The Human Genome: Structure and Function of Genes and ... · tion of genes and chromosomes at the molecular level. More recently, this has been supplemented by ... 20 THOMPSON & THOMPSON

Over the past 20 years, remarkable progress has beenmade in our understanding of the structure and func-tion of genes and chromosomes at the molecularlevel. More recently, this has been supplemented byan in-depth understanding of the organization of thehuman genome at the level of its DNA sequence.These advances have come about in large measurethrough the applications of molecular genetics andgenomics to many clinical situations, thereby provid-ing the tools for a distinctive new approach to med-ical genetics. In this chapter, we present an overviewof the organization of the human genome and theaspects of molecular genetics that are required for anunderstanding of the genetic approach to medicine.This chapter is not intended to provide an extensivedescription of the wealth of new information aboutgene structure and regulation. To supplement theinformation discussed here, Chapter 4 describesmany experimental approaches of modern moleculargenetics that are becoming critical to the practice andunderstanding of human and medical genetics.

The increased knowledge of genes, and of theirorganization in the genome, has had an enormousimpact on medicine and on our perception of humanphysiology. As Nobel laureate Paul Berg statedpresciently at the dawn of this new era:

Just as our present knowledge and practice of medicinerelies on a sophisticated knowledge of human anatomy,physiology, and biochemistry, so will dealing with dis-ease in the future demand a detailed understanding ofthe molecular anatomy, physiology, and biochemistry ofthe human genome. . . . We shall need a moredetailed knowledge of how human genes are organized and how they function and are regulated. We shall alsohave to have physicians who are as conversant with themolecular anatomy and physiology of chromosomes and genes as the cardiac surgeon is with the structureand workings of the heart.

DNA STRUCTURE: A BRIEFREVIEW

DNA is a polymeric nucleic acid macromoleculecomposed of three types of units: a five-carbon sugar,deoxyribose; a nitrogen-containing base; and a phos-phate group (Fig. 3–1). The bases are of two types,purines and pyrimidines. In DNA, there are twopurine bases, adenine (A) and guanine (G), and twopyrimidine bases, thymine (T) and cytosine (C).Nucleotides, each composed of a base, a phosphate,and a sugar moiety, polymerize into long polynu-cleotide chains by 5�–3� phosphodiester bondsformed between adjacent deoxyribose units (Fig.3–2). In the human genome, these polynucleotidechains (in their double-helix form) are hundreds ofmillions of nucleotides long, ranging in size from ap-proximately 50 million base pairs (for the smallestchromosome, chromosome 21) to 250 million basepairs (for the largest chromosome, chromosome 1).

The anatomical structure of DNA carries thechemical information that allows the exact transmis-sion of genetic information from one cell to itsdaughter cells and from one generation to the next.At the same time, the primary structure of DNAspecifies the amino acid sequences of the polypep-tide chains of proteins, as described later in thischapter. DNA has elegant features that give it theseproperties. The native state of DNA, as elucidatedby James Watson and Francis Crick in 1953, is adouble helix (Fig. 3 – 3). The helical structureresembles a right-handed spiral staircase in which itstwo polynucleotide chains run in opposite direc-tions, held together by hydrogen bonds betweenpairs of bases: A of one chain paired with T of theother, and G with C (see Fig. 3 – 3). Consequently,

The Human Genome:Structure and Function ofGenes and Chromosomes

3

17

knowledge of the sequence of nucleotide bases onone strand automatically allows one to determinethe sequence of bases on the other strand. Thedouble-stranded structure of DNA molecules allowsthem to replicate precisely by separation of thetwo strands, followed by synthesis of two new

complementary strands, in accordance with thesequence of the original template strands (Fig. 3 – 4).Similarly, when necessary, the base complementarityallows efficient and correct repair of damaged DNAmolecules.

THE CENTRAL DOGMA:DNA : RNA : PROTEIN

Genetic information is contained in DNA in thechromosomes within the cell nucleus, but proteinsynthesis, during which the information encoded inthe DNA is used, takes place in the cytoplasm. Thiscompartmentalization reflects the fact that the humanorganism is a eukaryote. This means that humancells have a genuine nucleus containing the DNA,which is separated by a nuclear membrane from thecytoplasm. In contrast, in prokaryotes like the intesti-nal bacterium Escherichia coli, DNA is not enclosedwithin a nucleus. Because of the compartmentaliza-tion of eukaryotic cells, information transfer from thenucleus to the cytoplasm is a very complex processthat has been a focus of attention among molecularand cellular biologists.

The molecular link between these two relatedtypes of information (the DNA code of genes and theamino acid code of proteins) is ribonucleic acid(RNA). The chemical structure of RNA is similar tothat of DNA, except that each nucleotide in RNA hasa ribose sugar component instead of a deoxyribose;in addition, uracil (U) replaces thymine as one ofthe pyrimidines of RNA (Fig. 3–5). An additionaldifference between RNA and DNA is that RNA inmost organisms exists as a single-stranded molecule,whereas DNA exists as a double helix.

18 THOMPSON & THOMPSON GENETICS IN MEDICINE

Figure 3–1. The four bases ofDNA and the general structure of anucleotide in DNA. Each of thefour bases bonds with deoxyribose(through the nitrogen shown inred) and a phosphate group to formthe corresponding nucleotides.

Cytosine (C)Guanine (G)

BaseO

Phosphate Deoxyribose

O

O O O_

P CH2

C

OH

HH H

H

C CHC

5'

3'

Adenine (A) Thymine (T)

N

HC

Purines PyrimidinesNH2

N

CC

CN

N

CH

H

O

N

CC

CN

N

CH

H

CH2N

HN

_

O

O

CH3

CH

CC

CN

H

HN

NH2

OCH

CCH

CN

H

N

Figure 3–2. A portion of a DNA polynucleotide chain, showingthe 3 �–5� phosphodiester bonds that link adjacent nucleotides.

Base 3OH2C

C

OH

HH H

H

C CHC

5'

3'

O

O

O OP_

Base 2OH2C

CH

H H

H

C CHC

5'

3'

O

O

O OP_

Base 1OH2C

CH

H H

H

C CHC

5'

3'

O

O

O OP_

_

3' end

5' end

The informational relationships among DNA,RNA, and protein are intertwined: DNA directs thesynthesis and sequence of RNA, RNA directs thesynthesis and sequence of polypeptides, and specificproteins are involved in the synthesis and metabo-lism of DNA and RNA. This flow of information isreferred to as the “central dogma” of molecularbiology.

