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Moreover, the coding regions of the genome (the exons) are typically found in shortsegments (average size about 145 nucleotide pairs) floating in a sea of DNAwhose exact nucleotide sequence is of little consequence. This arrangementmakes it very difficult to identify all the exons in a stretch of DNA sequence.Even harder is the determination of where a gene begins and ends and exactlyhow many exons it spans.Accurate gene identification requires approaches that extract informationfrom the inherently low signal-to-noise ratio of the human genome.
We shalldescribe some of them in Chapter 8. Here we discussonly one general approach,which is based on the observation that sequencesthat have a function are relatively conserved during evolution, whereas those without a function are free tomutate randomly. The strategy is therefore to compare the human sequencewith that of the corresponding regions of a related genome, such as that of themouse.
Humans and mice are thought to have diverged from a common mammalian ancestor about 80 x 106years ago,which is long enough for the majorityof nucleotides in their genomes to have been changed by random mutationalevents.Consequently,the only regions that will have remained closely similar inthe two genomes are those in which mutations would have impaired functionand put the animals carrying them at a disadvantage, resulting in their elimination from the population by natural selection.
Such closely similar regions areknown as conserued regions. The conserved regions include both functionallyimportant exons and regulatory DNA sequences. In contrast, nonconseruedregionsrepresent DNA whose sequenceis unlikely to be critical for function.The power of this method can be increased by comparing our genome withthe genomes of additional animals whose genomes have been completelysequenced,including the rat, chicken, chimpanzee, and dog. By revealing in thisway the results of a very long natural "experiment," lasting for hundreds of millions of years, such comparative DNA sequencing studies have highlighted themost interesting regions in these genomes.The comparisons reveal that roughly5% of the human genome consists of "multi-species conserved sequences,"asdiscussed in detail near the end of this chapter.
Unexpectedly, only about onethird of these sequences code for proteins. Some of the conserved noncodingsequences correspond to clusters of protein-binding sites that are involved ingene regulation, while others produce RNA molecules that are not translatedinto protein. But the function of the majority of these sequences remainsunknor.tm. This unexpected discovery has led scientists to conclude that weunderstand much less about the cell biology of vertebrates than we had previously imagined. Certainly, there are enormous opportunities for new discoveries, and we should expect many surprises ahead.Comparative studies have revealed not only that humans and other mammalsshare most of the same genes,but also that large blocks of our genomes containthese genes in the same order, a feature calIed conseruedsynteny.As a result, Iargeblocks of our chromosomes can be recognized in other species.
This allows thechromosome painting technique to be used to reconstruct the recent evolutionarv historv of human chromosomes (Fieure 4-18).Figure 4-17 Representationof thenucleotidesequencecontent of thecompletelysequencedhuman genome.retroviral-likeelements,The LlNEs,SlNEs,aremobileand DNA-onlytransposonsgeneticelementsthat havemultipliedinthemselvesour genomeby replicatingand insertingthe new copiesin differentpositions.Thesemobilegeneticelementsin Chapter5 (seeTable5-3,arediscussedp. 318).Simplesequencerepeatsare(lessthan 14shortnucleotidesequencesnucleotidepairs)that arerepeatedagainSegmentaland againfor long stretches.arelargeblocksof theduplicationsgenome(1000-200,000nucleotidepairs)that are presentat two or more locationsin the genome.The most highlyrepeatedhaveblocksof DNAin heterochromatinnot yet been completelysequenced;of human DNAthereforeabout 10oloin thisarenot representedsequencesdiagram.(DatacourtesyofE.Margulies.)208Chapter4: DNA,Chromosomes,and GenomesA N C E S T OCRH R O M O S O M Ea n c e s t o rD N Aof humanc h r o m o s o m e32 i n v e r soin 5a n c e s t o rD N Aof humanc h r o m o s o m e2 1f usionI(A)"6'B-rylemurorangutannu m a nabcdabcdabcd(B)ChromosomesExistin DifferentStatesThroughout the Life of a CellFigure4-18 A proposedevolutionaryhistoryof human chromosome3 and itsrelativesin other mammals.(A)Theorderof chromosome3 segmentshypothesizedto be presenton achromosomeof a mammalianancestorisshown$rellowbox).The minimumchangesin this ancestralchromosomenecessaryto accountfor the appearanceof eachof the threemodern(Thechromosomesareindicated.present-daychromosomesof humansand Africanapesareidenticalat thisresolution.)The smallcirclesdepicted inthe modernchromosomesreoresentthepositionsof centromeres.A fissionandinversionthat leadsto a changeinchromosomeorganizationis thoughttooccuronceevery5-10 x 106yearsinmammals.(B)Someof the chromosomepaintingexperimentsthat led to thediagramin (A).Eachimageshowsthechromosomemostcloselyrelatedtohumanchromosome3, paintedgreenbyhybridizationwith differentsegmentsofDNA,lettereda, b, c, and d alongthebottom of the figure.Theseletterscorrespondto the coloredsegmentsofthe diagramsin (A),as indicatedon the(From5.
