Human Molecular Genetics, Fourth Edition by Garland Science

new edition

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Human Molecular Genetics

LARGE INSERT

CLONING AND

CLONING SYST

EMS FOR PRO

DUCING SING

MCS

Tom Strachan, Newcastle University, UK Andrew Read, University of Manchester, UK

lacl

LE-STRANDED

DNA

177

lacZ¢

ori M13 vector ~7.3 kb

singlestranded recombinant DNA

inser t target DNA fragments isolation of DNA doublestranded recombinant DNA

Human Molecular Genetics is an established and class-proven textbook for upper-level undergraduates and graduate students which provides an authoritative and integrated approach to the molecular aspects of human genetics. The Fourth Edition has been completely updated so genomic technologies are integrated throughout, and next generation sequencing is included. Genetic testing, screening, approaches to therapy, personalized medicine, and disease models have been brought together in one section. Coverage of cell biology including stem cells and cell therapy, studying gene function and structure, comparative genomics, model organisms, noncoding RNAs and their functions, and epigenetics have all been expanded.

recombinant phage particles exit from cells

transfect doub stranded recom lebinant DNA into host cells

replication of double-strande d recombinant DNA

switch to single stranded DNA synthesis of strand only

transcription

Figure 6.9 Prod ucing single-st phage coat prote randed recombin pUC19 (see Figur ins ant DNA. M13 e 6.4). Target DNA vectors use the enters the norm is inserted in the same lacZ assay al cycle of phag multiple cloning for recombinan e replication to single-strande site (MCS). The t screening as generate num d DNA (+ stran double-strande erou d only) d M13 recombinan s copie the cell without . After packaging s of the genome, t DNA lysis. Single-str with M13 phag before switching anded recombinan e coat protein, removing the to production protein. mature recom t DNA is recov of binan ered by t phage particles Phagemid vec precipitating the exit from extruded phag tors e particles and chemically A small segm ent

of a single-stran inserted into ded filamento a plasmid to us bacteriopha form 6.10 shows the ge genome can pBluescript vect a hybrid vector known as be phage genome a contains all the or as an example. The cho phagemid; Figure and assembly sen segm cis-a cting elements into phage part required for DNA ent of the eral kilobase icle s. The y replication permit successf s long, unlike ul cloning of M13 vectors, unstable. Afte inserts sevin which inse r transformat rts ion of a suit phagemid, the 37 size tend to be able E. coli stra of this bacterial cell S in with a reco s are superin such as phage fecte MOSOME mbinant f1, OF CHRO d with a filamentous secreted from which is requ TIONfor helper phage, providing the NCired the sup D FU AN coa erin E t pro fect UR recombinanST tein ed cells will be . Phage particle RUCTgem t pha a mixture of s ids. The mix used directly helper phage ed single-stran for DNA sequ and ded DNA enc synthesis is desi ing because the pop gned to A primer for initi ulation can be adjacent to the specifically to ating DNA stra en the DNbind , whclon a sequ at us nd ing s enc site. cle e of the phagem oe underg phase nu id vector re ein the inter A double helix neFigu ot 6.10s.pBluescri pt, a phagemid sto pr emid DN . Even in roromatin , the 2 nm thick ing of basic hi nm phag thick ch vectors each contain two vector. The pBluescript series of nd form ch one (ori) origins of replic afand a second one from iling to extended form directed by bi coiled into a 30 ted ation, a standard by a sc the e superco or ly replic plasm ﬁ en ar lame ation gh pp id ntous phage f1. allows the prod ry hi is su g that at is th The f1 origin of uction of singl the genes for phag is in a ve levels of coilin t is formed th s looping and en oe etch of e coat proteins. Targe e-stranded DNA, but the vector o a str lacks least tw nm thick filam n fiber underg r, whicnd t DNA is inserted h is a aging:vecto then used to trans into the phagemid 10 A). arou A pack mati s form trans 2.8 ro rn DN host form re ch First, a tu ed E. of e coli cells. Supe neswith a helper M13 s (Figu unit stocells rinfection of an two DNA ber. Th be packen matin fi nhistone protein e fundamental in just less th e coreto hi t within phag phage allows the recombinan jac aged d ch of th singl th t phagemid e protein coats . Ad B)e-str anded DNA can , and the recom fold of no cleosome is d DNA is coile o molecules ea igure 2.8 gth be recovered as binant in Figure 6.9. The nu uble-strande A; the len eins) (F eins (tw ne prot conserved prot bp) of linker DN ns of the of do sto hi bp gio 7 ht re 14 ly 14 eig core of , all high ort length (8–1 and between cleosomes to l H4 ra d nt sh ce an nu ing B, H3, ted by a n species s show A packag H2A, H2 es are connec both betwee e preparation el of DN s om bl is first lev nucleos ker DNA varie raphs of suita (see 2.8C). Th lin osomes es n microg arance (Figure nal activity. of the e nucle th typ Electro pe m tio us e. ap ip m ads nscr geno rude fro undergo vario ing of be n, and allows tra histones prot orylatio tails can re have a str one that still ly of the co in the histone lation, phosph romatin in a s ch is the on terminal tails ety tional ino acid d to the tably ac The Ntranscrip s that ecific am ification, no s can be boun of Sp . el B) lev od ne protein e local Figure 2.8 nslational m re histo different tra n and th of the co of post- n. As a result, n condensatio variant forms tio ati methyla affects chrom s encode ne gene to enable way that ditional histo uncoiling gene . Ad e high-level activity nucleosom