Genetic information is stored in DNA by means ofa code (the genetic code, discussed later) in which thesequence of adjacent bases ultimately determines thesequence of amino acids in the encoded polypeptide.First, RNA is synthesized from the DNA templatethrough a process known as transcription. The RNA,carrying the coded information in a form called mes-senger RNA (mRNA), is then transported from thenucleus to the cytoplasm, where the RNA sequence isdecoded, or translated, to determine the sequence ofamino acids in the protein being synthesized. Theprocess of translation occurs on ribosomes, which arecytoplasmic organelles with binding sites for all of theinteracting molecules, including the mRNA, involvedin protein synthesis. Ribosomes are themselves madeup of many different structural proteins in associationwith a specialized type of RNA known as ribosomalRNA (rRNA). Translation involves yet a third type ofRNA, transfer RNA (tRNA), which provides themolecular link between the coded base sequence of themRNA and the amino acid sequence of the protein.

The Human Genome: Structure and Function of Genes and Chromosomes 19

A

Hydrogen bonds

C

C

T

C

5'

5'

3'

3'

3.4 Å

34 Å

G

G

G

20 Å

Figure 3–3. The structure of DNA.Left, A two-dimensional representationof the two complementary strands ofDNA, showing the AT and GC basepairs. Note that the orientation of thetwo strands is antiparallel. Right, Thedouble-helix model of DNA, as pro-posed by Watson and Crick. The hori-zontal “rungs” represent the pairedbases. The helix is said to be right-handed because the strand going fromlower left to upper right crosses overthe opposite strand. (Based on WatsonJD, Crick FHC [1953] Molecularstructure of nucleic acids—A structurefor deoxyribose nucleic acid. Nature171:737–738.)

Figure 3–4. Replication of a DNA double helix, resulting in twoidentical daughter molecules, each composed of one parentalstrand (black) and one newly synthesized strand (red ).

G

G

CG

CG

C G

C GC G

C

C G

G

C

CG

CG

CG

CG

CG

CG

CG

TA

TA

TA

TA TA

TATA

T AT A

T A

T A

T AT A

T A

T A T A

T A

T A

T A

TATA

TA

CG

CG

CG

5'

5'3'

5' 3' 5' 3'

3'

Figure 3–5. The pyrimidine uracil and the structure of a nu-cleotide in RNA. Note that the sugar ribose replaces the sugardeoxyribose of DNA. Compare with Figure 3–1.

BaseO

Phosphate Ribose

O

O O O_

P CH2

C

OH

HH H

OH

C CHC

5'

3'

_

O

OCH

CCH

CN

H

HN

Uracil (U)

Because of the interdependent flow of informationrepresented by the central dogma, one can begin dis-cussion of the molecular genetics of gene expressionat any of its three informational levels: DNA, RNA,or protein. We begin by examining the structure ofgenes as a foundation for discussion of the geneticcode, transcription, and translation.

Gene Structure and Organization

In its simplest form, a gene can be visualized as a seg-ment of a DNA molecule containing the code for theamino acid sequence of a polypeptide chain and the

regulatory sequences necessary for expression. Thisdescription, however, is inadequate for genes in thehuman genome (and indeed in most eukaryoticgenomes), because few genes exist as continuous codingsequences. Rather, the vast majority of genes areinterrupted by one or more noncoding regions. Theseintervening sequences, called introns, are initiallytranscribed into RNA in the nucleus but are notpresent in the mature mRNA in the cytoplasm. Thus,information from the intronic sequences is not nor-mally represented in the final protein product. Intronsalternate with coding sequences, or exons, that ulti-mately encode the amino acid sequence of the protein(Fig. 3–6). Although a few genes in the human genome


Figure 3–6. General structure of a typical human gene. Individual features are labeled in the figure and discussed in the text.Examples of three medically important human genes are presented at the bottom of the figure. Individual exons are numbered.Different mutations in the �-globin gene cause a variety of important hemoglobinopathies. Mutations in the factor VIII genecause hemophilia A. Mutations in the hypoxanthine phosphoribosyltransferase (HPRT ) gene lead to Lesch-Nyhan syndrome.

"Upstream"

Start oftranscription

5' 3'

"Downstream"

Termination codonExons (coding sequences)

Introns(intervening sequences)

initiatorcodon

Promoter

5' untranslatedregion 3' untranslated

region

Polyadenylationsignal

Direction of transcription

"CAT"

"TATA"

"TATA"

"GC-rich"

0 0.5 1.0 1.5 2.0 kb

0 50 100 150 200 kb

0 25 50 kb

321Beta Globin

Factor VIII

HPRT

1 7 - 13 2614 23 - 2515 - 222 - 6

1 4 5 6 7 982 3

have no introns, most genes contain at least one andusually several introns. Surprisingly, in many genes, thecumulative length of introns makes up a far greaterproportion of a gene’s total length than do the exons.Whereas some genes are only a few kilobases (kb,where 1 kb � 1000 base pairs) in length, others, likethe factor VIII gene shown in Figure 3–6, stretch onfor hundreds of kb. There are a few exceptionally largegenes, including the X-linked dystrophin gene (muta-tions in which lead to Duchenne muscular dystrophy)that spans more than 2 million base pairs (2000 kb), ofwhich less than 1 percent consists of coding exons.

STRUCTURAL FEATURES OF A TYPICAL HUMAN

GENE

A schematic representation of a portion of chromoso-mal DNA containing a typical gene is shown in Figure3–6, along with the structure of several medically rele-vant genes. Together, they illustrate the range offeatures that characterize human genes. In Chapters 1and 2, we briefly defined “gene” in general terms. Atthis point, we can provide a molecular definition of agene. In typical circumstances, we define a gene as a se-quence of chromosomal DNA that is required for productionof a functional product, be it a polypeptide or a func-tional RNA molecule. As is clear from Figure 3–6, agene includes not only the actual coding sequences butalso adjacent nucleotide sequences required for theproper expression of the gene—that is, for the produc-tion of a normal mRNA molecule, in the correctamount, in the correct place, and at the correct timeduring development or during the cell cycle.

