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Molecular Medical Parasitology
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Molecular Medical Parasitology Edited by J. Joseph Marr, M.D. President, BioMed., Estes Park, Colorado 80517, USA Timothy W. Nilsen, Ph.D. Case Western Reserve University School of Medicine, Cleveland, Ohio 43606, USA Richard W. Komuniecki, Ph.D. Department of Biology, University of Toledo, Toledo, Ohio 43606, USA Amsterdam • Boston • London • New York • Oxford • Paris San Diego • San Francisco • Singapore • Sydney • Tokyo
This book is printed on acid-free paper. Copyright 2003, Elsevier Science Ltd. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Academic Press An Imprint of Elsevier Science 84 Theobald’s Road, London WC1X 8RR, UK http://www.academicpress.com Academic Press An Imprint of Elsevier Science 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com ISBN 0–12–473346–8 Library of Congress Catalog Number: 2002111022 A catalogue record for this book is available from the British Library Cover ﬁgure: Adapted from The International Journal for Parasitology, Vol. 31, Issue 12 (October 2001), 1343–1353, Figure 1A, courtesy of Dr K. A. Joiner and The Australian Society for Parasitology Inc. Typeset by Charon Tec Pvt. Ltd, Chennai, India Printed and bound in Great Britain by MPG Books, Bodmin, Cornwall 02 03 04 05 06 07 MP 9 8 7 6 5 4 3 2 1
Contents List of contributors vii Preface xi I. MOLECULAR BIOLOGY 1 1. Parasite genomics 3 Mark Blaxter 2. RNA processing in parasitic organisms: trans-splicing and RNA editing 29 Jonatha M. Gott and Timothy W. Nilsen 3. Transcription 47 Arthur Günzl 4. Post-transcriptional regulation 67 Christine Clayton 5. Antigenic variation in African trypanosomes and malaria 89 George A.M. Cross 6. Genetic and genomic approaches to the analysis of Leishmania virulence 111 Stephen M. Beverley II. BIOCHEMISTRY AND CELL BIOLOGY: PROTOZOA 123 7. Energy metabolism Part I: Anaerobic protozoa 125 Miklós Müller Part II: Aerobic protists – trypanosomatidae 140 Fred R. Opperdoes and Paul A.M. Michels Part III: Energy metabolism in the Apicomplexa 154 Michael J. Crawford, Martin J. Fraunholz and David S. Roos v
vi CONTENTS 8. Amino acid and protein metabolism 171 Juan José Cazzulo 9. Purine and pyrimidine transport and metabolism 197 Nicola S. Carter, Nicolle Rager and Buddy Ullman 10. Trypanosomatid surface and secreted carbohydrates 225 Salvatore J. Turco 11. Intracellular signaling 241 Larry Ruben, John M. Kelly and Debopam Chakrabarti 12. Plastids, mitochondria, and hydrogenosomes 277 Geoffrey Ian McFadden III. BIOCHEMISTRY AND CELL BIOLOGY: HELMINTHS 295 13. Helminth surfaces: structural, molecular and functional properties 297 David P Thompson and Timothy G. Geary . 14. Carbohydrate and energy metabolism in parasitic helminths 339 Richard Komuniecki and Aloysius G.M. Tielens 15. Neurotransmitters 359 Richard J. Martin, Jennifer Purcell, Tim Day and Alan P Robertson . IV. MEDICAL APPLICATIONS 395 16. Drug resistance in parasites 397 Marc Ouellette and Steve A. Ward 17. Medical implications of molecular parasitology 433 Richard D. Pearson, Erik L. Hewlett and William A. Petri, Jr. Index 463 Colour plates appear between pages 252 and 253
List of contributors Mark Blaxter, Ph.D. Juan José Cazzulo, Ph.D. Institute of Cell, Animal and Population Instituto de Investigaciones Biotecnológicas Biology Universidad Nacional de General San Martín Darwin Building Av. General Paz y Albarellos King’s Buildings INTI, Ediﬁcio 24, Casilla de Correo 30 University of Edinburgh (1650) San Martín, Provincia de Buenos Aires West Mains Road Argentina Edinburgh EH9 3JT UK Debopam Chakrabarti, Ph.D. Associate Professor Stephen M. Beverley, Ph.D. Department of Molecular Biology & Professor and Chairman Microbiology, University of Central Florida 760B McDonnell Science Building 12722 Research Parkway Box 8230 Orlando, Fl 32826 Department of Molecular Microbiology USA Washington University School of Medicine 660 South Euclid Avenue Christine Clayton, B.A., Ph.D. St. Louis, MO 63110-1093 Zentrum für Molekulare Biologie USA Im Neuenheimer Feld 282 D-69120 Heidelberg Nicola S. Carter, Ph.D. Germany Assistant Professor Department of Biochemistry and Molecular Michael J. Crawford, Ph.D. Biology Department of Biology Oregon Health and Sciences University University of Pennsylvania 3181 S. W. Sam Jackson Park Road 415 South University Avenue Portland, OR 97239 Philadelphia, PA 19104-6018 USA USA vii