The pedagogical features include new Key Concepts at the beginning of each chapter and annotated further reading at the conclusion of each chapter, to help readers navigate the wealth of information in this subject.

MCS Plac

lacZ

f1 origin pBluescript vecto r 2.9 kb ori

Amp R

expression

(A) e

H1 histon

ain

looped dom

n of formatio cture stru solenoid

2 nm DNA

10 nm

30 nm chromatin fiber

octameric e cor histone

scaffold

teins

tone pro

of nonhis

DNA

H1 histon

(D) (C) histone octamer

(B)

IMMUNE

H L a or b

g or d

SYSTEM CE

LLS: FU

NCTION

THROUG

RSITY

to a histones ds basic ized helix bin is organ A double chromatin fiber thick DN ling ofb2the N the M al uncoi The 2 nm erphase loc ns. e int es tei uir In ton r. e pro n req fibe e his N omatin B-c gene expressio sic histon er of cor chr ba am nm of nm oct 10 g ell of of 30 an b2 M els N recept h lev (A) Bindin into theFc d round crograph or/ wn in TCEle Ig appe recteins. Hig romatin. is further coiled R ctron mi A wr N that sho hase ch t acidic pro eptoro turns of DN tails. (C) ponds to ein M tw to interp me filament tha class Gru nhistone erminal I nst le helix box corres of almost old of no from oso ding N-t A doub consists to a scaff . Garland ck nucle C ir protru e outer red ber. [(A) adapted MH From DN ll, 5th ed 10 nm thi of DNA attached (B) A nucleosome histones and the the solenoid [th fi omatin of the Ce lor & Francisclass II Figure 2.8 ling, forming a y of of ts. kb chr n CD en log 00 ure tur nm 1 In coi l filam cee ll-med struct ~50–2 g on the 30 lecular Bio ence/Tay MHC undergo ure of ins with cleosoma a-helical CD4 ber showin g the struct (2008) Mo ed. Garland Sci City. (D) adapted iaiste 10 nm nu te the extensive ped doma J et dal.im omatin fi fr men izin 5th into loo give the CD8 mCe ). No 30 nm chr rtant in organ ag —Kansas unll,ity nson A, of the tsLew n fiber to B, and H4 tion view of the of fologrey ig , Tssoceurills CD28 is impo erts B, Joh chromati rsity of Mi H3A, H3 lecular Bio sec on.] er DNA, ., and Alb M(20 nivepr rg, Un icr08) h of H2, ooMo (D) Cross Foundati recogniz ds to link c American Inc rgJak lchei Waterbo (two eac anob e ns e Weot is J et al. ism filaments. tone, which bin ce Th tifi l Lew of m ar en ma lls s A, sy fro n out of re rmissisu m Sci containi on ch as his couerte nucleoso pe s B, Johnso sas City. (C) th ission fro ional H1 vir ac th ert rm dit us Wi ng h pe . ad Alb e of es th ei133 Wi antibod from r surfa that pe —Kan . 39, (A)]. Th . 267, 68. adapted Missouri Figure ce em. Resfo ne ie Am of (B) Ch . s . y s. tra de Sci ce, T rsit LLC 4.2 reign an te cells dicate cells ev ren (1992) Unive Francis the immu 2 Some repres tion Confe terborg, Taylor & immun tigens. TCRs d T-cell recept olved to deal and multip entative lch Federa Jakob Wa Science/ ly with oglobu immuno noglobulin su with th ors (TCR s are he gs, RW We figures by in them perfami of lin globulin (see Fi Proceedin LLC from ly. The gure 4.2 s and othe terodimers th s) that reco is need; they structur do g A (1985) e held tog main is a ba r cell re ca gn 2). from Klu There rrel-li (pink do ether by ceptor at are evolutio ize sequen rry on ar t). e a disulfi ke s ce tw of the chains narily domain Multiple copie de bridg . Unlik o types of TC immun related s from s are e of fou e thi oglobu to bear th R, com the im s str nd in ma importa lin supe both m posed em ny protei uctural nt either been de are limited unoglobulin rfamily ns wi other me immune system of s mb function th superfam ers of the im derived graded intra to recognizing on B cells, m a and b chains munoglo s, but os cellula ily can ha from th t fo or ab re of ign such as bulin rly TC proteins ve differe g is degr Ignt functi adation . Peptides of proteins, an Rs and the ce and d (Section CAM cell adhe ons, d only peptid (Box 4.4) th lls that an appr bind to sion mo 4.2). Sh e af at lecules aded bo variable to a T ce held in a cleft are then tra major histoc opriate size ter they have xes do nsporte ompatib and se on its ou ll carry proteins. mains of immu show the qu d activat ne system b2M, b ing that ing an approp ter surface, th to the surfa ility complex ence 2 -microg ce of lobulin. riate T cell. (MH e MH