MLilleretancestralchromosome.al.,Proc.NatlAcad.Sci.U.5.A.97:206-211,2000.With permissionfrom NationalAcademyof Sciences.)We have seen how genesare arranged in chromosomes, but to form a functionalchromosome, a DNA molecule must be able to do more than simply carry genes:it must be able to replicate, and the replicated copies must be separatedand reliably partitioned into daughter cells at each cell division. This process occursthrough an ordered series of stages,collectively known as the cell cycle, whichprovides for a temporal separation between the duplication of chromosomesand their segregation into two daughter cells.
The cell cycle is briefly summarized in Figure 4-19, and it is discussed in detail in Chapter 17. Only certainn u c t e a re n v e t o p eMTTOSTSIn r e r p n a s ec hr o m o s o m em itoticchromosomeI NTERPHASEM PHASEINTERPHASEFigure4-19 A simplifiedview of the eucaryoticcell cycle,Duringinterphase,the cellis activelyexpressingits genesanois thereforesynthesizingproteins.Also,duringinterphaseand beforecelldivision,the DNAis replicatedand eachchromosomeis duplicatedto producetwo closelypaireddaughterchromosomes(a cellwith onlytwo chromosomesisillustratedhere).OnceDNAreplicationis complete,the cellcan enterM phase,when mitosisoccursand the nucleusisdividedinto two daughternuclei.Duringthis stage,the chromosomescondense,the nuclearenvelopebreaksdown,andthe mitoticspindleformsfrom microtubulesand other proteins.Thecondensedmitoticchromosomesarecapturedby themitoticspindle,and one completesetof chromosomesis then pulledto eachend of the cellby separatingeachdaughterchromosomepair.A nuclearenvelopere-formsaroundeachchromosomeset,and in the finalstepof M phase,the celldividesto producetwo daughtercells,Mostof the time in the cellcycleis spentin interphase;M phaseis briefincomparison,occupyingonly aboutan hour in manymammaliancells.CHROMOSOMALDNAAND ITSPACKAGINGINTHECHROMATINFIBER,:'l$9";,;:'"'Figure4-20 A comparisonof extendedinterphasechromatinwith thechromatin in a mitotic chromosome.(A)A scanningelectronmicrographof amitoticchromosome:a condensedduolicatedchromosomein whichthearestilllinkedtwo new chromosomestogether(seeFigure4-21).Theregionindicatesthe positionconstricteddescribedin Figureof the centromere,4-21.(B)An electronmicrographshowingan enormoustangleofchromatinspillingout of a lysedinterphasenucleus.Note the differenceinscales.(A,courtesyof TerryD.
Allen;B,courtesyof VictoriaFoe.)(A)1 lr.(B)l0 pmparts of the cycle concern us in this chapter. During interphase chromosomesare replicated, and during mitosis they become highly condensed and then areseparated and distributed to the two daughter nuclei. The highly condensedchromosomes in a dividing cell are knorm as mitotic chromosomes (Figure4-2OA).This is the form in which chromosomes are most easily visualized; infact, the images of chromosomes shor,rmso far in the chapter are of chromosomes in mitosis.
During cell division, this condensed state is important for theaccurate separation of the duplicated chromosomes by the mitotic spindle, asdiscussedin Chapter 17.During the portions of the cell cycle when the cell is not dividing, the chromosomes are extended and much of their chromatin exists as long, thin tangledthreads in the nucleus so that individual chromosomes cannot be easily distinguished (Figure 4-208).We shall refer to chromosomes in this extended state asinterphasechromosomes.Since cells spend most of their time in interphase, andthis is where their genetic information is being read out, chromosomes are ofgreatestinterest to cell biologists when they are least visible.EachDNAMoleculeThatFormsa LinearChromosomeMustContaina Centromere,OriginsTwoTelomeres,and ReplicationA chromosome operates as a distinct structural unit: for a copy to be passed onto each daughter cell at division, each chromosome must be able to replicate,and the newly replicated copies must subsequently be separated and partitioned correctly into the two daughter cells.These basic functions are controlledby three types of specialized nucleotide sequencesin the DNA, each of whichbinds specific proteins that guide the machinery that replicates and segregateschromosomes (Figure 4-21) .Experiments in yeasts, whose chromosomes are relatively small and easy tomanipulate, have identified the minimal DNA sequence elements responsiblefor each of these functions.
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