viii LIST OF CONTRIBUTORS George A.M. Cross, Ph.D. Arthur Günzl, Ph.D. Andre and Bella Meyer Professor Associate Professor of Genetics and Laboratory of Molecular Parasitology Developmental Biology (Box 185) Center for Microbial Pathogenesis The Rockefeller University University of Connecticut Health Center 1230 York Avenue 262 Farmington Avenue New York, NY 10021-6399 Farmington, CT 06030-8130 USA USA Tim Day, Ph.D. Erik L. Hewlett, M.D. Assistant Professor Professor of Medicine and Pharmacology Biomedical Sciences University of Virginia School of Medicine College of Veterinary Medicine Box 800419 School of Medicine Christensen Drive University of Virginia Iowa State University Charlottesville, VA 22908 Ames, IA 50011 USA USA John M. Kelly, Ph.D. Pathogen Molecular Biology and Martin J. Fraunholz, Ph.D. Biochemistry Unit Department of Biology Department of Infectious and Tropical University of Pennsylvania Diseases 415 South University Avenue London School of Hygiene and Tropical Philadelphia, PA 19104-6018 Medicine USA Keppel Street London WC1E 7HT Timothy G. Geary, Ph.D. UK Discovery Research Pharmacia Corp. Richard W. Komuniecki, Ph.D. 301 Henrietta St. Distinguished University Professor of Mailstop 7923-25-423 Biological Sciences Kalamazoo, MI 49006 Department of Biological Sciences USA University of Toledo 2801 West Bancroft St. Jonatha M. Gott, Ph.D. Toledo, OH 43606 Center for RNA Molecular Biology USA Case Western Reserve University School of Medicine J. Joseph Marr, M.D. 10900 Euclid Avenue 180 Centennial Dr. Cleveland, OH 44106-4960 Estes Park, CO 80517 USA USA
LIST OF CONTRIBUTORS ix Richard. J. Martin, Ph.D. Fred R. Opperdoes, Ph.D. Professor and Chair Research Unit for Tropical Diseases and Biomedical Sciences Laboratory of Biochemistry College of Veterinary Medicine Christian de Duve Institute of Cellular Christensen Drive Pathology Iowa State University Catholic University of Louvain Ames, IA 50011-1250 Avenue Hippocrate 74–75 USA B-1200 Brussels Belgium Paul A.M. Michels, Ph.D. Research Unit for Tropical Diseases Christian de Duve Institute of Cellular Marc Ouellette, Ph.D. Pathology MRC Scientist Catholic University of Louvain Burroughs Wellcome Fund Scholar Avenue Hippocrate 74–75 Centre de Recherche en Infectiologie B-1200 Brussels CHUQ, pavillon CHUL Belgium 2705 Boul. Laurier Québec, QuéG1V 4G2 Canada Geoffrey Ian McFadden, Ph.D. ARC Professorial Fellow Plant Cell Biology Research Centre Richard D. Pearson, M.D. School of Botany Professor of Medicine and Pathology University of Melbourne Division of Infectious Diseases and Victoria 3010 International Health, Box 801378 Australia Departments of Internal Medicine and Pathology Miklós Müller, M.D. University of Virginia Laboratory of Biochemical Parasitology School of Medicine The Rockefeller University University of Virginia Health System 1230 York Avenue Charlottesville, VA 22908 New York, NY 10021-6399 USA USA Timothy W. Nilsen, Ph.D. William A. Petri, Jr., M.D., Ph.D. Professor and Director Professor of Medicine, Microbiology, and Center for RNA Molecular Biology Pathology Case Western Reserve University University of Virginia Health System School of Medicine MR4 Building, Room 2115 10900 Euclid Avenue P.O. Box 801340 Cleveland, OH 44106-4960 Charlottesville, VA 22908-1340 USA USA
