 genes) that may maintain the right stoichiometry of Mn=K(kl In complexes upon segmental duplication. \k2/ The examples discussed above show that the appear- ance of dominant phenotypes may have simple physio- FIGURE 4.—Doubling the genomic content associated with an increase in nuclear and cellular volume can warrant success- logical explanations in terms of dosage imbalances and ful duplication of certain dosage-sensitive genes.Consider that that the resilience of a system to such alterations can M is an inactive monomer and that Mn is the active multimer be adjusted by selection. They illustrate also key points composed of n monomers. Synthesis (kJ, degradation (k2), concerning duplicability of dosage-sensitive genes. The and interaction of the monomers are represented with chemi- fact that there are rules governing changes of gene cal (a) and differential equations (b). For simplicity, let the association/dissociation reactions between monomers and dosage does not exclude the possibility of compensation oligomers be faster than synthesis/degradation.We can define by up-or downregulation of partners in the same path- a pseudo-equilibrium constant Kin the steady state (i.e.,when way as long as fitness is not severely compromised by dM/dt = 0). As usual, increasing dosage of M(i.e., by 1.5x) the initial dosage alteration. Indeed, coevolution of cis- will be represented by 1.5 x ki.If the initial volume does not regulatory sequences is commonplace (WRAY et al. change,this will imply a(1.5)"-fold increase of active M!Thus, maintaining a balance with another oligomer, say N, after 2003),which may explain gene synexpression(coregula- strict coduplication in the same volume is possible only if the tion within modules in time and space; NIERHs and number of monomers involved in M. and N are identical POLLET 1999), as a way to ensure balance. A scenario (n=x),for similar KM and KN.A polyploidization event increas- involving massive duplications is crucial to produce par- ing cell volume proportionally is more likely to restore balance alogous pathways (probably more complex than those whatever the K's, n,or x are.Similar arguments explain imbal- ance after a heterozygous deletion of M or N. studied by TEICHMANN and BABu 2004) and raw mate- rial for evolution in cases where single-gene duplication is difficult or impossible. tion events in many eukaryotes including yeast, plants, I thank Nils Bluthgen for his kind help concerning simulations of and vertebrates (MAKALOWSKI 2001;BLANC and WOLFE the MAPK pathway and for helpful comments on the manuscript.I 2004•KELLIs et al. 2004).This process is expected to be thank Jim Collins for his comments concerning both the original and the revised manuscript and Sandrine Caburet,Indram Bose,and Vidya followed by deletion or rearrangements leading in part Nanjundiah for their interesting suggestions. to preferential retention of"interacting"genes to avoid imbalances.After genome duplication,the retained par- alogs may diverge concertedly in sequence and pattern of expression,producing new paralogous network mod- LITERATURE CITED ules unable to interfere stoichiometrically. Indeed, BHALLA,U.,and R.IYENGAR,1999 Emergent properties of networks BLANc and WOLFE (2004) have found many groups of of biological signaling pathways.Science 283:381-387. BIRCHLER,J.A.,U. BHADRA,M. P. BHADRA and D. L.AUGER, 2001 paralogs in Arabidopsis bearing this signature. In line Dosage-dependent gene regulation in multicellular eukaryotes: with the ideas presented here, they suggest that the implications for dosage compensation, aneuploid syndromes, impact of polyploidy on the evolution of networks may and quantitative