Initial sequencing and analysis of the human genome - Vitagenes

More documents

Recommendations

Info

articles20±35 Mb. The increase is most pronounced in the male meioticmap. The effect can be seen, for example, from the higher slope atboth ends of chromosome 12 (Fig. 15). Regional and sex-speci®ceffects have been observed for chromosome 21 (refs 110, 134).Why is recombination higher on smaller chromosome arms? Ahigher rate would increase the likelihood of at least one crossoverduring meiosis on each chromosome arm, as is generally observedin human chiasmata counts 135 . Crossovers are believed to benecessary for normal meiotic disjunction of homologous chromosomepairs in eukaryotes. An extreme example is the pseudoautosomalregions on chromosomes Xp and Yp, which pair during malemeiosis; this physical region of only 2.6 Mb has a genetic length of50 cM (corresponding to 20 cM per Mb), with the result that acrossover is virtually assured.Mechanistically, the increased rate of recombination on shorterchromosome arms could be explained if, once an initial recombinationevent occurs, additional nearby events are blocked by positivecrossover interference on each arm. Evidence from yeast mutants inwhich interference is abolished shows that interference plays a keyrole in distributing a limited number of crossovers among thevarious chromosome arms in yeast 136 . An alternative possibility isthat a checkpoint mechanism scans for and enforces the presence ofat least one crossover on each chromosome arm.Variation in recombination rates along chromosomes andbetween the sexes is likely to re¯ect variation in the initiation ofmeiosis-induced double-strand breaks (DSBs) that initiate recombination.DSBs in yeast have been associated with openchromatin 137,138 , rather than with speci®c DNA sequence motifs.With the availability of the draft genome sequence, it should bepossible to explore in an analogous manner whether variationin human recombination rates re¯ects systematic differences inchromosome accessibility during meiosis.Repeat content of the human genomeA puzzling observation in the early days of molecular biology wasthat genome size does not correlate well with organismal complexity.For example, Homo sapiens has a genome that is 200 times aslarge as that of the yeast S. cerevisiae, but 200 times as small as that ofRecombination rate (cM per Mb)32.521.510.500 20 40 60 80 100 120 140 160Length of chromosome arm (Mb)Figure 16 Rate of recombination averaged across the euchromatic portion of eachchromosome arm plotted against the length of the chromosome arm in Mb. For largechromosomes, the average recombination rates are very similar, but as chromosome armlength decreases, average recombination rates rise markedly.Amoeba dubia 139,140 . This mystery (the C-value paradox) was largelyresolved with the recognition that genomes can contain a largequantity of repetitive sequence, far in excess of that devoted toprotein-coding genes (reviewed in refs 140, 141).In the human, coding sequences comprise less than 5% of thegenome (see below), whereas repeat sequences account for at least50% and probably much more. Broadly, the repeats fall into ®veclasses: (1) transposon-derived repeats, often referred to as interspersedrepeats; (2) inactive (partially) retroposed copies of cellulargenes (including protein-coding genes and small structural RNAs),usually referred to as processed pseudogenes; (3) simple sequencerepeats, consisting of direct repetitions of relatively short k-merssuch as (A) n , (CA) n or (CGG) n ; (4) segmental duplications, consistingof blocks of around 10±300 kb that have been copied fromone region of the genome into another region; and (5) blocks oftandemly repeated sequences, such as at centromeres, telomeres,the short arms of acrocentric chromosomes and ribosomal geneclusters. (These regions are intentionally under-represented in thedraft genome sequence and are not discussed here.)Repeats are often described as `junk' and dismissed as uninteresting.However, they actually represent an extraordinary trove ofinformation about biological processes. The repeats constitute arich palaeontological record, holding crucial clues about evolutionaryevents and forces. As passive markers, they provide assaysfor studying processes of mutation and selection. It is possible torecognize cohorts of repeats `born' at the same time and to followtheir fates in different regions of the genome or in different species.As active agents, repeats have reshaped the genome by causingectopic rearrangements, creating entirely new genes, modifying andreshuf¯ing