The adjacent nucleotide sequences provide themolecular “start” and “stop” signals for the synthesisof mRNA transcribed from the gene. At the 5� end ofthe gene lies a promoter region, which includessequences responsible for the proper initiation oftranscription. Within the 5� region are several DNAelements whose sequence is conserved among manydifferent genes. This conservation, together withfunctional studies of gene expression in many labora-tories, indicates that these particular sequence

elements play an important role in regulation. Thereare several different types of promoter found in thehuman genome, with different regulatory propertiesthat specify the developmental patterns, as well as thelevels of expression, of a particular gene in differenttissues. The roles of individual conserved promoterelements, identified in Figure 3–6, are discussed ingreater detail in the section on “Fundamentals ofGene Expression.” Both promoters and other regula-tory elements (located either 5� or 3� of a gene or inits introns) can be sites of mutation in genetic diseasethat can interfere with the normal expression ofa gene. These regulatory elements, includingenhancers, silencers, and locus control regions(LCRs), are discussed more fully later in this chapter.

At the 3� end of the gene lies an untranslated regionof importance that contains a signal for addition of asequence of adenosine residues (the so-called polyAtail) to the end of the mature mRNA. Although it isgenerally accepted that such closely neighboring regu-latory sequences are part of what is called a “gene,” theprecise dimensions of any particular gene will remainsomewhat uncertain until the potential functions ofmore distant sequences are fully characterized.

GENE FAMILIES

Many genes belong to families of closely relatedDNA sequences, recognized as families because ofsimilarity of the nucleotide sequence of the genesthemselves or of the amino acid sequence of theencoded polypeptides.

One small, but medically important gene family iscomposed of genes that encode the protein chainsfound in hemoglobins. The �-globin and �-globingene clusters, on chromosomes 16 and 11, respec-tively, are shown in Figure 3–7 and are believed tohave arisen by duplication of a primitive precursorgene about 500 million years ago. These two clusterscontain genes coding for closely related globin chainsexpressed at different developmental stages, fromembryo to adult. The individual genes within eachcluster are more similar in sequence to one another


Figure 3–7. Chromosomal organization of the two clusters of human globin genes. Functional genes are indicated in red.Pseudogenes are indicated by the open boxes. (Redrawn from Nienhuis AW, Maniatis T [1987] Structure and expression of glo-bin genes in erythroid cells. In Stamatoyannopoulos G, Nienhuis AW, Leder P, Majerus PW [eds] The Molecular Basis ofBlood Diseases. WB Saunders, Philadelphia, pp. 28–65.)

0 4020 60 kb

5' 3'

Chromosome 16

Chromosome 11

5' 3'α - like genes

β - like genes

ζ ψζ ψα α2 α1

ε Gγ Aγ ψβ δ β

than to genes in the other cluster; thus, each cluster isbelieved to have evolved by a series of sequential geneduplication events within the past 100 million years.The exon-intron patterns of the globin genes appearto have been remarkably conserved during evolution;each of the functional globin genes shown in Figure3–7 has two introns at similar locations, although thesequences contained within the introns have accumu-lated far more nucleotide base changes over time thanhave the coding sequences of each gene. The controlof expression of the various globin genes, in thenormal state as well as in the many inherited hemo-globinopathies, is considered in more detail both laterin this chapter and in Chapter 11.

Several of the globin genes do not produce any RNAor protein product and therefore are unlikely to haveany function. DNA sequences that closely resembleknown genes but are nonfunctional are called pseudo-genes. Pseudogenes are widespread in the genome andare thought to be byproducts of evolution, representinggenes that were once functional but are now vestigial,having been inactivated by mutations in coding orregulatory sequences. In some cases, as in the pseudo-�-globin and pseudo-�-globin genes, the pseudogenespresumably arose through gene duplication, followedby the introduction of numerous mutations into theextra copies of the once-functional gene. In other cases,pseudogenes have been formed by a process, calledretrotransposition, that involves transcription, genera-tion of a DNA copy of the mRNA, and, finally, integra-tion of such DNA copies back into the genome.Pseudogenes created by retrotransposition lack intronsand are called processed pseudogenes. They are notnecessarily or usually on the same chromosome (orchromosomal region) as their progenitor gene.

The largest known gene family in the humangenome is the so-called immunoglobulin superfam-ily, which includes many hundreds of genes involvedin cell surface recognition events in the immune andnervous system, such as genes on chromosomes 2, 14,and 22 that encode the immunoglobulin heavy andlight chains themselves; genes on chromosome 6 thatmake up the major histocompatibility complex; geneson chromosomes 7 and 14 whose products make upthe T-cell receptor; and genes that are expressedprimarily in neural tissues, such as genes for celladhesion molecules or for myelin-associated glyco-proteins. The structure and function of many of thesegenes are examined in detail in Chapter 14.

FUNDAMENTALS OF GENE EXPRESSION

The flow of information from gene to polypeptideinvolves several steps (Fig. 3–8). Initiation of tran-scription of a gene is under the influence of promoters

and other regulatory elements, as well as specific pro-teins known as transcription factors that interact withspecific sequences within these regions. Transcriptionof a gene is initiated at the transcriptional “start site”on chromosomal DNA just upstream from the codingsequences and continues along the chromosome, foranywhere from several hundred base pairs to morethan a million base pairs, through both introns andexons and past the end of the coding sequences. Aftermodification at both the 5� and 3� ends of the primaryRNA transcript, the portions corresponding to intronsare removed, and the segments corresponding to exonsare spliced together. After RNA splicing, the resultingmRNA (now colinear with only the coding portions ofthe gene) is transported from the nucleus to thecytoplasm, where the mRNA is finally translated intothe amino acid sequence of the encoded polypeptide.Each of the steps in this complex pathway is prone toerror, and mutations that interfere with the individualsteps have been implicated in a number of inheritedgenetic disorders (see Chapters 5, 11, and 12).

Transcription

Transcription of protein-coding genes by RNA poly-merase II (one of several classes of RNA polymerases)is initiated upstream from the first coding sequence atthe transcriptional start site, the point correspondingto the 5� end of the final RNA product (see Figs. 3–6and 3–8). Synthesis of the primary RNA transcriptproceeds in a 5�-to-3� direction, whereas the strand ofthe gene being transcribed is actually read in a 3�-to-5� direction with respect to the direction of thedeoxyribose phosphodiester backbone (see Fig. 3–2).Because the RNA synthesized corresponds both inpolarity and in base sequence (substituting U for T)to the 5�-to-3� strand of DNA, this nontranscribedDNA strand is sometimes called the “coding,” or“sense,” DNA strand. The 3�-to-5� transcribedstrand of DNA is then referred to as the “noncod-ing,” or “antisense,” strand. Transcription continuesthrough both intron and exon portions of the gene,beyond the position on the chromosome that eventu-ally corresponds to the 3� end of the mature mRNA.Whether transcription ends at a predetermined 3�termination point is unknown.