traits.Dev.Biol. 234:275-288. BLANC,G.,and K.WOLFE,2004 Functional divergence of duplicated be more important than the cumulative effect of the genes formed by polyploidy during Arabidopsis evolution. Plant duplication of individual genes.Consider also that yeast Cell 16: 1679-1691. alone contains five different MAPK pathways: the hap- BLUTHGEN, N., and H. HERZEL, 2003 How robust are switches in intracellular signaling cascades?J.Theor.Biol.225:293-300. loid mating, invasive growth, and cell wall remodeling CAFFREY,D.R.,L.A.O'NEILL and D.C.SHIELDS,1999 The evolution pathways and two pathways involved in stress responses of the MAP kinase pathways:coduplication of interacting proteins (WIDMANN et al. 1999).They share a common evolution- leads to new signaling cascades.J.Mol.Evol.49:567-582. CHFN,K. C.,A. CSIKASz-NAGY,B.GYORFFY,J.VAL, B. NOVAK et at., ary origin (CAFFREY et al. 1999) but according to the 2000 Kinetic analysis of a molecular model of the budding yeast perspective outlined above, all of them cannot result cell cycle.Mol.Biol. Cell 11:369-391. NORTHAMPTON CODE ERBUSES ALLOWED Uses Allowed By Right: • Single-,two-,three-family dwellings • Attached(to a single-family)accessory dwelling unit not to exceed 900 square feet gross living area. See§ 350-10.10. 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See below for lot layout standards and§350-10.5 for other required criteria. • Parking off-site and combined parking.See§§350-8.5 and 350-8.7. 350 Attachment 7:4 09-01-2014 Untitled 3-Multivariate Multivariate Pairwise Correlations Variable by Variable Correlation Count Lower 95% Upper 95% Signif Prob "- i► 5S 5.8S 0.7612 47 0.6066 0.8603 5e-10 5S 28S -0.0024 47 -0.2893 0.2850 0.9874 18S_seg2 18S 0.6241 47 0.4105 0.7728 2.8e-6 18S_seg2 5.8S 0.4520 47 0.1895 0.6543 0.0014 18S_seg2 28S 0.5339 47 0.2914 0.7119 0.0001 18S_seg2 5S 0.2153 47 -0.0766 0.4732 0.1461 5.8S_seg2 18S 0.5036 47 0.2530 0.6908 0.0003 5.8S_seg2 5.8S 0.8015 47 0.6681 0.8850 1e-11 5.8S_seg2 28S 0.1628 47 -0.1305 0.4299 0.2742 5.8S_seg2 5S 0.5784 47 0.3492 0.7423 2.1e-5 . 5.8S_seg2 18S_se92 0.6762 47 0.4828 0.8067 1.8e-7 28S_seg2 18S 0.4847 47 0.2295 0.6776 0.0006 28S_seg2 5.8S 0.4533 47 0.1910 0.6552 0.0014 28S_seg2 28S 0.6278 47 0.4156 0.7752 2.3e-6 28S_seg2 5S 0.2862 47 -0.0011 0.5298 0.0512 -28S_seg2 18S_seg2 0.9187 47 0.8579 0.9542 9e-20 - 28S_seg2 5.8S_seg2 0.6055 47 0.3854 0.7605 6.5e-6 5S_seg2 18S -0.0322 47 -0.3164 0.2574 0.83 •5S_seg2 5.8S 0.6866 47 0.4975 0.8134 9.9e-8 5S_seg2 28S -0.0563 47 -0.3380 0.2347 0.7071 4bw&& 5S_seg2 5S 0.8794 47 0.7923 0.9314 4e-16 5S_seg2 18S_seg2 0.3829 47 0.1075 0.6037 0.0079 5S_seg2 5.8S_seg2 0.5928 47 0.3684 0.7520 1.1e-5 5S_seg2 28S_seg2 0.3985 47 0.1258 0.6153 0.0055 18S_seg3 18S 0.3772 47 0.1009 0.5994 0.009 18S_seg3 5.8S 0.3278 47 0.0449 0.5621 0.0245 18S_seg3 28S 0.5751 47 0.3449 0.7401 2.4e-5 18S_seg3 5S 0.3458 47 0.0651 0.5758 0.0173 18S_seg3 18S_seg2 0.7163 47 0.5402 0.8322 1.5e-8 18S_seg3 5.8S_seg2 0.4596 47 0.1987 0.6597 0.0012 18S_seg3 28S_seg2 0.7424 47 0.5785 0.8486 2.3e-9 18S_seg3 5S_seg2 0.3345 47 0.0523 0.5672 0.0216 5.8S_seg3 18S 0.2702 47 -0.0184 0.5172 0.0662 5.8S_seg3 5.8S 0.7896 47 0.6497 0.8777 4e-11 5.8S_seg3 28S -0.0154 47 -0.3012 0.2730 0.9183 5.8S_seg3 5S 0.6530 47 0.4503 0.7917 6.5e-7 5.8S_seg3 18S_seg2 0.5024 47 0.2515 0.6900 0.0003 5.8S_seg3 5.8S_seg2 0.8306 47 0.7137 0.9025 5e-13 5.8S_seg3 28S_seg2 0.4488 47 0.1856 0.6520 0.0016 �..5.8S_seg3 5S_seg2 0.6259 47 0.4130 0.7740 2.5e-6 5.8S_seg3 18S_seg3 0.5864 47 0.3598 0.7477 1.5e-5 28S_seg3 18S 0.4154 47 0.1456 0.6277 0.0037 28S_seg3 5.8S 0.2785 47 -0.0094 0.5238 0.058 28S_seg3 28S 0.6543 47 0.4521 0.7926 6.1e-7 28S_seg3 5S 0.2630 47 -0.0262 