x LIST OF CONTRIBUTORS Jenny Purcell, Ph.D. David P. Thompson, Ph.D. Department of Preclinical Discovery Research Veterinary Sciences R. (D.) S. V. S., Summerhall Pharmacia Corp. University of Edinburgh 301 Henrietta St. Edinburgh EH91QH Mailstop 7923-25-410 UK Kalamazoo, MI 49007 USA Nicolle Rager, B.A. Senior Research Assistant Aloysius G.M. Tielens, Ph.D. Department of Biochemistry and Molecular Department Biochemistry and Cell Biology Biology Faculty of Veterinary Medicine Oregon Health and Sciences University Utrecht University 3181 S. W. Sam Jackson Park Road P.O. Box 80176 Portland, OR 97239 3508 TD Utrecht USA The Netherlands Alan P. Robertson, Ph.D. Salvatore J. Turco, Ph.D. Adjunct Assistant Professor Anthony S. Turco Professor of Biochemistry Biomedical Sciences Department of Biochemistry College of Veterinary Medicine University of Kentucky Medical Center Christensen Drive Lexington, KY 40536 Iowa State University USA Ames, IA 50011 USA Buddy Ullman, Ph.D. David S. Roos, Ph.D. Department of Biochemistry and Merriam Professor of Biology Molecular Biology Director, Genomics Institute Oregon Health and Sciences University University of Pennsylvania 3181 S. W. Sam Jackson Park Road 415 South University Avenue Portland, OR 97239 Philadelphia, PA 19104-6018 USA USA Steve Ward Larry Ruben, Ph.D. Walter Myers Professor of Parasitology Professor and Chairman Liverpool School of Tropical Medicine Department of Biological Sciences Pembroke Place Southern Methodist University Liverpool L35 5QA 6501 Airline UK Dallas, TX 75275 USA
Preface Parasitology was born as the tropical stepchild some discussions, traditional taxonomy, which of medicine but has become a well recognized grouped certain organisms according to simi- scientiﬁc and medical discipline in its own right larities in morphology or disease processes, has in our increasingly globally conscious world. not been adhered to rigorously. This has been It began as a descriptive medical curiosity but done judiciously in order to emphasize the the remarkable adaptive mechanisms evinced universality of biochemical and molecular by these astoundingly versatile organisms have biological mechanisms. Wherever appropriate, stimulated signiﬁcant research. Many advances information from one chapter has been cross- in basic science have come from the study of referenced to another in order to strengthen this increasingly fascinating, phylogenetically the important molecular relationships among diverse group of organisms. Parasitology, in the groups. past decade, has undergone another conse- The ﬁrst section, entitled Molecular Biology, quential metamorphosis. The entry of molecu- opens with a chapter on genomics that is the lar biology with its elucidation of the genetics, stage on which the next ﬁve chapters play. genomics, and proteomics of these organisms These chapters include RNA editing and pro- has provided increasingly sophisticated expla- cessing, transcription, and post-transcriptional nations of their capacities to persist under events and describe the interplay of molecular intense ecological and physiological pressures. biology and physiology that is manifest in such Molecular Medical Parasitology had its incep- speciﬁc topics as antigenic variation of African tion in an earlier volume entitled Biochemistry trypanosomes and the genetics of virulence. and Molecular Biology of Parasites. This earlier The second section encompasses the bio- work has been subsumed in the present text. chemistry and cell biology of the protozoa Molecular Medical Parasitology presents para- and then the helminths. Energy metabolism, sitology in the context of current molecular biol- probably the most thoroughly studied aspect ogy, biochemistry and cell biology. Throughout of the biochemistry of these organisms, is pre- the text, emphasis has been placed on the com- sented ﬁrst in each part. In the sub-section on monality of biochemical and cellular biological protozoa, chapters on amino acid and nucleic processes among these varied organisms. In acid metabolism are followed by speciﬁc topics xi