The primary RNA transcript is processed by addi-tion of a chemical “cap” structure to the 5� end of theRNA and cleavage of the 3� end at a specific pointdownstream from the end of the coding information.This cleavage is followed by addition of a polyA tailto the 3� end of the RNA; the polyA tail appears toincrease the stability of the resulting polyadenylatedRNA. The location of the polyadenylation point isspecified in part by the sequence AAUAAA (or avariant of this), usually found in the 3� untranslated


portion of the RNA transcript. These post-transcrip-tional modifications take place in the nucleus, as doesthe process of RNA splicing. The fully processedRNA, now called mRNA, is then transported tothe cytoplasm, where translation takes place (see Fig.3–8).

Translation and the Genetic Code

In the cytoplasm, mRNA is translated into protein bythe action of a variety of tRNA molecules, each spe-cific for a particular amino acid. These remarkablemolecules, each only 70 to 100 nucleotides long, havethe job of transferring the correct amino acids totheir positions along the mRNA template, to beadded to the growing polypeptide chain. Proteinsynthesis occurs on ribosomes, macromolecularcomplexes made up of rRNA (encoded by the 18Sand 28S rRNA genes) and several dozen ribosomalproteins (see Fig. 3–8).

The key to translation is a code that relates specificamino acids to combinations of three adjacent basesalong the mRNA. Each set of three bases constitutesa codon, specific for a particular amino acid (Table

3–1). In theory, almost infinite variations are possiblein the arrangement of the bases along a polynu-cleotide chain. At any one position, there are fourpossibilities (A, T, C, or G); thus, there are 4n possiblecombinations in a sequence of n bases. For threebases, there are 43, or 64, possible triplet combina-tions. These 64 codons constitute the genetic code.

Because there are only 20 amino acids and 64possible codons, most amino acids are specified bymore than one codon; hence the code is said to bedegenerate. For instance, the base in the third posi-tion of the triplet can often be either purine (A or G)or either pyrimidine (T or C) or, in some cases, anyone of the four bases, without altering the codedmessage (see Table 3–1). Leucine and arginine areeach specified by six codons. Only methionine andtryptophan are each specified by a single, uniquecodon. Three of the codons are called stop (or non-sense) codons because they designate termination oftranslation of the mRNA at that point.

Translation of a processed mRNA is always initi-ated at a codon specifying methionine. Methionine istherefore the first encoded (amino-terminal) aminoacid of each polypeptide chain, although it is usuallyremoved before protein synthesis is completed. The


Figure 3–8. Flow of information from DNA to RNA to protein for a hypothetical gene with three exons and two introns.Steps include transcription, RNA processing and splicing, RNA transport from the nucleus to the cytoplasm, and translation.

Ribosomes

Completed polypeptide

Growing polypeptidechain

A A A A 3'

A A A A 3'

A A A A 3'

3'

3'3'

3'3

5'

5'

5'

5'

5'

5'

5'

4. Translation

5. Protein assembly

3. Transport

Cytoplasm

Nucleus

Transcribed strand

Non-transcribed strand

1. Transcription CAP

Exons: 1 2

RNA

Poly (A) addition

2. RNA processing and splicing

codon for methionine (the initiator codon, AUG)establishes the reading frame of the mRNA; eachsubsequent codon is read in turn to predict the aminoacid sequence of the protein.

The molecular links between codons and aminoacids are the specific tRNA molecules. A particularsite on each tRNA forms a three-base anticodon thatis complementary to a specific codon on the mRNA.Bonding between the codon and anticodon brings theappropriate amino acid into the next position on theribosome for attachment by formation of a peptidebond to the carboxyl end of the growing polypeptidechain. The ribosome then slides along the mRNAexactly three bases, bringing the next codon into linefor recognition by another tRNA with the next aminoacid. Thus, proteins are synthesized from the aminoterminus to the carboxyl terminus, which correspondsto translation of the mRNA in a 5�-to-3� direction.

As mentioned earlier, translation ends when a stopcodon (UGA, UAA, or UAG) is encountered in thesame reading frame as the initiator codon. (Stopcodons in either of the other two, unused reading

frames are not read and therefore have no effect ontranslation.) The completed polypeptide is thenreleased from the ribosome, which becomes availableto begin synthesis of another protein.

Post-Translational Processing

Many proteins undergo extensive post-translationalmodifications. The polypeptide chain that is theprimary translation product is folded and bonded intoa specific three-dimensional structure that is deter-mined by the amino acid sequence itself. Two ormore polypeptide chains, products of the same geneor of different genes, may combine to form a singlemature protein complex. For example, two �-globinchains and two �-globin chains associate noncova-lently to form the tetrameric �2�2 hemoglobinmolecule. The protein products may also be modifiedchemically by, for example, addition of phosphate orcarbohydrates at specific sites. Other modificationsmay involve cleavage of the protein, either to removespecific amino-terminal sequences after they have


TABLE 3–1

The Genetic Code

Second BaseYears

First ThirdBase U C A G Base

UUU phe UCU ser UAU tyr UGU cys UUUC phe UCC ser UAC tyr UGC cys C

UUUA leu UCA ser UAA stop UGA stop AUUG leu UCG ser UAG stop UGG trp G

CUU leu CCU pro CAU his CGU arg UCUC leu CCC pro CAC his CGC arg C

CCUA leu CCA pro CAA gln CGA arg ACUG leu CCG pro CAG gln CGG arg G

AUU ile ACU thr AAU asn AGU ser UAUC ile ACC thr AAC asn AGC ser C

AAUA ile ACA thr AAA lys AGA arg AAUG met ACG thr AAG lys AGG arg G

GUU val GCU ala GAU asp GGU gly UGUC val GCC ala GAC asp GGC gly C

GGUA val GCA ala GAA glu GGA gly AGUG val GCG ala GAG glu GGG gly G

Abbreviations for amino acids:ala (A) alanine leu (L) leucinearg (R) arginine lys (K) lysineasn (N) asparagine met (M) methionineasp (D) aspartic acid phe (F) phenylalaninecys (C) cysteine pro (P) proline

gln (Q) glutamine ser (S) serineglu (E) glutamic acid thr (T) threoninegly (G) glycine trp (W) tryptophanhis (H) histidine tyr (Y) tyrosineile (I) isoleucine val (V) valine

Other abbreviation:stop termination codon

Codons are shown in terms of messenger RNA, which are complementary to the corresponding DNA codons.

functioned to direct a protein to its correct locationwithin the cell (e.g., proteins that function within thenucleus or mitochondria) or to split the molecule intosmaller polypeptide chains. For example, the twochains that make up mature insulin, one 21 and theother 30 amino acids long, are originally part of an82 amino-acid primary translation product, calledproinsulin.