0.5115 0.0741 28S_seg3 18S_seg2 0.7339 47 0.5661 0.8434 4.4e-9 28S_seg3 5.8S_seg2 0.4468 47 0.1831 0.6505 0.0016 28S_seg3 28S_seg2 0.8440 47 0.7350 0.9105 9e-14 28S_seg3 5S_seg2 0.2839 47 -0.0035 0.5280 0.0531 28S_seg3 18S_seg3 0.9223 47 0.8640 0.9562 3e-20 28S_seg3 5.8S_seg3 0.5024 47 0.2515 0.6900 0.0003 5S_seg3 18S -0.1541 47 -0.4226 0.1392 0.3009 5S_seg3 5.8S 0.4683 47 0.2092 0.6659 0.0009 5S_seg3 28S 0.1788 47 -0.1142 0.4432 0.229 5S_seg3 5S 0.7640 47 0.6109 0.8621 4e-10 5S_seg3 18S_seg2 0.2365 47 -0.0543 0.4904 0.1095 5S_seg3 5.8S_seg2 0.3442 47 0.0633 0.5746 0.0178 5S_seg3 28S_seg2 0.4162 47 0.1465 0.6283 0.0036 5S_seg3 5S_seg2 0.5988 47 0.3764 0.7560 8.7e-6 5S_seg3 18S_seg3 0.6055 47 0.3854 0.7605 6.5e-6 5S_seg3 5.8S_seg3 0.5344 47 0.2921 0.7123 0.0001 5S_seg3 28S_seg3 0.5241 47 0.2788 0.7051 0.0002 -.8-.6-.4-.2 0 .2 .4 .6 .8 Page 2 of 2 s Q +John +l 2-car ,;:Garage . 4, -Al wa- lop gg l a � T �i jw R Isar Driveway F r Ong `' �, Untitled 3-Multivariate Multivariate Correlations 18S 5.8S 18S 1.0000 0.2679 5.8S 0.2679 1.0000 28S 0.6117 0.0155 5S -0.0071 0.7612 18S_seg2 0.6241 0.4520 5.8S_seg2 0.5036 0.8015 28S_seg2 0.4847 0.4533 5S_seg2 -0.0322 0.6866 18S_seg3 0.3772 0.3278 5.8S_seg3 0.2702 0.7896 28S_seg3 0.4154 0.2785 5S_seg3 -0.1541 0.4683 Scatterplot Matrix 350 250 150 50 100 50 0 150 50 100 50 0 60 20 25 15 5 -5 60 20 30 20 10 0 70 50 30 10 10 0 60 20 30 15 0 28S 0.6117 0.0155 1.0000 -0.0024 0.5339 0.1628 0.6278 -0.0563 0.5751 -0.0154 0.6543 0.1788 5S 18S_seg2 -0.0071 0.6241 0.7612 0.4520 -0.0024 0.5339 1.0000 0.2153 0.2153 1.0000 0.5784 0.6762 0.2862 0.9187 0.8794 0.3829 0.3458 0.7163 0.6530 0.5024 0.2630 0.7339 0.7640 0.2365 5.8S_seg2 28S_seg2 0.5036 0.4847 0.8015 0.4533 0.1628 0.6278 0.5784 0.2862 0.6762 0.9187 1.0000 0.6055 0.6055 1.0000 0.5928 0.3985 0.4596 0.7424 0.8306 0.4488 0.4468 0.8440 0.3442 0.4162 5S_seg2 18S_seg3 -0.0322 0.3772 0.6866 0.3278 -0.0563 0.5751 0.8794 0.3458 0.3829 0.7163 0.5928 0.4596 0.3985 0.7424 1.0000 0.3345 0.3345 1.0000 0.6259 0.5864 0.2839 0.9223 0.5988 0.6055 Page 1 of 2 5.8S_seg3 28S_seg3 5S_seg; 0.2702 0.4154 -0.1541 0.7896 0.2785 0.468 -0.0154 0.6543 0.1788 0.6530 0.2630 0.764C 0.5024 0.7339 0.2365 0.8306 0.4468 0.3442 0.4488 0.8440 0.4162 0.6259 0.2839 0.5988 0.5864 0.9223 0.6055 1.0000 0.5024 0.5344 0.5024 1.0000 0.5241 0.5344 0.5241 1.0000 18S 50 150 �w L' t•• :�.f: ;ray•. �If ri }/�. a.'{wt' +•'. •SLY �Fa�: •Ifi �R 5.8S 18S +Y' V., a ,'ir -0.0209 .T+' iy• •!I +ti:. •{. f� .T�• ,,{{' 1• t � .' 0.3937 jY,I� T ' 5.8S 28S 5.8S 0.0155 47 -0.2729 0.3013 0.9176 Z+{a. '7�'�i{.• i• :r1�.. afw .:�.r ' � .wf�.�r' JKf ..lira' 4� r . ,ey fr+�+"k �.r• 28S �r� •t.r f�i }• ��. ;y. �:'jt• •t a ��'i �r•• � ! «r{iv • . . t ix 5S • #' +�� { + ••+ �Ff 18S_seg2 �+ + •} - Y.• k + ��} i4r Y;r-' I• q '� �'.� �• �� 4� •.. {r:r 5.8S_seg2 a;% , Ive �y�fC. •s;- ti '• '• '. 28S seg2 •: �,� �• �•; • 5S_seg2 j�.• ,�t�L � �, ��• { i� +L{'. ti��' r�•. '� 18S_seg3 ;ti r a;• •r ;•. .. XW . .. ± ![���. r.. ti., 5.8S_seg3 ••• �g .v=:;. t��y: L+� ';yT' r a � 3:b•: •� •�'• •j, •' � •�•� .4i •,�,� 28S_seg3 :•'t as 1'` �l /f ;A '� 5S_seg3 . ar. IN I IQ iM%}• s.�n:�• .�� �r.':� wirlf� � w�.?> 50 200 0 50 100 50 150 0 50 20 60 -5 5 15 20 60 0 10 25 10 40 70 0 5 15 .20 .60 0 10 25 Pairwise Correlations Variable by Variable Correlation Count Lower 95% Upper 95% Signif Prob -.8-.6-.4-.2 0 .2 .4 .6 .8 5.8S 18S 0.2679 47 -0.0209 0.5154 0.0687 28S 18S 0.6117 47 0.3937 0.7645 4.9e-6 28S 5.8S 0.0155 47 -0.2729 0.3013 0.9176 .A is City of Northampton HOME BUSINESS APPLICATION (To be used when seeing clients etc) Applicant Name: 1 ctt_1 v''A UIf� ai"`C, Applicant Address: A v\ y e me po, MA 0(0 6 0 Applicant Email: '000,dfglegoAva4vkAo ,cows PHONE: ;- (011, W1 ADDRESS of Home Business: -` L(v\C J v\ A Vhf yo%ka V,(,too b 1A A o I olp b ASSESSOR ID of Business-Map: Lot: DESCRIBE WHERE AND WHAT PORTION OF RESIDENCE Business will Occupy: �6✓f �1�, 5{L�l EXPLAIN WORK. Detail nature of Home Business including type of business: 4v, A (.i C e p 4.e j c oLa q1 u se 0,P y-�' ✓ v 5'eutp 4b coed O coi l-ti W p l '-9 f u✓ D•' V,���vac t U ��a sera �� HOURS Your Business will operate: Vcy ubl- C C? 0 DAYS PER WEEK: Vu V1 K[(t r _F HOW MANY CLIENTS Seen per week: ?