new edition

www.garlandscience.com

177

Phagemid vectorsA small segment of a single-stranded fi lamentous bacteriophage genome can be

inserted into a plasmid to form a hybrid vector known as a phagemid; Figure

6.10 shows the pBluescript vector as an example. The chosen segment of the

phage genome contains all the cis-acting elements required for DNA replication

and assembly into phage particles. They permit successful cloning of inserts sev-

eral kilobases long, unlike M13 vectors, in which inserts of this size tend to be

unstable. After transformation of a suitable E. coli strain with a recombinant

phagemid, the bacterial cells are superinfected with a fi lamentous helper phage,

such as phage f1, which is required for providing the coat protein. Phage particles

secreted from the superinfected cells will be a mixture of helper phage and

recombinant phagemids. The mixed single-stranded DNA population can be

used directly for DNA sequencing because the primer for initiating DNA strand

synthesis is designed to bind specifi cally to a sequence of the phagemid vector

adjacent to the cloning site.

lacZ¢lacl

ori

M13 vector~7.3 kb

double-stranded

recombinantDNA

single-stranded

recombinantDNA

inserttarget DNAfragments

transfect double-stranded recombinantDNA into host cells

replication ofdouble-strandedrecombinant DNA

switch to single-stranded DNAsynthesis of

strand only

transcription

phage coat proteins

recombinant phageparticles exit from cells

isolation of DNA

MCS

Figure 6.9 Producing single-stranded recombinant DNA. M13 vectors use the same lacZ assay for recombinant screening as

pUC19 (see Figure 6.4). Target DNA is inserted in the multiple cloning site (MCS). The double-stranded M13 recombinant DNA

enters the normal cycle of phage replication to generate numerous copies of the genome, before switching to production of

single-stranded DNA (+ strand only). After packaging with M13 phage coat protein, mature recombinant phage particles exit from

the cell without lysis. Single-stranded recombinant DNA is recovered by precipitating the extruded phage particles and chemically

removing the protein.

lacZPlac

ori

pBluescript vector2.9 kb

MCS

AmpR

f1origin

Figure 6.10 pBluescript, a phagemid vector. The pBluescript series of phagemid vectors each contain two origins of replication, a standard plasmid

one (ori) and a second one from the fi lamentous phage f1. The f1 origin of

replication allows the production of single-stranded DNA, but the vector lacks

the genes for phage coat proteins. Target DNA is inserted into the phagemid

vector, which is then used to transform host E. coli cells. Superinfection of transformed cells with a helper M13 phage allows the recombinant phagemid

DNA to be packaged within phage protein coats, and the recombinant single-stranded DNA can be recovered as in Figure 6.9.

LARGE INSERT CLONING AND CLONING SYSTEMS FOR PRODUCING SINGLE-STRANDED DNA

Human molecular Genetics

tom stracHan, Newcastle University, UKandrew read, University of Manchester, UK

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.

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.

Garland scienceapril 2010784 pages 135 tables

550 illustrationsPaperback: 978-8153-4149-9

£49.00

125

In cell-mediated immunity, T cells recognize cells containing

fragments of foreign proteinsMicroorganisms such as viruses that penetrate cells and multiply within them

are out of reach of antibodies. T cells evolved to deal with this need; they carry on

their surfaces dedicated T-cell receptors (TCRs) that recognize sequences from

foreign antigens. TCRs are heterodimers that are evolutionarily related to both

immunoglobulins and other cell receptors of the immunoglobulin superfamily

(see Figure 4.22).There are two types of TCR, composed either of a and b chains or of g and d

chains. Unlike the immunoglobulins on B cells, most ab TCRs and the cells that

bear them are limited to recognizing foreign proteins, and only after they have

been degraded intracellularly. Peptides of an appropriate size and sequence

derived from this degradation bind to major histocompatibility complex (MHC)

proteins (Box 4.4) that are then transported to the surface of the cell. With the

peptide held in a cleft on its outer surface, the MHC protein presents the peptide

to a T cell carrying an appropriate TCRab receptor (antigen presentation), thereby

activating that T cell.

Fc receptor

B-cellreceptor/Ig TCRclass IMHC

CD1CD4

CD8CD28

class IIMHC

L

L

H

H

a or ba

b2M

g or d

b2M

a

a b

Fcreceptor

effector cell

host cell

host cell

e.g. macrophageactivation

(C)

(A)

(B)

–Ab

+Ab

–Ab

+Ab

dockingprotein

antibody

cell receptor

cellreceptor

bacterialtoxins

Figure 4.22 Some representatives of

the immunoglobulin superfamily. The

immunoglobulin domain is a barrel-like

structure held together by a disulfi de bridge

(pink dot). Multiple copies of this structural

domain are found in many proteins with

important immune system functions, but

other members of the immunoglobulin

superfamily can have diff erent functions,

such as Ig-CAM cell adhesion molecules

(Section 4.2). Shaded boxes show the

variable domains of immune system proteins. b2M, b2-microglobulin.