Gene Expression in Action: The Beta-Globin Gene

The flow of information outlined in the precedingsections can best be appreciated by reference to aparticular well-studied gene, the �-globin gene. The�-globin chain is a 146 amino-acid polypeptide,encoded by a gene that occupies approximately 1.6 kbon the short arm of chromosome 11. The gene hasthree exons and two introns (see Fig. 3–6). The �-globin gene, as well as the other genes in the �-globincluster (see Fig. 3–7), is transcribed in a centromere-to-telomere direction. This orientation, however, isdifferent for other genes in the genome and dependson which strand of the chromosomal double helix isthe coding strand for a particular gene.

DNA sequences required for accurate initiation oftranscription of the �-globin gene are located in thepromoter within approximately 200 base pairsupstream from the transcription start site. The dou-ble-stranded DNA sequence of this region of the �-globin gene, the corresponding RNA sequence, andthe translated sequence of the first 10 amino acids aredepicted in Figure 3–9 to illustrate the relationshipsamong these three information levels. As mentionedpreviously, it is the 3�-to-5� strand of the DNA thatserves as template and is actually transcribed, but it isthe 5�-to-3� strand of DNA that most directly corre-sponds to the 5�-to-3� sequence of the mRNA (and,in fact, is identical to it except that U is substitutedfor T). Because of this correspondence, the 5�-to-3�DNA strand of a gene (i.e., the strand that is nottranscribed) is the strand generally reported in thescientific literature or in databases.

In accordance with this convention, the completesequence of approximately 2.0 kb of chromosome 11that includes the �-globin gene is shown in Figure3–10. (It is sobering to reflect that this page ofnucleotides represents only 0.000067 percent of thesequence of the entire human genome!) Within these2.0 kb are contained most, but not all, of thesequence elements required to encode and regulatethe expression of this gene. Indicated in Figure 3–10are many of the important structural features of the �-globin gene, including conserved promotersequence elements, intron and exon boundaries, RNAsplice sites, the initiator and termination codons, andthe polyadenylation signal, all of which are known tobe mutated in various inherited defects of the �-globin gene (see Chapter 11).

INITIATION OF TRANSCRIPTION

The �-globin promoter, like many other genepromoters, consists of a series of relatively short func-tional elements that are thought to interact withspecific proteins (generically called transcriptionfactors) that regulate transcription, including, in thecase of the globin genes, those proteins that restrictexpression of these genes to erythroid cells, the cellsin which hemoglobin is produced. One importantpromoter sequence is the “TATA box,” a conservedregion rich in adenines and thymines that is approxi-mately 25 to 30 base pairs upstream of the start site oftranscription (see Figs. 3–6 and 3–10). The TATAbox appears to be important for determining the posi-tion of the start of transcription, which in the �-globin gene is approximately 50 base pairsupstream from the translation initiation site (see Fig.3–9). Thus, in this gene there are about 50 base pairsof sequence that are transcribed but are not trans-lated. In other genes, this 5� transcribed but untrans-lated region (called the 5� UTR) can be much longerand can, in fact, be interrupted by one or more in-trons. A second conserved region, the so-called CATbox (actually CCAAT), is a few dozen base pairs far-ther upstream (see Fig. 3–10). Both experimentally


Figure 3–9. Structure and nucleotide sequence of the 5� end of the human �-globin gene on the short arm of chromosome 11.Transcription of the 3�- to-5� (lower) strand begins at the indicated start size to produce �-globin mRNA. The translationalreading frame is determined by the AUG initiator codon (***); subsequent codons specifying amino acids are indicated in red.The other two potential frames are not used.

DNA

mRNA

Start

A C A U U U G C U U C U G A C A C A A C U G U G U U C A C U A G C A A C C U C A A A C A G A C A C C A U G G U G C A C C U G A C U C C U G A G G A G A A G U C U G C C

Transcription

5'

3'5' . . .

. . .3'

3'

5'

. . .

. . .

. . .

. . .

Translation

Reading frame

globin V a l H i s L e u T h r P r o G l u G l u L y s S e r A l aβ

induced and naturally occurring mutations in eitherof these sequence elements, as well as in other regula-tory sequences even farther upstream, lead to a sharpreduction in the level of transcription, therebydemonstrating the importance of these elements for

normal gene expression. Many mutations in theseregulatory elements have been identified in patientswith the disorder �-thalassemia (see Chapter 11).

Not all gene promoters contain the two specificelements described. In particular, genes that are


Figure 3–10. Nucleotide sequence of the complete human �-globin gene. The sequence of the 5�-to-3� strand of the gene isshown. Capital letters represent sequences corresponding to mature mRNA. Lowercase letters indicate introns and flanking se-quences. The CAT and TATA box sequences in the 5� flanking region are boxed. The ATG initiator codon (AUG in mRNA)and the TAA stop codon (UAA in mRNA) are shown in red. The amino-acid sequence of �-globin is shown above the codingsequence; the three-letter abbreviations in Table 3–1 are used here. The GT and AG dinucleotides important for RNA splicingat the intron/exon junctions are boxed. (From Lawn RM, Efstratiadis A, O’Connell C, et al [1980] The nucleotide sequence ofthe human �-globin gene. Cell 21: 647–651.)

* * *

constitutively expressed in most or all tissues (called“housekeeping” genes) often lack the CAT andTATA boxes that are more typical of tissue-specificgenes. Promoters of many housekeeping genes oftencontain a high proportion of cytosines and guaninesin relation to the surrounding DNA (see thepromoter of the hypoxanthine phosphoribosyltrans-ferase gene in Fig. 3 – 6). Such CG-rich promotersare often located in regions of the genome calledCG (or CpG) islands, so named because of theunusually high concentration of the dinucleotide 5�-CG-3� that stands out from the more general AT-rich chromosomal landscape. Some of the CG-richsequence elements found in these promoters arethought to serve as binding sites for specifictranscription factors.