� 2 ANY EMPLOYEES who are not a resident of the home? /VO How many on-site parking spaces are available in addition to personal vehicle spaces: Will the owner of the home business occupy the main residential building as his/her bona fide residence? vo Please attach diagram of the parcel with driveway access and parking areas shown. Also Attach Google Earth or other aerial photo image of street with parcel identified. Application Approved Application Requires Permit from the Zoning Board of Appeals Signed: Date: City of Northampton Building Commissioner Y. 1 GVI OPEN a ACCESS Freely available online PLoS Adaptive Copy Number Evolution in Malaria Parasites Shalini Nair',Becky Miller 2,Marion Barends3,4,Anchalee Jaidee3,Agar Patel, Mayfong Mayxay6'7, Paul Newton", Fran4ois Nosten3.4,8, Michael T. Ferdig2, Tim J. C. Anderson'* 1 Southwest Foundation for Biomedical Research(SFBR),San Antonio,Texas,United States of America,2 Department of Biological Sciences,Eck Institute for Global Health, University of Notre Dame,Notre Dame,Indiana,United States of America,3 Shoklo Malaria Research Unit(SMRU),Mae Sot,Tak,Thailand,4 Faculty of Tropical Medicine, Mahidol University,Bangkok,Thailand,5 Roche NimbleGen,Inc.,Madison,Wisconsin,United States of America,6 Wellcome Trust—Mahosot Hospital—Oxford Tropical Medicine Research Collaboration,Mahosot Hospital,Vientiane,Lao People's Democratic Republic,7 Department of Post Graduates and Research, Faculty of Medical Science,National University of Laos,Vientiane,Lao People's Democratic Republic,8Centre for Tropical Medicine and Vaccinology,Churchill Hospital,Oxford,United Kingdom Abstract Copy number polymorphism(CNP)is ubiquitous in eukaryotec 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 (gchi),shows extensive CNP.We provide compelling evidence that gchi CNP is an adaptive consequence of selection by antifolate drugs,which target enzymes downstream in this pathway.(1)We compared gchi 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 synonymous3NPs.(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.gchi 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 gch 1 in parasites from Thailand, c with rapid recent spread of chromosomes carrying multiple copies of gchi.(4)We found that parasites bearing dhfr-164L hich causes high-level resistance to antifolate drugs,carry significantly(p=0.00003) higher copy numbers of c n parasites bearing 1641,indicating functional association between genes located on different chromosomes but m'l-e-d in thp----hin&hem6QLQthway.These results demonstrate that CNP at gchi is adaptiveve awe ass�o�iiiisns with fr-164L strongly suggest a compensatory function.More generally,these data demonstrate how selection affects multiple enzymes in a�si�ng�le biochhe ical jjgbWay,and suggest that investigation of structural variation may provide a fast-track to locating generunder ylT ng'adaptatlon. Citation:Nair S,Miller B,Barends M,Jaidee A,Patel J,et al.