xii PREFACE of special interest including surface antigens, environmental pressures. It has become a intracellular signaling, and intracellular signiﬁcant medical problem in recent years. organelles, each with a special emphasis on The chapter on therapy discusses the implica- the commonalities and notable differences tions of the basic science presented in the ear- in the genomics of the organisms involved. In lier sections as well as speciﬁcs of treatment. the sub-section on helminths, the chapter on This is the ﬁrst parasitology text that inte- energy metabolism is followed by an impor- grates current molecular biology, biochem- tant chapter on neurotransmitters and their istry, and cell biology with the control of these receptors. These are critical to the parasite in heterogeneous organisms. The authors are maintaining its niche in the host and, from a among the best in their respective ﬁelds and medical perspective, are major therapeutic the knowledgeable scientist will recognize targets. This section concludes with a chapter their contributions. They have written clearly, on the structure and function of helminth sur- comprehensively, and well. Presentations by faces with emphasis the anatomy and physiol- these seasoned investigators should be of ogy of these critical interfaces that protect the interest to the experienced scientist, the grad- parasites from most host defenses. uate student, and the physician. Throughout the volume, the authors and We must list ﬁrst among the acknowledge- editors have emphasized the actual or poten- ments, our authors. Much credit, however, tial medical importance of major biochemical must go to Ms. Claire Minto, an extraordinary or molecular biological advances. These con- editor, who has been an exceptional resource siderations are expanded in the third section. in the preparation of this book. The ﬁrst chapter is on drug resistance, which, in fact, is a medical manifestation of molecu- J. Joseph Marr, M.D. lar biology and biochemistry bringing about Richard W. Komuniecki, Ph.D. alterations in the cell biology as a result of Timothy W. Nilsen, Ph.D.
S E C T I O N I MOLECULAR BIOLOGY
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C H A P T E R 1 Parasite genomics Mark Blaxter Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, UK INTRODUCTION genomes: how does the organism self-assemble given this set of genotypic data? These two Genomics, like parasitology, is a research ﬁeld sectors overlap, as resource generation neces- that thrives on the intersection between differ- sarily underlies the testing of hypotheses of ent disciplines. Parasitologists study a phylo- genome-wide function. While the methodolo- genetically disparate assemblage of organisms gies used to analyse the genomes of proto- chosen from global diversity on the basis of zoan, nematode and platyhelminth genomes their trophic relationships to other ‘host’ organ- may differ because of the ways the genomes isms, and use the tools and paradigms of bio- of these organisms are organized, the aims of chemistry, molecular biology, physiology and programs on individual species are in general behaviour (amongst others) to illuminate the the same: biology of these important taxa. Genomics uses data arising from karyotypic analysis, genetic 1. The determination of the complete sequence and physical mapping of traits and anonymous of the chromosomal (and plastid) genome of markers, DNA sequencing and bioinformatic the organism prediction of function-structure relationships. 2. The identiﬁcation of the coding genes (both The meld of parasitology and genomics is thus protein and RNA) on the sequence (also necessarily and productively hybrid. termed ‘gene discovery’) Genomics research in parasitology can 3. The prediction of function of each of the be divided, pragmatically, into two sectors. genes, and the prediction of function of One is a drive to generate resources: clone operator/promoter/control regions in the banks, sequence, annotated genes, functional non-coding DNA genomics platforms. The other is a hypothesis- 4. The integration of functional, sequence and driven search for pattern and process in architectural information into biological the structure, expression and evolution of models of the structure of the chromosomes Molecular Medical Parasitology Copyright © 2003 Elsevier Science Ltd. ISBN 0–12–473346–8 3 All rights of reproduction in any form reserved.