In addition to the sequences that constitute apromoter itself, there are other sequence elementsthat can markedly alter the efficiency of transcrip-tion. The best characterized of these “activating”sequences are called enhancers. Enhancers are se-quence elements that can act at a distance (oftenseveral kb) from a gene to stimulate transcription.Unlike promoters, enhancers are both position- andorientation-independent and can be located either5� or 3� of the transcription start site. Enhancerelements function only in certain cell types and thusappear to be involved in establishing the tissuespecificity and/or level of expression of many genes,in concert with one or more transcription factors. Inthe case of the �-globin gene, several tissue-specificenhancers are present within both the gene itself andin its flanking regions. The interaction of enhancerswith particular proteins leads to increased levels oftranscription.

Normal expression of the �-globin gene duringdevelopment also requires more distant sequencescalled the locus control region (LCR), locatedupstream of the �-globin gene (see Fig. 3–7), whichis required for appropriate high-level expression. Asexpected, mutations that disrupt or delete eitherenhancer or LCR sequences interfere with or prevent�-globin gene expression (see Chapter 11).

RNA SPLICING

The primary RNA transcript of the �-globin genecontains two exons, approximately 100 and 850 basepairs in length, that need to be spliced together. Theprocess is exact and highly efficient; 95 percent of �-globin transcripts are thought to be accuratelyspliced to yield functional globin mRNA. The splicingreactions are guided by specific DNA sequences at boththe 5� and the 3� ends of introns. The 5� sequence con-sists of nine nucleotides, of which two (the dinucleotideGT located in the intron immediately adjacent to the

splice site) are virtually invariant among splice sites indifferent genes (see Fig. 3–10). The 3� sequenceconsists of about a dozen nucleotides, of which, again,two, the AG located immediately 5� to the intron/exonboundary, are obligatory for normal splicing. The splicesites themselves are unrelated to the reading frame ofthe particular mRNA. In some instances, as in the caseof intron 1 of the �-globin gene, the intron actuallysplits a specific codon (see Fig. 3–10).

The medical significance of RNA splicing is illus-trated by the fact that mutations within the conservedsequences at the intron/exon boundaries commonlyimpair RNA splicing, with a concomitant reductionin the amount of normal, mature �-globin mRNA;mutations in the GT or AG dinucleotides mentionedearlier invariably eliminate normal splicing of theintron containing the mutation. A number of splicesite mutations, identified in patients with �-tha-lassemia, are discussed in detail in Chapter 11.

POLYADENYLATION

The mature �-globin mRNA contains approximately130 base pairs of 3� untranslated material (the 3�UTR) between the stop codon and the location of thepolyA tail (see Fig. 3–10). As in other genes, cleavageof the 3� end of the mRNA and addition of the polyAtail is controlled, at least in part, by an AAUAAAsequence approximately 20 base pairs before thepolyadenylation site. Mutations in this polyadenyla-tion signal in patients with �-thalassemia (as well asmutations in the corresponding polyadenylation signalin the �-globin gene in patients with �-thalassemia)document the importance of this signal for proper 3�cleavage and polyadenylation (see Chapter 11). The 3�untranslated region of some genes can be quite long,up to several kb. Other genes have a number of alter-native polyadenylation sites, selection among whichmay influence the stability of the resulting mRNA andthus the steady-state level of each mRNA.

STRUCTURE OF HUMANCHROMOSOMES

The composition of genes in the human genome, aswell as the determinants of their expression, is speci-fied in the DNA of the 46 human chromosomes. Aswe saw in an earlier section, each human chromosome isbelieved to consist of a single, continuous DNA doublehelix; that is, each chromosome in the nucleus is along, linear double-stranded DNA molecule. Chro-mosomes are not naked DNA double helices,however. The DNA molecule of a chromosome existsas a complex with a family of basic chromosomalproteins called histones and with a heterogeneousgroup of acidic, nonhistone proteins that are much


less well characterized, but that appear to be criticalfor establishing a proper environment to ensure nor-mal chromosome behavior and appropriate geneexpression. Together, this complex of DNA andprotein is called chromatin.

There are five major types of histones that play acritical role in the proper packaging of the chromatinfiber. Two copies each of the four core histones H2A,H2B, H3, and H4 constitute an octamer, aroundwhich a segment of DNA double helix winds, likethread around a spool (Fig. 3–11). Approximately140 base pairs of DNA are associated with each his-tone core, making just under two turns around theoctamer. After a short (20 to 60 base-pair) “spacer”segment of DNA, the next core DNA complexforms, and so on, giving chromatin the appearance ofbeads on a string. Each complex of DNA with corehistones is called a nucleosome, which is the basicstructural unit of chromatin. The fifth histone, H1,appears to bind to DNA at the edge of each nucleo-some, in the internucleosomal spacer region. Theamount of DNA associated with a core nucleosome,together with the spacer region, is about 200 basepairs.

During the cell cycle, as we saw in Chapter 2,chromosomes pass through orderly stages of conden-sation and decondensation (see Fig. 2–5). In theinterphase nucleus, chromosomes and chromatin arequite decondensed in relation to the highly con-densed state of chromatin in metaphase. Nonetheless,even in interphase chromosomes, DNA in chromatin

is substantially more condensed than it would be as anative, protein-free double helix.

The long strings of nucleosomes are themselvesfurther compacted into a secondary helical chromatinstructure that appears under the electron microscopeas a thick, 30-nm-diameter fiber (about three timesthicker than the nucleosomal fiber) (see Fig. 3–11).This cylindrical “solenoid” fiber (from the Greek sole-noeides, “pipe-shaped”) appears to be the fundamentalunit of chromatin organization. The solenoids arethemselves packed into loops or domains attached atintervals of about 100 kb or so to a nonhistone pro-tein scaffold or matrix. It has been speculated thatloops are, in fact, functional units of DNA replicationor gene transcription, or both, and that the attach-ment points of each loop are fixed along the chromo-somal DNA. Thus, one level of control of geneexpression may depend on how DNA and genes arepackaged into chromosomes and on their associationwith chromatin proteins in the packaging process.

