Research:

Plasmodium falciparum is a single celled parasite that causes the most deadly of the 4 kinds of human malaria. There is currently no vaccine to prevent this disease, and about 200 million people suffer from malaria each year. In fact, malaria causes 2 million deaths each year, most of them children in sub-Saharan Africa. There is an acute need for effective chemotherapeutic agents for prophylaxis and treatment of falciparum malaria. Drugs that target dihydrofolate reductase (DHFR), a key enzyme in the synthesis of deoxythymidine, histidine, and methionine and sulfonamides that target dihydropteroate synthase (DHPS), required for the synthesis of folate have been extremely effective in the past . In most cases, combinations of these drugs have been used, because the drugs act synergistically. However, the incredibly rapid selection of resistant P. falciparum populations has made the drugs virtually useless in many regions. The parasites are resistant to the drugs because they carry alleles of the DHFR or DHPS genes that encode mutant forms of the target enzyme.

The P. falciparum parasites can be grown in the lab, but their culture is expensive and labor intensive. To simplify study of these target enzymes from P. falciparum, we have engineered a series of strains of the budding yeast, Saccharomyces cerevisiae. These yeast lack endogenous DHFR or DHPS, but can grow because we have transformed them with the P. falciparum version of one of these enzymes. This approach has allowed us to use the yeast system to study the function of the parasite enzymes in a simple, inexpensive way. Normally, yeast are insensitive to antimalaria drugs, but these engineered yeast strains are now sensitive to inhibitors of the P. falciparum enzymes. We are studying the mutations in the DHFR and DHPS genes that confer resistance to inhibitors of these enzymes. We have used this approach to identify new mutations that can encode drug-resistant enzymes, and to screen a panel of potential new inhibitors for their effectiveness against the parasite enzymes. In addition, we have used the polymerase chain reaction to amplify DHFR genes from blood samples collected between 1984 and 2001 in Kenya. Yeast strains that express these alleles have been engineered, and used to characterize the drug sensitivity profiles of the parasites that infected these patients. These data are being used to reconstruct the history of the selection for drug-resistant alleles that has occurred since the introduction of Fansidar into use in Kenya.

We have recently extend these studies to include the DHFR and DHPS genes from a number of other related pathogens (Plasmodium vivax, Cryptosporidium parvum, Toxoplasma gondii)  and the bacterium that causes tuberculosis, Mycobacterium tuberculosis. We have two overall goals in our work. First, to use the basic techniques of genetics and molecular biology to understand the mechanism of inhibition of DHFR and DHPS by antifolate drugs. This will allow the design of alternative drugs that are effective against parasites that are resistant to currently available drugs. Second, to use this simple yeast system to understand the selection pressures that have resulted in mutations that confer drug resistance.  Our work is highly collaborative.  We have strong collaborations with colleagues at the Wellcome Trust Research Laboratory in Nairobi, Kenya, the National Institute for Medical Research, Amani-Tanga, Tanzania, the Liverpool School of Tropical Medicine, the London School of Hygiene and Tropical Medicine, and the University of Manchester Institute of Science and Technology, in the UK, Jacobus Pharmaceutical Company in Princeton, NJ, the Dana Farber Cancer Institute, Boston, MA and the CSIRO in Melbourne, Australia. We hope to apply our  understanding of antifolate drugs to design deployment and use strategies that will slow the selection of drug-resistant parasites in the future.

Selected Publications:

Certain, L and Sibley, CH. 2007. Plasmodium falciparum: A novel method for analyzing haplotypes in mixed infections. Experimental Parasutology, 115:233-241.

Sandefur, CI, Wooden, JM, Quaye, IK, Sirawaraporn, W and Sibley, CH . 2007. Pyrimethamine-resistant dihydrofolate reductase enzymes of P. falciparum are not enzymatically compromised in vitro. Molecular and Biochemical Parasitology.154:1-5.

VN Hawkins, H Joshi, K Rungsihirunrat, K Na-Bangchang, CH Sibley: 2007 Antifolates can have a role in the treatment of Plasmodium vivax. Trends Parasitol 23: 213-222.