4 PARASITE GENOMICS and of the interaction between the expressed THE SIZE OF THE PROBLEM parts of the genome 5. Investigating natural variation in the Bacterial genomes are relatively small (0.6 to genome in the context of the host, popula- 15 Mb) compared to those of eukaryotes tion structure, drug treatment and other (10 Mb to Ͼ10 000 Mb). Parasitic eukaryote selective forces. genomes range from ϳ9 Mb (Theileria) to 5000 Mb (Ascaris) and above (Table 1.1). The Along this difﬁcult path additional goals can be number of genes encoded by a genome is found, such as the identiﬁcation of candidate roughly proportional to its size, but is modi- sequences, genes, or gene products that may ﬁed by the presence of intronic DNA and of be of utility in diagnosis, surveillance, drug tar- junk, or non-coding repetitive DNA. For exam- geting or vaccine component development. ple, while the genome of the nematode Caeno- Genomics and genome sequencing is still a rhabditis elegans is 100 Mb, and contains 20 000 young ﬁeld. The ﬁrst genomes sequenced protein coding genes, the human genome is were those of parasites: viruses infecting bac- 3000 Mb (30-fold larger) but encodes only teria (phiX174 and lambda phage are land- 30 000–40 000 genes. The average gene density marks). Progress to whole genome sequencing in C. elegans is thus about one gene per 5 kb, of self-reproducing organisms had as stepping- while in humans it is one gene per Ͼ70 kb. stones the determination of the complete Protozoa have relatively small genomes that genome sequence of the human mitochon- are often rich in non-coding repeats, and are drion (again relevant to parasitology as mito- likely to have in the region of 6000 to 15 000 chondria arise from an ancient symbiotic protein coding genes. Parasitic nematodes event). The ﬁrst genome sequence determined have genomes of a similar size to C. elegans in for a self-reproducing organism was that of the main, but several species have much larger Helicobacter pylori, an important human- DNA contents per haploid genome. In Ascaris pathogenic bacterium. In the ﬁeld of bacterial and related taxa, the genome is both highly genomics, the focus has remained on patho- repetitive and much larger than that of C. genic species, and most of the over 100 elegans. Overall, parasitic nematodes are likely sequenced genomes are from human patho- to have similar gene complements to C. elegans gens. For parasitology, these genomes give (20 000). The genomes of platyhelminths are insight into the differences at the level of the much less well known, but Schistosoma species genome between free-living bacteria (such as have genomes of ϳ270 Mb that are rich in Escherichia coli) and endoparasitic bacteria repetitive sequences. Again the gene count is (such as the Chlamydiae and Rickettsiales). likely to be in the 20 000 range. Arthropod Importantly, it is now technically feasible to parasites have larger genomes than the model sequence the genomes of eukaryotes with arthropod, Drosophila melanogaster, which large genomes (Ͼ20 Mb) and thus several par- has 15 000 genes in a 160 Mb genome. For asite genome projects are underway. As with example, Anopheles has a genome of 280 Mb the sequencing of the nematode Caenorhabdi- but is expected to have a gene count similar to tis elegans, the ﬂy Drosophila melanogaster D. melanogaster. and the human genomes, this will in turn The multitude and phylogenetic diversity of bring about a revolution in the way parasite parasites means that genomic approaches to biology research is done. parasite biology and control need to be carefully MOLECUL AR BIOLOGY