The various hierarchical levels of packaging seenin an interphase chromosome are illustrated schemat-ically in Figure 3–11. The enormous amount ofDNA packaged into a chromosome can be appreci-ated when chromosomes are treated to remove mostof the chromatin proteins in order to observe theprotein scaffold (Fig. 3–12). When DNA is releasedfrom chromosomes treated this way, long loops ofDNA can be visualized, and the residual scaffoldingcan be seen to reproduce the outline of a typicalmetaphase chromosome.


Figure 3–11. Hierarchical levels of chromatin packaging in a human chromosome.

Double helix Nucleosome fiber("beads-on-a-string")

Solenoid Interphasenucleus

2 nm ~10 nm ~30 nm

Histoneoctamer

~200bpof DNA

Each loop contains~100,000 bp of DNA

Portion of aninterphasechromosome

The Mitochondrial Chromosome

A small but important subset of genes encoded in thehuman genome resides in the cytoplasm in the mito-chondria. Mitochondrial genes exhibit exclusively

maternal inheritance (see Chapter 5). Human cellshave hundreds of mitochondria, each containing anumber of copies of a small circular molecule, themitochondrial chromosome. The mitochondrialDNA molecule is only 16 kb in length (less than 0.03


Figure 3–12. Electron micrograph of a protein-depleted human metaphase chromosome, showing the residual chromosomescaffold and loops of DNA. Individual DNA fibers can be best seen at the edge of the DNA loops. Bar�2�. (From Paulson JR,Laemmli UK [1977] The structure of histone-depleted metaphase chromosomes. Cell 12:817–828. Reprinted by permission ofthe authors and Cell Press.)

percent of the length of the smallest nuclear chromo-some!) and encodes only a few dozen genes. Al-though the products of these genes function in mito-chondria, it should be emphasized that the vastmajority of proteins found in mitochondria are, infact, the products of nuclear genes. Mutations inmitochondrial genes have been demonstrated inseveral maternally inherited, as well as sporadic,disorders (see Chapter 12).

ORGANIZATION OF THE HUMANGENOME

Regions of the genome with similar characteristics ororganization, replication, and expression are notarranged randomly but, rather, tend to be clusteredtogether. This functional organization of the genomecorrelates remarkably well with its structural organi-zation as revealed by metaphase chromosome band-ing (introduced in Chapter 2 and discussed in detailin Chapter 9). The overall significance of this func-tional organization is that chromosomes are not just arandom collection of different types of genes andother DNA sequences. Some chromosome regions,or even whole chromosomes, are quite high in genecontent (“gene-rich”), whereas others are low (“gene-poor”). Certain types of sequence are characteristic ofthe different physical hallmarks of human chromo-somes. The clinical consequences of abnormalities ofgenome structure reflect the specific nature of thegenes and sequences involved. Thus, abnormalities ofgene-rich chromosomes or chromosomal regions

tend to be much more severe clinically than similar-sized defects involving gene-poor parts of thegenome.

As the Human Genome Project nears completion, itis apparent that the organization of DNA in thehuman genome is far more complex than was antici-pated, as illustrated in Figure 3–13 for the fully char-acterized region of chromosome 17 in the vicinity ofthe BRCA1 gene, mutations in which are responsiblefor some forms of familial breast cancer (see Chapter16). Of the DNA in the genome, less than 10 percentactually encodes genes. Only about one half to threequarters of the total linear length of the genomeconsists of so-called single-copy or unique DNA—that is, DNA whose nucleotide sequence is representedonly once (or at most a few times) per haploid genome.The rest of the genome consists of several classes ofrepetitive DNA and includes DNA whose nucleotidesequence is repeated, either perfectly or with somevariation, hundreds to millions of times in the genome.Whereas most (but not all) of the estimated 50,000genes in the genome are represented in single-copyDNA, sequences in the repetitive DNA fractioncontribute to maintaining chromosome structure.

Single-Copy DNA Sequences

Although single-copy DNA makes up most of theDNA in the genome, much of its function remains amystery because, as mentioned, sequences actuallyencoding proteins (i.e., the coding portion of genes)constitute only a small proportion of all the single-


Figure 3–13. Sequence organization of the region of the human genome near the BRCA1 gene that is responsible for somecases of familial, early-onset breast cancer. Four genes and two pseudogenes are found in this 117-kb sequence from chromo-some 17, and their locations and structures are indicated above the chromosome. Nearly half of the sequence consists of variousinterspersed repetitive elements, including 170 copies of Alu repeats and 8 copies of L1 repeats that are indicated below thechromosome. BRCA1 contains a GC-rich promoter, with a number of specific transcription-factor binding sites. The RHO7gene encodes a member of a GTP-binding protein family. VAT1 encodes a membrane protein found in synaptic vesicles. BothRHO7 and VAT1 also have GC-rich promoters. IFP35 encodes a protein whose expression can be induced by interferon. (Basedon Smith TM, Lee MK, Szabo CI et al [1996] Complete genomic sequence and analysis of 117 kb human DNA containing thegene BRCA1. Genome Research 6:1029–1049.)

Genes :

Exons :

Repeat DNA :

pseudogene pseudogene

Rho7 VAT1 IFP35

16111 5 - 7 6113 - 24.... ....

BRCA1

10 kb

copy DNA. Long stretches of unique DNA sequences( � 25 kb) are quite rare in the genome. Most single-copy DNA is found in short stretches (several kb orless), interspersed with members of various repetitiveDNA families (see Fig. 3–13).

Repetitive DNA Families

Several different categories of repetitive DNA arerecognized. A useful distinguishing feature iswhether the repeated sequences (“repeats”) are clus-tered in one or a few locations or whether they aredispersed throughout the genome, interspersed withsingle-copy sequences along the chromosome. Clus-tered repeated sequences constitute an estimated 10to 15 percent of the genome and consist of arrays ofvarious short repeats organized tandemly in a head-to-tail fashion. The different types of such tandemrepeats are collectively called satellite DNAs, sonamed because many of the original tandem repeatfamilies could be purified by density centrifugationfrom the rest of the genome as “satellite” fractions ofDNA.