Figure 4.23 Aspects of antibody function.

(A) Inhibiting viral infection. Viruses infect

cells by fi rst using docking proteins to

bind to certain receptors on the plasma

membrane of host cells. Antibodies (Ab) can

bind to the viral docking proteins to prevent

them from binding to host cell receptors.

(B) Neutralizing toxins. Antibodies bind to

toxins released from invading microbes and

so stop them from binding to cell receptors.

(C) Activating eff ector cells. Antibodies can

bind and coat the surfaces of microbes and

large target cells. Various immune system

eff ector cells (notably macrophages, natural

killer cells, neutrophils, eosinophils, and

mast cells) carry Fc receptors that enable

them to bind to the Fc region on IgA, IgG,

or IgE antibodies. Antibody binding to an Fc

receptor activates the eff ector cell and can

lead to cell killing by phagocytosis, or release

of lytic enzymes, death signals, and so on.

IMMUNE SYSTEM CELLS: FUNCTION THROUGH DIVERSITY

37

supercoiling to form chromatin. Even in the interphase nucleus, when the DNA

is in a very highly extended form, the 2 nm thick DNA double helix undergoes at

least two levels of coiling that are directed by binding of basic histone proteins.

First, a 10 nm thick fi lament is formed that is then coiled into a 30 nm thick chro-

matin fi ber. The chromatin fi ber undergoes looping and is supported by a scaf-

fold of nonhistone proteins (Figure 2.8A).

The nucleosome is the fundamental unit of DNA packaging: a stretch of

147 bp of double-stranded DNA is coiled in just less than two turns around a

central core of eight histone proteins (two molecules each of the core histones

H2A, H2B, H3, and H4, all highly conserved proteins) (Figure 2.8B). Adjacent

nucleosomes are connected by a short length (8–114 bp) of linker DNA; the length

of the linker DNA varies both between species and between regions of the

genome. Electron micrographs of suitable preparations show nucleosomes to

have a string of beads appearance (Figure 2.8C). This fi rst level of DNA packaging

is the only one that still allows transcriptional activity.

The N-terminal tails of the core histones protrude from the nucleosomes (see

Figure 2.8B). Specifi c amino acids in the histone tails can undergo various types

of post-translational modifi cation, notably acetylation, phosphorylation, and

methylation. As a result, different proteins can be bound to the chromatin in a

way that affects chromatin condensation and the local level of transcriptional

activity. Additional histone genes encode variant forms of the core histones that

N

N

N

N

NN

N

N

2 nmDNA

H1 histone

10 nm

nucleosome

(A)

(B)

(C)

(D)

octameric

histone core

formation of

solenoid structure

30 nmchromatin

fiber

uncoiling to enable

high-level gene

expression

looped domain

scaffold of nonhistone proteins

H1 histone

histone

octamer

DNA

STRUCTURE AND FUNCTION OF CHROMOSOMES

Figure 2.8 From DNA double helix to interphase chromatin. (A) Binding of basic histone proteins. The 2 nm thick DNA double helix binds basic histones to

undergo coiling, forming a 10 nm thick nucleosome fi lament that is further coiled into the 30 nm chromatin fi ber. In interphase the chromatin fi ber is organized

into looped domains with ~50–200 kb of DNA attached to a scaff old of nonhistone acidic proteins. High levels of gene expression require local uncoiling of the

chromatin fi ber to give the 10 nm nucleosomal fi laments. (B) A nucleosome consists of almost two turns of DNA wrapped round an octamer of core histones

(two each of H2, H3A, H3B, and H4). Note the extensive a-helical structure of histones and their protruding N-terminal tails. (C) Electron micrograph of 10 nm

nucleosomal fi laments. (D) Cross section view of the 30 nm chromatin fi ber showing one turn of the solenoid [the outer red box corresponds to that shown in

(A)]. The additional H1 histone, which binds to linker DNA, is important in organizing the structure of the 30 nm chromatin fi ber. [(A) adapted from Grunstein M

(1992) Sci. Am. 267, 68. With permission from Scientifi c American Inc., and Alberts B, Johnson A, Lewis J et al. (2008) Molecular Biology of the Cell, 5th ed. Garland

Science/Taylor & Francis LLC. (B) adapted from Alberts B, Johnson A, Lewis J et al. (2008) Molecular Biology of the Cell, 5th ed. Garland Science/Taylor & Francis

LLC from fi gures by Jakob Waterborg, University of Missouri—Kansas City. (C) courtesy of Jakob Waterborg, University of Missouri—Kansas City. (D) adapted

from Klug A (1985) Proceedings, RW Welch Federation Conference, Chem. Res. 39, 133. With permission from The Welch Foundation.]