(2008)Adaptive Copy Number Evolution in Malaria Parasites.PLoS Genet 4(10):e1000243.doi:10.1371/ jou rna I.pgen.1000243 Editor:Molly Przeworski,University of Chicago,United States of America Received July 25,2008;Accepted September 29,2008;Published October 31,2008 Copyright:©2008 Nair et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited. Funding:This work was funded by grant from NIH(R01 A1075145 and A148071)to TJCA.The molecular work at the Southwest Foundation for Biomedical Research was conducted in facilities constructed with support from Research Facilities Improvement Program grant C06 RRO13556 from the National Center for Research Resources,NIH.The funders played no role in the design and conduct of the study,in the collection,analysis,and interpretation of the data,and in the preparation,review,or approval of the manuscript. Competing Interests:The authors have declared that no competing interests exist. *E-mail:tanderso @sfbrgenetics.org Introduction A spate of studies over the past five years have described widespread copy number variation (CNP) within the genomes of humans,mice,Drosophila and other eukaryotes[1-5].The existence of large regions of t e genome that vary in copy number between individuals has lead to a reconsideration of the importance of structural variation for our understanding of genetic and phenotypic variation [6]. However, it is unclear whether CNP evolution is predominantly neutral, or whether ve or ne_2 n p si ificant r` s in. rng the patterns observed [1].The fact that CNPs tend to be enriched for parcu ag�eclasses,and for genes showing evidence for positive selection at the nucleotide level, strongly suggests the action of positive selection [7], although the alternative explanation of purifying selection against CNP in particular gene classes cannot be discounted. Furthermore, that CNPs explain —20% of variance in transcript abundance in humans suggests that they have the potential to make a significant PLoS Genetics I www.plosgenetics.org contribution to disease susceptibility and adaptive evolution [8]. However, despite these indirect lines of evidence for positive selection,adaptive copy number evolution has been demonstrated or hypothesized in only a few cases. In humans there are two notable examples. Gonzales et al [9] showed that protection from HIV is associated with CNP at the CCUL1 gene.This CNP shows extreme geographical variation which further supports the action of selection by HIV (or, more likely, by an older human pathogen) [10].Similarly,Perry et al [1 l] showed higher copy number of the amylase gene in populations with high starch diets. CNP is also widespread in the malaria parasite genome [12,13]. Malaria parasites are exposed to strong selection from the human immune response and treatment with antimalarial drugs.They have relatively small genomes(23 Mb)and haploid genetics,and can be grown and genetically manipulated in the laboratory,so provide a useful eukaryotic organism for investigating the functional role of CNP. One CNP on chromosome (chr.) 5 is known to underlie a multidrug resistance phenotype: chromosomes carrying this CNP October 2008 1 Volume 4 1 Issue 10 1 ell 000243 10. Do any signs exist on the property? YES NO IF YES, describe size, type and location: Are there any proposed changes to or additions of signs intended for the property? YES NO Lof' IF YES, describe size, type and location: 11. Will the construction activity disturb (clearing, grading, excavation, or filling) over acre or is it part of a common plan of development that will disturb over 1 acre? YES NO IF YES, then a Northampton Storm Water Management Permit from the DPW is required. 