TCRab

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the cell. C) receptor C protein pr With th esen (antigen e presenta ts the peptide tion), th ereby (C)

docking protein antibody cell rec

eptor

host cel

Fc receptor

l effector

(B) –Ab

Garland Science April 2010 784 pages 135 tables 550 illustrations Paperback: 978-8153-4149-9 £49.00

H DIVE

+Ab

bacterial toxins

cell receptor

host cel

cell

e.g. ma cropha activatio ge n

Figure 4.2 (A) Inhibi 3 Aspects of antibod ting vir y functi al infec cells by tio on first usi ng docki n. Viruses infec . bind to t ng protei cer ns to membran tain receptors on the e of host plasma bind to cells. An the viral tibodies do them fro (Ab) can m bindin cking proteins to (B) Neutr g to ho st cell rec prevent ali eptors. toxins rel zing toxins. An tib eased fro so stop m invad odies bind to the ing micro (C) Activ m from bindin g to cel bes and ating eff l receptor ec bind an d coat the tor cells. Antib s. odies can large tar surfaces ge of micro effector t cells. Various bes and im cel killer cel ls (notably ma mune system cro ls, mast cel neutrophils, eo phages, natur al ls) sinophil s, and them to carry Fc recep tor bin or IgE an d to the Fc reg s that enable tib ion on IgA receptor odies. Antibod y bindin , IgG, activates lead to g to an the eff cell killin Fc ector cel g l of lytic enzymes, by phagocyto and can sis, or rel death sig ease nals, an d so on .

Contents PART 1: THE BASICS OF DNA, CHROMOSOMES, CeLLS, AND DEVELOPMENT 1. 2. 3. 4. 5.

Nucleic Acid Structure and Gene Expression Chromosome Structure and Function Genes in Pedigrees and Populations Cells and Cell-Cell Communication Principles of Development

PART 2: ANALYZING THE STRUCTURE AND EXPRESSION OF GENES AND GENOMES 6. Amplifying DNA: Cell-based DNA Cloning and PCR 7. Nucleic Acid Hybridization: Principles and Applications 8. Analyzing the Structure and Expression of Genes and Genomes

PART 3: INVESTIGATING THE HUMAN GENOME AND ITS RELATIONSHIP TO OTHER GENOMES 9. Organization of the Human Genome 10. Model Organisms, Comparative Genomics and Evolution 11. Human Gene Expression 12. Studying Gene Function in the Post-Genome Era

PART 4: HUMAN GENETIC VARIATION AND DISEASES 13. Human Genetic Variability and its Consequences 14. Genetic Mapping of Mendelian Characters 15. Mapping Genes Conferring Susceptibility to Complex Disease 16. Identifying Human Disease Genes and Susceptibility Factors 17. Cancer Genetics 125

PART 5: APPLIED HUMAN MOLECULAR GENETICS 18. Genetic Testing of Individuals 19. Pharmacogenetics, Personalized Medicine, and Population Screening 20. Genetic Manipulation of Animals for Modeling Disease and Investigating Gene Function 21. Genetic Approaches to Treating Disease

Hallmark Features l

Clear, yet detailed, full-color illustrations.

Special

Consistent

Topic Boxes covering ethical, legal and social issues.

emphasis on the explanation of principles rather than the presentation of a large number of facts.

Extensive

glossary.

Index

Color-coded

includes names of diseases and disorders. sections for ease of use.