Satellite DNA families vary with regard to theirlocation in the genome, the total length of the tan-dem array, and the length of the constituent repeatunits that make up the array. In general, satellitearrays can stretch several million base pairs or morein length and constitute up to several percent of theDNA content of an individual human chromosome.Many satellite sequences are important as moleculartools that have revolutionized clinical cytogeneticanalysis because of their relative ease of detection(see Chapter 9). Some human satellite sequences arebased on repetitions (with some variation) of a shortsequence such as a pentanucleotide. Long arrays ofsuch repeats are found in heterochromatic regionson the proximal long arms of chromosomes 1, 9, and16 and on nearly the entire long arm of the Y chro-mosome (see Chapter 9). Other satellite DNAs arebased on somewhat longer basic repeats. For exam-ple, the �-satellite family of DNA is composed oftandem arrays of different copies of an approxi-mately 171 base-pair unit, found at the centromericregion of each human chromosome. This repeatfamily is believed to play a role in centromerefunction, ensuring proper chromosome segregationin mitosis and meiosis.

In addition to satellite DNAs, another major classof repetitive DNA in the genome consists of relatedsequences that are dispersed throughout the genomerather than localized (see Fig. 3–13). Although manysmall DNA families meet this general description,two in particular warrant discussion because togetherthey make up a significant proportion of the genomeand because they have been implicated in genetic

diseases. The best-studied dispersed repetitiveelements belong to the so-called Alu family. Themembers of this family are about 300 base pairs inlength and are recognizably related to each other al-though not identical in sequence. In total, there areabout 500,000 Alu family members in the genome,making up at least several percent of human DNA.In some regions of the genome, however, includingnear the BRCA1 gene as seen in Figure 3–13, theymake up a much higher percentage of the DNA. Asecond major dispersed, repetitive DNA family iscalled the L1 family. L1 elements are long, repeti-tive sequences (up to 6 kb in length) that are found inabout 100,000 copies per genome. They are plentifulin some regions of the genome but relatively sparsein others.

Families of repeats dispersed throughout thegenome are clearly of medical importance. Both Aluand L1 sequences have been implicated as the causeof mutations in hereditary disease through theprocess of retrotransposition, introduced in an ear-lier section. At least a few copies of the L1 and Alufamilies are still transpositionally active and gener-ate copies of themselves that can integrateelsewhere in the genome, occasionally causinginsertional inactivation of a medically importantgene. The frequency of retrotransposition eventscausing genetic disease in humans is unknowncurrently, but they may account for as many as 1 in500 mutations. In addition, aberrant recombinationevents between different copies of dispersed repeatscan also be a cause of mutation in some geneticdiseases (see Chapter 6).

VARIATION IN GENE EXPRESSIONAND ITS RELEVANCE TOMEDICINE

The regulated expression of the estimated 50,000genes encoded in human chromosomes involves a setof complex interrelationships among different levels ofcontrol, including proper gene dosage (controlledby mechanisms of chromosome replication and segre-gation), gene structure, and, finally, transcription,mRNA stability, translation, protein processing, andprotein degradation. For some genes, fluctuations inthe level of functional gene product, due either toinherited variation in the structure of a particular geneor to changes induced by nongenetic factors such asdiet or the environment, are of relatively little impor-tance. For other genes, changes in the level of expres-sion can have dire clinical consequences, reflecting theimportance of those gene products in particularbiological pathways. The nature of inherited variationin the structure and function of chromosomes andgenes, and the influence of this variation on the


expression of specific traits, is the very essence ofmedical and molecular genetics and is dealt with insubsequent chapters.

General References

Abel T, Maniatis T (1994) Mechanisms of eukaryotic gene regula-tion. In Stamatoyannopoulos G, Nienhuis AW, Majerus PW,Varmus H, (eds) The Molecular Basis of Blood Diseases. WBSaunders, Philadelphia. pp. 33–70.

Alberts B, Bray D, Lewis J, et al. (1994) Molecular Biology of theCell, 3rd ed. Garland Publishing, New York.

Bernardi G (1995) The human genome, organization, and evolu-tionary history. Ann Rev Genet 29:445–476.

Lewin B (2000) Genes VII, 7th ed. Oxford University Press, Ox-ford, England.

Semenza G (1999) Transcription Factors and Human Disease. Ox-ford University Press, New York.

Singer M, Berg P (1997) Exploring Genetic Mechanisms. Univer-sity Science Books, Sausalito, California.

Wolffe A (1998) Chromatin Structure and Function, 3rd ed. Acad-emic Press, San Diego.

References Specific to Particular Topics

Berg P (1981) Dissections and reconstructions of genes and chro-mosomes (Nobel Prize lecture). Science 213:296–303.

Kazazian HH, Moran JV (1998) The impact of L1 retrotrans-posons on the human genome. Nature Genetics 19:19–24.

Lawn RM, Efstratiadis A, O’Connell C, et al (1980) The nu-cleotide sequence of the human �-globin gene. Cell21:647–651.

Smith TM, Lee MK, Szabo CI, et al (1996) Complete genomic se-quence and analysis of 117 kb of human DNA containing thegene BRCA1. Genome Research 6:1029–1049.

Wallace DC (1999) Mitochondrial diseases in man and mouse. Sci-ence 283:1482–1488.

Problems

1. The following amino acid sequence represents part of aprotein. The normal sequence and four mutant formsare shown. By consulting Table 3–1, determine the dou-ble-stranded sequence of the corresponding section ofthe normal gene. Which strand is the strand that RNApolymerase “reads”? What would the sequence of the re-sulting mRNA be? What kind of mutation is each mu-tant protein most likely to represent?

Normal -lys-arg-his-his-tyr-leu-Mutant 1 -lys-arg-his-his-cys-leu-Mutant 2 -lys-arg-ile-ile-ile-Mutant 3 -lys-glu-thr-ser-leu-ser-Mutant 4 -asn-tyr-leu-

2. The following items are related to each other in a hier-archical fashion. What are these relationships? Chromo-some, base pair, nucleosome, G-band, kb pair, intron,gene, exon, chromatin, codon, nucleotide.

3. The following schematic drawing illustrates a chromo-some 5 in which the most distal band (band p15) on theshort arm is deleted. This deleted chromosome is associ-ated with the cri du chat syndrome (see Chapters 9 and10). Given what you know about the organization ofchromosomes and the genome, approximately howmuch DNA is deleted? How many genes?


4. Most of the human genome consists of sequences thatare not transcribed and do not directly encode geneproducts. For each of the following, consider ways inwhich these genome elements can contribute to humandisease: introns, Alu or L1 repetitive sequences, locuscontrol regions, pseudogenes.

Documents

The Human Genome: Structure and Function of Genes and ... · tion of genes and chromosomes at the molecular level. More recently, this has been supplemented by ... 20 THOMPSON & THOMPSON