Sibley CH, Barnes KI and Plowe CV .2007. The rationale and plan for creating a World Antimalarial Resistance Drug Network (WARN). Malar J 6:118.

Price RN, Dorsey G, Ashley EA, Barnes KI, Baird JK, d’Alessandro U, Guerin PJ, Laufer MK, Naidoo I, Nosten F, Olliaro P, Plowe CV, Ringwald P, Sibley CH, Stepniewska K and White NJ .2007. World Antimalarial Resistance Network (WARN) I: Clinical efficacy of antimalarial therapy. Malar J 6:119.

Plowe CV, Roper C, Barnwell JW, Happi CT, Joshi HH, Mbacham W, Meshnick SR, Mugittu K, Naidoo I, Price RN, Shafer RW, Sibley CH, Sutherland CJ, Zimmerman PA and Rosenthal PJ. 2007. World Antimalarial Resistance Network (WARN) III: Molecular markers for drug resistant malaria. Malar J 6:121.

Bacon DJ, Jambou R, Fandeur T, Le Bras J, Wongsrichchanalai C, Fukuda MM, Ringwald P, Sibley CH and Kyle DE. 2007. World Antimalarial Resistance Network (WARN) II: In vitro antimalarial drug susceptibility. Malar J 6:120.

Barnes KI, Lindegardh N, Ogundahunsi O, Olliaro P, Plowe CV, Randrianarivelojosia M, Gbotosho GO, Watkins WM, Sibley CH and White NJ. 2007. World Antimalarial Resistance Network (WARN) IV: Clinical pharmacology. Malar J 6:122.

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Research:

The Trask group studies large-scale facets of genome organization. Our work relies on continued development of fluorescence in situ hybridization (FISH), a means of fluorescently tagging specific DNA sequences in chromosomes or nuclei, and flow cytometry, a technology for isolating specific chromosomes for molecular analyses based on their DNA content.

One aspect of genomic organization under study is the arrangement of DNA within the interphase nucleus. Two meters of DNA are packed within each nucleus in interphase, the stage when transcription, repair, and replication occur. FISH is used to mark sites of sequences lying at known distances from each other on the same chromosome (or on different chromosomes). By comparing interphase distances between these points to predictions of various physical models, such as that of a random-walk, we hope to learn which arrangements, if any, are dictated by functional constraints and which can be explained by the physical forces acting on these large molecules.

The structure, function, and evolution of some of the more complex and variable regions of the human genome are also under investigation. One project focuses on the subtelomeric regions of human chromosomes. These regions are a patchwork of sequence-blocks that are duplicated near the ends of multiple chromosomes. They exhibit remarkable polymorphism: the number and location of large blocks can vary among individuals. Because these segments can contain genes, the compositional variability of subtelomeric DNA may have phenotypic consequences. A combination of molecular and cytogenetic techniques is currently being used to unravel the structure and function of these highly dynamic regions of the genome.

In addition, we are studying the large and complex duplications encompassing members of the olfactory receptor gene family. Members of this large gene family are distributed over 40 sites in the human genome, yet each sensory neuron expresses only one gene. In order to determine how the expressed repertoire of olfactory receptors has evolved and is regulated, we are analyzing the genomic organization and function of these genes in mouse and man.

Selected Publications:

McPherson JD, Marra M, Hillier L, Waterston RH, Chinwalla A, Wallis  J, Sekhon M, Wylie K, Mardis ER, Wilson RK, Fulton R, Kucaba TA,  Wagner-McPherson C, Barbazuk WB, Gregory SG, Humphray SJ, French L,  Evans RS, Bethel G, Whittaker A, Holden JL, McCann OT, Dunham A,  Soderlund C, Scott CE, Bentley DR, Schuler G, Chen HC, Jang W, Green  ED, Idol JR, Maduro VV, Montgomery KT, Lee E, Miller A, Emerling S,  Kucherlapati, Gibbs R, Scherer S, Gorrell JH, Sodergren E, Clerc- Blankenburg K, Tabor P, Naylor S, Garcia D, de Jong PJ, Catanese JJ,  Nowak N, Osoegawa K, Qin S, Rowen L, Madan A, Dors M, Hood L, Trask  B, Friedman C, Massa H, Cheung VG, Kirsch IR, Reid T, Yonescu R,  Weissenbach J, Bruls T, Heilig R, Branscomb E, Olsen A, Doggett N,  Cheng JF, Hawkins T, Myers RM, Shang J, Ramirez L, Schmutz J,  Velasquez O, Dixon K, Stone NE, Cox DR, Haussler D, Kent WJ, Furey T,  Rogic S, Kennedy S, Jones S, Rosenthal A, Wen G, Schilhabel M,  Gloeckner G, Nyakatura G, Siebert R, Schlegelberger B, Korenberg J,  Chen XN, Fujiyama A, Hattori M, Toyoda A, Yada T, Park HS, Sakaki Y,  Shimizu N, Asakawa S, Kawasaki K, Sasaki T, Shintani A, Shimizu A,  Shibuya K, Kudoh J, Minoshima S, Ramser J, Seranski P, Hoff C,  Poustka A, Reinhardt R, Lehrach H. A physical map of the human  genome. Nature 409:934-941, 2001.

Olivier M, Aggarwal A, Allen J, Almendras AA, Bajorek ES, Beasley EM,  Brady SD, Bushard JM, Bustos VI, Chu A, Chung TR, De Witte A, Denys  ME, Dominguez R, Fang NY, Foster BD, Freudenberg RW, Hadley D,  Hamilton LR, Jeffrey TJ, Kelly L, Lazzeroni L, Levy MR, Lewis SC, Liu  X, Lopez FJ, Louie B, Marquis JP, Martinez RA, Matsuura MK, Misherghi  NS, Norton JA, Olshen A, Perkins SM, Perou AJ, Piercy C, Piercy M,  Qin F, Reif T, Sheppard K, Shokoohi V, Smick GA, Sun WL, Stewart EA,  Fernando J, Tejeda, Tran NM, Trejo T, Vo NT, Yan SC, Zierten DL, Zhao  S, Sachidanandam R, Trask BJ, Myers RM, Cox DR. A high-resolution  radiation hybrid map of the human genome draft sequence. Science  291:1298-1302, 2001.

Bailey JA, Yavor AM, Massa HF, Trask BJ, Eichler EE. Segmental  duplications: organization and impact within the current human genome  project assembly. Genome Res 11:1005-1017, 2001.

Kouros-Mehr H, Pintchovski S, Melnyk J, Chen YJ, Friedman C, Trask B,  Shizuya H. Identification of non-functional human VNO receptor genes  provides evidence for vestigiality of the human VNO. Chem Senses  26:1167-1174, 2001.

Lane RP, Roach JC, Lee IY, Boysen C, Smit A, Trask BJ, Hood L.  Genomic analysis of the olfactory receptor region of the mouse and  human T-cell receptor alpha/delta loci. Genome Res 12:81-87, 2002.

Lane RP, Cutforth T, Axel R, Hood L, Trask BJ. Sequence analysis of  mouse vomeronasal receptor gene clusters reveals common promoter  motifs and a history of recent expansion. Proc Natl Acad Sci U S A  99:291-296, 2002.

Young JM, Friedman C, Williams EM, Ross JA, Tonnes-Priddy L, Trask  BJ. Different evolutionary processes shaped the mouse and human  olfactory receptor gene families. Hum Mol Genet 11:535-546, 2002.

Mefford HC, Trask BJ. The complex structure and dynamic evolution of  human subtelomeres. Nat Rev Genet 3:91-102, 2002.

Gygi MP, Ferguson MD, Mefford HC, Lund KP, O’Day C, Zhou P, Friedman  C, van den Engh G, Stolowitz ML, Trask BJ. Use of fluorescent  sequence-specific polyamides to discriminate human chromosomes by  microscopy and flow cytometry. Nucleic Acids Res 30:2790-2799, 2002.