125

In cell-mediated immunity, T cells recognize cells containing

fragments of foreign proteinsMicroorganisms such as viruses that penetrate cells and multiply within them

are out of reach of antibodies. T cells evolved to deal with this need; they carry on

their surfaces dedicated T-cell receptors (TCRs) that recognize sequences from

foreign antigens. TCRs are heterodimers that are evolutionarily related to both

immunoglobulins and other cell receptors of the immunoglobulin superfamily

(see Figure 4.22).There are two types of TCR, composed either of a and b chains or of g and d

chains. Unlike the immunoglobulins on B cells, most ab TCRs and the cells that

bear them are limited to recognizing foreign proteins, and only after they have

been degraded intracellularly. Peptides of an appropriate size and sequence

derived from this degradation bind to major histocompatibility complex (MHC)

proteins (Box 4.4) that are then transported to the surface of the cell. With the

peptide held in a cleft on its outer surface, the MHC protein presents the peptide

to a T cell carrying an appropriate TCRab receptor (antigen presentation), thereby

activating that T cell.

Fc receptor

B-cellreceptor/Ig TCRclass IMHC

CD1CD4

CD8CD28

class IIMHC

L

L

H

H

a or ba

b2M

g or d

b2M

a

a b

Fcreceptor

effector cell

host cell

host cell

e.g. macrophageactivation

(C)

(A)

(B)

–Ab

+Ab

–Ab

+Ab

dockingprotein

antibody

cell receptor

cellreceptor

bacterialtoxins

Figure 4.22 Some representatives of

the immunoglobulin superfamily. The

immunoglobulin domain is a barrel-like

structure held together by a disulfi de bridge

(pink dot). Multiple copies of this structural

domain are found in many proteins with

important immune system functions, but

other members of the immunoglobulin

superfamily can have diff erent functions,

such as Ig-CAM cell adhesion molecules

(Section 4.2). Shaded boxes show the

variable domains of immune system proteins. b2M, b2-microglobulin.

Figure 4.23 Aspects of antibody function.

(A) Inhibiting viral infection. Viruses infect

cells by fi rst using docking proteins to

bind to certain receptors on the plasma

membrane of host cells. Antibodies (Ab) can

bind to the viral docking proteins to prevent

them from binding to host cell receptors.

(B) Neutralizing toxins. Antibodies bind to

toxins released from invading microbes and

so stop them from binding to cell receptors.

(C) Activating eff ector cells. Antibodies can

bind and coat the surfaces of microbes and

large target cells. Various immune system

eff ector cells (notably macrophages, natural

killer cells, neutrophils, eosinophils, and

mast cells) carry Fc receptors that enable

them to bind to the Fc region on IgA, IgG,

or IgE antibodies. Antibody binding to an Fc

receptor activates the eff ector cell and can

lead to cell killing by phagocytosis, or release

of lytic enzymes, death signals, and so on.

IMMUNE SYSTEM CELLS: FUNCTION THROUGH DIVERSITY

contentsPart 1: tHe Basics oF dna, cHromosomes, cells, and deVeloPment

1. Nucleic Acid Structure and Gene Expression 2. Chromosome Structure and Function 3. Genes in Pedigrees and Populations 4. Cells and Cell-Cell Communication 5. 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

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 Featuresl Clear, yet detailed, full-color illustrations.

l Special topic Boxes covering ethical, legal and social issues.

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

l extensive glossary.

l index includes names of diseases and disorders.

l Color-coded sections for ease of use.

chimeric mice,Hsa21 in ES cell-

derived tissue

HMG4 20.21

739 or 1141human cell line withNeo tagged Hsa21

arrest 739 or 1141cells in metaphase

harvest microcells irradiatemicrocells

PEG fuse, selectG418 resistant ES

cell coloniesinject recipient

blastocysts withtranschromosomic

ES cellstranschromosomic

ES cell, freelysegregating Hsa21

wild-typeES cell

(A)

(B) MmuX

Hsa21

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 resourcesAll of the figures from Human Molecular Genetics, Fourth edition will be available upon publication in PowerPoint® and JPeG formats via Classwire.

Garland science classwireinstructors 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 textbooks.

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.

new in tHe FourtH editionl 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.

tHe autHorstom 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 Fellowof the Academy of Medical Sciences. Andrew has been particularly concerned with making thebenefits of dnA technology available to people with genetic problems. He established one of the firstdnA diagnostic laboratories in the UK over 20 years ago (it is now one of two national GeneticsReference 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.

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