12. ALL INFORMATION MUST BE COMPLETED, or PERMIT CAN BE DENIED DUE TO LACK OF INFORMATION This column reserved for use by the Building Department EXISTING PROPOSED REQUIRED BY ZONING Lot size , 3 G [t✓. ! v�0 ( lti�rn f Frontage Setbacks Front Side L: R: L: R: L: R: Rear Building Height Building Square Footage 7(� OU %kVj t4 01 _���%AV%A r %Open Space: (lot area minus building Et paved parking #of Parking Spaces rn #of Loading Docks Fill: (volume Et location 13. Certification: I hereby certify that the information contained herein is true and accurate to the best of my knowledge. r Date: I 11111 1L Applicant's Signature NOTE:Issuance of a zoning permit does not relieve an applicant's burden to comply with all zoning requirements and obtain all required permits from the Board of Health,Conservation Commission, Historic and Architectural Boards,Department of Public Works and other applicable permit granting authorities. R':\Documents\FORMS\original\Building-Inspector\Zoning-Permit-Application-passive.doc 8/4/2004 Author Summary Recent comparative genomic hybridization studies have revealed extensive copy number variation in eukaryotic genomes. The first gene in the Plasmodium folate biosynthesis pathway, GTP-cyclohydrolase I (gchl), shows extensive copy number polymorphism (CNP).We provide compelling evidence that gch 1 CNP is adaptive and most likely results from selection by antifolate drugs, which target enzymes downstream in this pathway. Gch 1 CNP shows extreme geographical differentiation; hitchhiking reduces diversity and increases LD in flanking sequence, indicating recent rapid spread within Thailand, while amplicon structure reveals multiple origins and parallel evolution. Furthermore, strong association between ele- vated copy number and a critical mutation dhfr-1164L that underlies high-level antifolate resistance indicates func- tional linkage and fitness epistasis between genes on different chromosomes. These data reveal hidden com- plexity in the evolutionary response to antifolate treat- ment and demonstrate that analysis of structural variation can provide a fast-track to locating genes that underlie adaptation. have risen to high frequencies in Southeast Asia [14,15] and manipulation of copy number alters response to multiple drugs[l6]. However,this one example of adaptive copy number variation in P. falciparum has been regarded as an exceptional case,and SNP based approaches have been prioritized as the primary tool for mapping functional genes in Plasmodium [17]. The first eGH study of P.falciparum in 16 laboratory isolates revealed a particularly interesting CNP containing GTP-cyclohy- drolase I(gchl) [12].This gene encodes the first and rate limiting enzyme in the folate metabolism pathway(Figure 1) [18,19].Two key enzymes in later stages of this pathway—dihydrofolate reductase (dhfr) (chr. 4) and dihydropteroate synthase (dhps) (chr. 8)—are targets of the antifolate drugs pyrimethamine and sulfadoxine, which are combined in the drug Fansidar (Roche). This drug replaced chloroquine as the first-line treatment against malaria in many countries,but resistance has spread rapidly where it has been deployed. Specific point mutations (N51I, C59R, S 108N,I164L)in parasite dhfr alter the binding of pyrimethamine to the enzyme's active site [20]. In addition to causing resistance, mutations in dhfr reduce enzyme efficacy and carry adverse fitness effects [21,22,but see 23]. Similarly,mutations in dhps(S436A/F, A437G, K540E, A581G, and A613T/S) underlie resistance to sulfadoxine [20]. Kidgell et al [l2] speculated that increased gene dosage might play a compensatory role in antifolate resistance by increasing flux in the pathway to compensate for reduced efficacy of dhfr and/or dhps genes bearing resistance mutations. This study was designed to determine whether CNP at gchI is a consequence of adaptive evolution. To do this, we