Tanaka H, Tapscott SJ, Trask BJ, Yao MC. Short inverted repeats  initiate gene amplification through the formation of a large DNA  palindrome in mammalian cells. Proc Natl Acad Sci U S A 99:8772-8777,  2002.

Sultana R, Yu CE, Yu J, Munson J, Chen D, Hua W, Estes A, Cortes F,  de la Barra F, Yu D, Haider ST, Trask BJ, Green ED, Raskind WH,  Disteche CM, Wijsman E, Dawson G, Storm DR, Schellenberg GD,  Villacres EC. Identification of a novel gene on chromosome 7q11.2  interrupted by a translocation breakpoint in a pair of autistic  twins. Genomics 80:129-134, 2002.

Trask BJ. Human cytogenetics: 46 chromosomes, 46 years and counting.  Nat Rev Genet 3:769-778, 2002.

Fan Y, Linardopoulou E, Friedman C, Williams E, Trask BJ. Genomic  structure and evolution of the ancestral chromosome fusion site in  2q13-2q14.1 and paralogous regions on other human chromosomes. Genome  Res 12:1651-1662, 2002.

Fan Y, Newman T, Linardopoulou E, Trask BJ. Gene content and function  of the ancestral chromosome fusion site in human chromosome  2q13-2q14.1 and paralogous regions. Genome Res 12:1663-1672, 2002.

Newman T, Trask BJ. Complex evolution of 7E olfactory receptor genes  in segmental duplications. Genome Res 13:781-793, 2003.

Heilig R, Eckenberg R, Petit JL, Fonknechten N, Da Silva C, Cattolico  L, Levy M, Barbe V, de Berardinis V, Ureta-Vidal A, Pelletier E, Vico  V, Anthouard V, Rowen L, Madan A, Qin S, Sun H, Du H, Pepin K,  Artiguenave F, Robert C, Cruaud C, Bruls T, Jaillon O, Friedlander L,  Samson G, Brottier P, Cure S, Segurens B, Aniere F, Samain S,  Crespeau H, Abbasi N, Aiach N, Boscus D, Dickhoff R, Dors M, Dubois  I, Friedman C, Gouyvenoux M, James R, Mairey-Estrada B, Mangenot S,  Martins N, Menard M, Oztas S, Ratcliffe A, Shaffer T, Trask B,  Vacherie B, Bellemere C, Belser C, Besnard-Gonnet M, Bartol-Mavel D,  Boutard M, Briez-Silla S, Combette S, Dufosse-Laurent V, Ferron C,  Lechaplais C, Louesse C, Muselet D, Magdelenat G, Pateau E, Petit E,  Sirvain-Trukniewicz P, Trybou A, Vega-Czarny N, Bataille E, Bluet E,  Bordelais I, Dubois M, Dumont C, Guerin T, Haffray S, Hammadi R,  Muanga J, Pellouin V, Robert D, Wunderle E, Gauguet G, Roy A, Sainte- Marthe L, Verdier J, Verdier-Discala C, Hillier L, Fulton L,  McPherson J, Matsuda F, Wilson R, Scarpelli C, Gyapay G, Wincker P,  Saurin W, Quetier F, Waterston R, Hood L, Weissenbach J. The DNA  sequence and analysis of human chromosome 14. Nature 421:601-607, 2003.

Young JM, Shykind BM, Lane RP, Tonnes-Priddy L, Ross JA, Walker M,  Williams EM, Trask BJ. Odorant receptor expressed sequence tags  demonstrate olfactory expression of over 400 genes, extensive  alternate splicing and unequal expression levels. Genome Biol 4:R71,  2003.