New in the Fourth Edition Extensive detail on all the major model organisms from the perspective of understanding development and gene function and for modeling disease, with comprehensive coverage of genetic modification of mice and other vertebrates. l Completely rewritten chapter on the study of gene function in the post-genome era reflects new technologies and progress since the previous edition such as ChIP-chip or ChIP-on-chip, ChIPSeq, and TAP-tagging. Up-to-date tables describe all the major publicly accessible databases for transcriptomics, proteomics, protein interactions, and conserved elements and protein domains. l Extensive coverage of the recent successes in identifying susceptibility factors in common diseases, including the problem of hidden heritability. l Expanded coverage of pharmacogenetics and personalized medicine. l Comprehensive treatment of comparative genomics applications for identifying both highly conserved noncoding DNA sequences and rapidly evolving sequences. l An extremely comprehensive coverage of genetic approaches to treating disease and the prospects for stem cell therapy, including potential applications of induced pluripotent stem cells. l Comprehensive up-to-date account of the diversity and functions of noncoding RNA. l Coverage of DNA sequencing technologies updated to reflect the most recent developments in next generation sequencing including massively parallel sequencing of amplified DNA and single-molecule sequencing. l Key Concepts at the beginning of each chapter and annotated further reading at the conclusion of each chapter help readers navigate the wealth of information in this subject. l End-of-chapter summaries provide readers with an overview of the material presented, allowing for review and assessment of their understanding. l

(A) wild-type ES cell arrest 739 or 1141 cells in metaphase harvest microcells

irradiate microcells

PEG fuse, select G418 resistant ES cell colonies transchromosomic ES cell, freely segregating Hsa21

739 or 1141 human cell line with Neo tagged Hsa21

inject recipient blastocysts with transchromosomic ES cells

chimeric mice, Hsa21 in ES cellderived tissue

MmuX (B) Figure 20.21a Making the transchromosomic mouse line Tc1 to model Down syndrome. (Courtesy of Elizabeth Fischer and Victor Tybulewicz, University College London)

fREE dOWNLOADABLE rESOURCES All of the figures from Human Molecular Genetics, Fourth Edition will be available upon publication in PowerPoint® and JPEG formats via Classwire. Hsa21

Garland Science Classwire Instructors who adopt Human Molecular Genetics, Fourth Edition for their course will have access to Garland Science Classwire. The Classwire course management system allows instructors to build websites for their courses easily. It also serves as an online archive for instructors’ resources. After registering for Classwire, instructors will be able to download all of the figures from Human Molecular Genetics, Fourth Edition which are available in PowerPoint® and JPEG formats. Instructors may also download resources from other Garland Science HMG4 textbooks. 20.21 Please visit the Garland Science website at www.classwire.com/garlandscience or email garlandUK@tandf.co.uk for additional information on Classwire. Classwire is a trademark of Chalkfree, Inc.

PRAISE FOR PREVIOUS EDITIONS OF HUMAN MOLECULAR GENETICS “This book is an excellent companion for students in human genetics or for researchers that want to gain background and knowledge in this field.” —Human Genetics Journal “The presentation is excellent, the text is easy to read and very up-to-date and the information boxes are an added bonus…the authors should be heartily congratulated on this venture which clearly deserves its success.” —Journal of Neuromuscular Disorders “I would strongly recommend it as the best text to introduce students and scientists to the molecular aspects of human genetics…I congratulate Strachan and Read on an outstanding job.” —Trends in Genetics “This is truly a Rolls Royce amongst textbooks…and it would be difficult to praise it highly enough…” —Molecular Medicine Today “…addresses the gap between introductory textbooks and the primary literature. There’s no other textbook quite like it.” —Nature

The Authors TOM STRACHAN is Scientific Director of the Institute of Human Genetics and Professor of Human

Molecular Genetics at Newcastle University, UK, and is a Fellow of the Academy of Medical Sciences and a Fellow of the Royal Society of Edinburgh. Tom’s early research interests were in multigene family evolution and interlocus sequence exchange, notably in the HLA and 21-hydroxylase gene clusters. While pursuing the latter, he became interested in medical genetics and disorders of development. His most recent research has focused on developmental control of the vertebrate cohesin regulators Nipbl and Mau-2.

ANDREW READ is Emeritus Professor of Human Genetics at Manchester University, UK and a Fellow

of the Academy of Medical Sciences. Andrew has been particularly concerned with making the benefits of DNA technology available to people with genetic problems. He established one of the first DNA diagnostic laboratories in the UK over 20 years ago (it is now one of two National Genetics Reference Laboratories), and was founder chairman of the British Society for Human Genetics, the main professional body in this area. His own research is on the molecular pathology of various hereditary syndromes, especially hereditary hearing loss. Drs. Strachan and Read were recipients of the European Society of Human Genetics Education Award.