examined the population genetics of gchl CNP in Laos and Thailand.These two neighboring countries in SE Asia have contrasting selection histories with antifolate drugs. In Western Thailand antifolate drugs were the first line treatment from 1970-80, and resistance mutations at both dhps and dhfr are fixed or at high frequency [24,25].By contrast,in Laos antifolate drugs were the second line drug until 2006, but were seldom used [26], and resistance mutations are at lower frequencies in both dltfr and dhps [25,27]. We provide compelling evidence—from patterns of population differentiation, hitchhiking, and amplicon structure—that the gchl CNP (chr. 12) in P. falciparum results from recent adaptive evolution that is most likely associated with antifolate treatment. ;r PbMGehefics I www.plosgenetics.org Adaptive Copy Number Evolution in Plasmodium GTP 7,8-d ihydroneopterin tdphosphate ptps 6-hydroxymethyl dihydropterin pppk 6-hydroxymethyl dihydropterin pyrophosphate m <— SDX 7,84hydropteroate dhfs 7,8-dihydrofolate , .M <— PYR tetrahydrofolate Figure 1.The folate bi thesis pathway of A falciparum.The steps catalyzed by chi fr nd dhps are highlighted.The positions at which antifolate drur ( ynmethamine (PYR) and Sulfadoxine (SDX)) target the pathway are marked. Abbreviations: pyruvoyltetrahydrop- terin synthase (ptps), hydroxymethyldihydropterin pyrophosphokinase (pppk),dihydrofolate synthase(dhfs).Modified from [18]. doi:10.1371/jou rnal.pgen.1000243.g001 Furthermore,we show strong association between gchl CNP and a critical mutation in dhfr, indicating functional linkage between genes on different chromosomes in the same biochemical pathway. Materials and Methods Sample Collection We collected 5 mL blood samples from P.falciparum infected patients visiting the malaria clinic at Mawker-Thai on the Thai- Burma border between December 2000 and September 2003. These patients had not visited the malaria clinic within the past 60 days, and had no history of prior malaria treatment during this time.The clinic serves people on both sides of the border;90% of patients travel from within a 10 km radius around the clinic (FranSois Nosten,personal communication).We collected samples from Phalanxay(Savannaket)in Laos between June 2002 and Sept 2003 from patients involved in clinical drug efficacy trials. Collection protocols were approved by the Ethical Committee of the Faculty of Tropical Medicine, Mahidol University, Bangkok, and by the Institutional Review Board at the University of Texas Health Science Center at San Antonio. Parasite DNA was prepared by phenol/chloroform extraction of whole blood, following removal of buffy coats as described previously [28]. Plasmodium Genetics and Biology Malaria parasites are obligately sexual protozoans. Haploid blood-stage parasites replicate asexually within the human host October 2008 1 Volume 4 1 Issue 10 1 el 000243 I , JAN 13 201 File No. /III IF U'lb'ng N PE"IT"PLICATION (§10.2) Northarnpto Please type or print all information and return this form to the Building Inspector's Office with the $15filing fee (check or money order)payable to the City of Northampton 1. Name of Applicant: A e S�Q h d rQ u/p m o Address: 13 L j"t D I i s V- - A/#f rt-o ph n'-h Telephone: O k o3 G 11 6 1 3 1 2. Owner of Property: S A 0,,k Address: Telephone: 3. Status of Applicant: Owner ��Contract Purchaser Lessee Other (explain) 4. Job Location: f I o 0 r Parcet Id: Zoning Map# Parcel# District(s): In Elm Street District In Central Business District (TO BE FILLED IN BY THE BUILDING DEPARTMENT) lM 5. Existing Use of Structure/Property: 1M 1 q 0 u C h 4NA 7 ~ YEA IH "IN 6. 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