Gibbs RA, Weinstock GM, Metzker ML, Muzny DM, Sodergren EJ, Scherer  S, Scott G, Steffen D, Worley KC, Burch PE, Okwuonu G, Hines S, Lewis  L, DeRamo C, Delgado O, Dugan-Rocha S, Miner G, Morgan M, Hawes A,  Gill R, Celera, Holt RA, Adams MD, Amanatides PG, Baden-Tillson H,  Barnstead M, Chin S, Evans CA, Ferriera S, Fosler C, Glodek A, Gu Z,  Jennings D, Kraft CL, Nguyen T, Pfannkoch CM, Sitter C, Sutton GG,  Venter JC, Woodage T, Smith D, Lee HM, Gustafson E, Cahill P, Kana A,  Doucette-Stamm L, Weinstock K, Fechtel K, Weiss RB, Dunn DM, Green  ED, Blakesley RW, Bouffard GG, De Jong PJ, Osoegawa K, Zhu B, Marra  M, Schein J, Bosdet I, Fjell C, Jones S, Krzywinski M, Mathewson C,  Siddiqui A, Wye N, McPherson J, Zhao S, Fraser CM, Shetty J, Shatsman  S, Geer K, Chen Y, Abramzon S, Nierman WC, Havlak PH, Chen R, Durbin  KJ, Egan A, Ren Y, Song XZ, Li B, Liu Y, Qin X, Cawley S, Worley KC,  Cooney AJ, D’Souza LM, Martin K, Wu JQ, Gonzalez-Garay ML, Jackson  AR, Kalafus KJ, McLeod MP, Milosavljevic A, Virk D, Volkov A, Wheeler  DA, Zhang Z, Bailey JA, Eichler EE, Tuzun E, Birney E, Mongin E,  Ureta-Vidal A, Woodwark C, Zdobnov E, Bork P, Suyama M, Torrents D,  Alexandersson M, Trask BJ, Young JM, Huang H, Wang H, Xing H, Daniels  S, Gietzen D, Schmidt J, Stevens K, Vitt U, Wingrove J, Camara F, Mar  Alba M, Abril JF, Guigo R, Smit A, Dubchak I, Rubin EM, Couronne O,  Poliakov A, Hubner N, Ganten D, Goesele C, Hummel O, Kreitler T, Lee  YA, Monti J, Schulz H, Zimdahl H, Himmelbauer H, Lehrach H, Jacob HJ,  Bromberg S, Gullings-Handley J, Jensen-Seaman MI, Kwitek AE, Lazar J,  Pasko D, Tonellato PJ, Twigger S, Ponting CP, Duarte JM, Rice S,  Goodstadt L, Beatson SA, Emes RD, Winter EE, Webber C, Brandt P,  Nyakatura G, Adetobi M, Chiaromonte F, Elnitski L, Eswara P, Hardison  RC, Hou M, Kolbe D, Makova K, Miller W, Nekrutenko A, Riemer C,  Schwartz S, Taylor J, Yang S, Zhang Y, Lindpaintner K, Andrews TD,  Caccamo M, Clamp M, Clarke L, Curwen V, Durbin R, Eyras E, Searle SM,  Cooper GM, Batzoglou S, Brudno M, Sidow A, Stone EA, Venter JC,  Payseur BA, Bourque G, Lopez-Otin C, Puente XS, Chakrabarti K,  Chatterji S, Dewey C, Pachter L, Bray N, Yap VB, Caspi A, Tesler G,  Pevzner PA, Haussler D, Roskin KM, Baertsch R, Clawson H, Furey TS,  Hinrichs AS, Karolchik D, Kent WJ, Rosenbloom KR, Trumbower H,  Weirauch M, Cooper DN, Stenson PD, Ma B, Brent M, Arumugam M,  Shteynberg D, Copley RR, Taylor MS, Riethman H, Mudunuri U, Peterson  J, Guyer M, Felsenfeld A, Old S, Mockrin S, Collins F.  Genome  sequence of the Brown Norway rat yields insights into mammalian  evolution. Nature 428:493-521, 2004.

Lane RP, Young J, Newman T, Trask BJ. Species specificity in rodent  pheromone receptor repertoires. Genome Res 14:603-608, 2004.

Sebat J, Lakshmi B, Troge J, Alexander J, Young J, Lundin P, Maner S,  Massa H, Walker M, Chi M, Navin N, Lucito R, Healy J, Hicks J, Ye K,  Reiner A, Gilliam TC, Trask B, Patterson N, Zetterberg A, Wigler M.  Large-scale copy number polymorphism in the human genome. Science  305:525-528, 2004.