TABLE 1.1 Parasite genomes: genome sizes, karyotype, gene number and genome project status of selected par Species Genome Karyotype Genome survey Genome Methods used size (2n) sequences in sequencing dbGSS status Nematode parasites Brugia malayi 100 Mb 12 18 000 Selected genome segments Ascaris suum 5000 Mb 48 – Haemonchus contortus 100 Mb 12 – Platyhelminth parasites Schistosoma mansoni 270 Mb 14 42 000 Physical map based Apicomplexan parasites Plasmodium falciparum 35 Mb 14 (also 18 000 GSS Completed Whole genome and from other chromosome-b plasmodial shotgun species) Theileria annulata 10 Mb 4 Genome sequencing initiated Trypanosomatid parasites Trypanosoma cruzi 35 Mb 35 21 000 Near completion Chromosome-b Haploid and physical map based Trypanosoma brucei 35 Mb 11 90 000 Near completion Chromosome-b shotgun and physical map based Leishmania major 33.6 Mb 36 15 000 Near completion Chromosome-b shotgun and physical map based Other protozoan parasites Entamoeba histolytica Ͻ20 Mb 14 80 000 Near completion Whole genome shotgun Giardia intestinalis 12 Mb 5 Near completion Whole genome shotgun Vectors of parasites and arthropod parasites Anopheles gambiae 280 Mb 3 60 000 Near completion Whole genome shotgun
6 PARASITE GENOMICS tailored to each target organism. The World (microsatellite sequence tagged sites or restric- Health Organisation in collaboration with the tion fragment length polymorphisms for exam- national funding agencies of both endemic and ple). The result is a linkage map showing the developed countries have therefore sponsored association of the markers and their relative genome projects on target organisms represent- order. This map is of utility in verifying the cor- ing the major human and animal parasitic dis- rectness of related genome maps made at the eases. Each project has used tools based on the physical (DNA) level, as markers placed adja- peculiarities of their system and the knowledge/ cent by genetics should also be adjacent in any skill base present in the interested community. physical map. Genetic mapping is necessarily The parasite genome projects are models of restricted to organisms that reproduce sexu- north–south, endemic–developed cooperation, ally, and operationally is further restricted by and, in this open spirit, most of the data pro- considerations of practicality (is it possible to duced is freely available through the internet to carry out controlled crosses and score prog- interested researchers. eny in the laboratory?). To overcome this need for sex, a method for genetic mapping without sexual recom- GENERATING GENOMICS bination has been developed, called HAPPY DATA mapping. HAPPY mapping is based on the observation that in a population of large DNA Genomics uses data from many sources. The fragments generated by random shearing of parasite genome projects use layers of related a complete genome, the chance that two data types to build ﬁrst physical and genetic sequence tagged markers will be on the same maps of the target genomes, followed by ﬁner individual molecule is proportional to their detail sequence and expression maps, ulti- separation on the genome. This mapping pro- mately yielding an annotated genome. Most of cedure uses PCR-based genotyping assays to the projects are still in the midst of the data screen sub-haploid quantities of sheared DNA generation part of the process (see http://www. for association between markers, and the asso- ebi.ac.uk/parasites/parasite-genome.html for ciation is then used to build a ‘genetic’ map as the latest news on the various parasite genome one would with real genetic data. The beneﬁt projects), and no simple summary will ade- of the HAPPY map is that the markers are quately cover all the projects. The ﬁeld is also cloned and sequenced at the outset, allowing changing extremely rapidly, and a summary rapid progression to complete physical map- given today may be rendered obsolete with ping (see below). tomorrow’s database release. Karyotyping Genetic maps Chromosomes are the units of genome organ- Genetic maps are available for many parasitic ization. Mapping of genes or other molecular organisms. The maps are built by examining markers to physical chromosomes is a useful the genotype of recombinant cross progeny of and often central step in genomics. At a gross marked parents. The markers can be pheno- level, chromosomes can be separated by mor- typic (eye color, resistance to ﬁlarial nema- phology (for example the ﬁlarial nematode sex tode infection) or anonymous genetic markers determining X and Y chromosomes) and by MOLECUL AR BIOLOGY