Loo LW, Grove DI, Williams EM, Neal CL, Cousens LA, Schubert EL,  Holcomb IN, Massa HF, Glogovac J, Li CI, Malone KE, Daling JR, Delrow  JJ, Trask BJ, Hsu L, Porter PL. Array comparative genomic  hybridization analysis of genomic alterations in breast cancer  subtypes. Cancer Res 64:8541-8549, 2004.

Young JM, Kambere M, Trask BJ, Lane RP. Divergent V1R repertoires in  five species: Amplification in rodents, decimation in primates, and a  surprisingly small repertoire in dogs. Genome Res 15:231-240, 2005.

Rowen L, Williams E, Glusman G, Linardopoulou E, Friedman C, Ahearn  ME, Seto J, Boysen C, Qin S, Wang K, Kaur A, Bloom S, Hood L, Trask  BJ. Interchromosomal segmental duplications explain the unusual  structure of PRSS3, the gene for an inhibitor-resistant trypsinogen.  Mol Biol Evol 22:1712-1720, 2005.

Linardopoulou EV, Williams EM, Fan Y, Friedman C, Young JM, Trask  BJ.  Human subtelomeres are hot spots of interchromosomal  recombination and segmental duplication.  Nature 437:94-100, 2005.

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Research

One of the main questions addressed in Dr. Furlong’s laboratory is the genetic variability of insecticide metabolism and sensitivity in humans. His research group has cloned and sequenced the cDNA’s from humans, rabbits, and mice that encode paraoxonase (PON1) an HDL-associated enzyme that inactivates the toxic metabolites of several toxic organophosphate pesticides and nerve agents. The molecular basis of the genetic polymorphism in humans that specifies high or low metabolism has been identified. Dr. Furlong has worked closely with Dr. Lucio Costa in developing a mouse model that has provided valuable information on the role of PON1 in protecting against insecticide exposure.

Recently Dr. Nickerson (Genome Sciences, UW) sequenced the PON1 genes of more than forty individuals and identified close to 200 new polymorphisms. The role of this enzyme in lipid metabolism is also being investigated. A collaborative effort with Dr. Gail Jarvik has shown that low plasma levels of PON1 are a risk factor for carotid artery disease. Since the PON1 family of enzymes appear to play an important role in protecting against oxidative stress, the role of these enzymes as risk factors for other diseases is also being investigated.

A second area of interest is the development of biosensors that have applications in medicine and environmental monitoring. They have developed a fully portable surface plasmon resonance (SPR)-based sensor system that can monitor up to 24 analytes at a time. 

Selected Publications

Shih, D.M., Gu, L., Y.-R. Xia, M. Mavab, W.-F. Li, S. Hama, L.W. Castellani, C.E. Furlong, L.G. Costa, A.M. Fogelman and A.J. Lusis. 1998. Serum paraoxonase knockout mice are susceptible to organophosphate insecticides and lipoprotein oxidation. Nature 394: 284-287.

Li, W.-F., L.G. Costa and C.E. Furlong. 1997. Paraoxonase (Pon1) gene in mice: sequencing, chromosomal location, and developmental expression. Pharmacogenet. 7: 137-144.

Davies, H., R.J. Richter, M. Keifer, C. Broomfield, J. Sowalla and C.E. Furlong. 1996. The effect of the human serum paraoxonase polymorphism is reversed with diazoxon, soman and sarin. Nature Genetics 14: 334-336.

Humbert, R., D.A. Adler, C.M. Disteche, C. Hassett, C.J. Omiecinski, and C.E. Furlong. 1993. The molecular basis of the human serum paraoxonase activity polymorphism. Nat. Genet. 3: 73-76.

Furlong, C.E. and J.A. Sundstrom. 1989. Immobilized cell bioreactors for producing immobilized protein bioreactors. Developments in Indust. Microbiol. 30: 141-148.
(J. Industrial Micro., Suppl. No. 4)

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