GENERATING GENOMICS DATA 7 differential banding staining with intercalary Yeast host cells are often more tolerant of dyes. In the protozoa, the chromosomes are skewed base-composition insert DNA, such as often too small to be resolved usefully by that from Plasmodium, and of repeat-rich microscopy, but are within the range that is insert DNA. resolved by pulsed ﬁeld gel electrophoresis The inserts of large-insert clone libraries can (PFGE). PFGE karyotypes are available for all be compared to each other and the overlap data of the major parasitic protozoan species, and used to build a map of the cloned genome, a comparative karyotyping of strains and related physical map. Overlap between clones can be species has yielded valuable information on predicted in two ways. One is derived from conservation of linkage, and patterns of genome restriction enzyme ﬁngerprinting of each clone. evolution. Fluorescence in situ hybridization A ﬁngerprint is the pattern of bands observed (FISH) involves the ‘painting’ of a chromo- when the clone is cut with one or two enzymes. somal copy of a gene with a ﬂuor-labelled Clones containing DNA from the same genomic probe in a preparation of metaphase cells. It is region will share more ﬁngerprint bands than useful in conﬁrming linkage of cloned markers, would be expected to occur by chance, and can and in joining otherwise unlinked segments of be overlapped on the basis of shared fragments. a physical map. For chromosomes separable The other method of building a physical map is by PFGE, Southern hybridization can be used by sequence-tagged site mapping, where the to similar effect. library is screened with probes by hybridization For many organisms, including the nema- or clones are identiﬁed using STS-based PCR. todes, the chromosomes are too large (Ͼ10 Mb) The two methods (ﬁngerprinting and STS to be separated by PFGE and too small to be mapping) can be, and usually are, combined in useful for FISH and banding studies. It is pos- the production of a map. Maps have been sible to separate these chromosomes using a produced or are in production for many para- ﬂuorescence-activated cell sorting instrument, sites. FISH hybridization to spread chromoso- though this technique has not been used yet in mal segments can also be used to build maps, parasite genomics. and for smaller genomes it is possible to con- struct restriction fragment-based maps using stretched chromosomes cut in situ on the slide. Physical maps Physical map construction is compromised It is often useful to have a genomic copy of a by the sheer volume of data that must be pro- gene of interest cloned. Large-insert genomic duced and analysed, and the known sorts of DNA clones can be constructed in a number of confounding errors that can occur. In ﬁnger- different vector–host systems. These range from printing, there is (usually known) error in band lambda bacteriophage (maximal insert capac- size estimation, and two bands can be scored ity ϳ21 kb of foreign DNA), through cosmids as matching by size despite being different in (ϳ35 kb), bacterial artiﬁcial chromosomes (BAC, sequence. The method is very sensitive to the ϳ200 kb) and yeast artiﬁcial chromosomes number of shared bands required to score a (YAC, ϳ3000 kb). Each vector–host combina- real overlap, as too high a score requirement tion also differs in copy number within the will result in failure to link overlapping clones, host cell: in general vectors maintained at low whereas too low a score will result in multiple, copy number tend to be more stable against incompatible overlaps being accepted. In STS recombination, rearrangement and deletion. mapping, errors can arise from the presence of MOLECUL AR BIOLOGY