existing genes, and modulating overall GC content. Theyalso shed light on chromosome structure and dynamics, andprovide tools for medical genetic and population genetic studies.The human is the ®rst repeat-rich genome to be sequenced, andso we investigated what information could be gleaned from thismajority component of the human genome. Although some of thegeneral observations about repeats were suggested by previousstudies, the draft genome sequence provides the ®rst comprehensiveview, allowing some questions to be resolved and new mysteries toemerge.Transposon-derived repeatsMost human repeat sequence is derived from transposableelements 142,143 . We can currently recognize about 45% of thegenome as belonging to this class. Much of the remaining`unique' DNA must also be derived from ancient transposableelement copies that have diverged too far to be recognized assuch. To describe our analyses of interspersed repeats, it is necessarybrie¯y to review the relevant features of human transposableelements.Classes of transposable elements. In mammals, almost all transposableelements fall into one of four types (Fig. 17), of which threetranspose through RNA intermediates and one transposes directlyas DNA. These are long interspersed elements (LINEs), shortinterspersed elements (SINEs), LTR retrotransposons and DNAtransposons.LINEs are one of the most ancient and successful inventions ineukaryotic genomes. In humans, these transposons are about 6 kblong, harbour an internal polymerase II promoter and encode twoopen reading frames (ORFs). Upon translation, a LINE RNAassembles with its own encoded proteins and moves to the nucleus,where an endonuclease activity makes a single-stranded nick andthe reverse transcriptase uses the nicked DNA to prime reversetranscription from the 39 end of the LINE RNA. Reverse transcriptionfrequently fails to proceed to the 59 end, resulting in manytruncated, nonfunctional insertions. Indeed, most LINE-derivedrepeats are short, with an average size of 900 bp for all LINE1 copies,and a median size of 1,070 bp for copies of the currently activeLINE1 element (L1Hs). New insertion sites are ¯anked by a smallNATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com © 2001 Macmillan Magazines Ltd879
articlesClasses of interspersed repeat in the human genomeLength CopynumberLINEs AutonomousORF1 ORF2 (pol)AAA 6–8 kb 850,000ABSINEs Non-autonomous AAA100–300 bp 1,500,000Fraction ofgenome21%13%Retrovirus-likeelementsAutonomousNon-autonomousgag pol (env)(gag)6–11 kb1.5–3 kb450,0008%DNAtransposonfossilsAutonomousNon-autonomoustransposase2–3 kb80–3,000 bp300,0003%Figure 17 Almost all transposable elements in mammals fall into one of four classes. See text for details.target site duplication of 7±20 bp. The LINE machinery is believedto be responsible for most reverse transcription in the genome,including the retrotransposition of the non-autonomous SINEs 144and the creation of processed pseudogenes 145,146 . Three distantlyrelated LINE families are found in the human genome: LINE1,LINE2 and LINE3. Only LINE1 is still active.SINEs are wildly successful freeloaders on the backs of LINEelements. They are short (about 100±400 bp), harbour an internalpolymerase III promoter and encode no proteins. These nonautonomoustransposons are thought to use the LINE machineryfor transposition. Indeed, most SINEs `live' by sharing the 39 endwith a resident LINE element 144 . The promoter regions of all knownSINEs are derived from tRNA sequences, with the exception of asingle monophyletic family of SINEs derived from the signalrecognition particle component 7SL. This family, which also doesnot share its 39 end with a LINE, includes the only active SINE in thehuman genome: the Alu element. By contrast, the mouse has bothtRNA-derived and 7SL-derived SINEs. The human genome containsthree distinct monophyletic families of SINEs: the active Alu,and the inactive MIR and Ther2/MIR3.LTR retroposons are ¯anked by long terminal direct repeats thatcontain all of the necessary transcriptional regulatory elements. Theautonomous elements (retrotransposons) contain gag and polgenes, which encode a protease, reverse transcriptase, RNAse Hand integrase. Exogenous retroviruses seem to have arisen fromendogenous retrotransposons by acquisition of a cellular envelopegene (env) 147 . Transposition occurs through the retroviral mechanismwith reverse transcription occurring in a cytoplasmic virus-likeparticle, primed by a tRNA (in contrast to the nuclear location andchromosomal priming of LINEs). Although a variety of LTR retrotransposonsexist, only the vertebrate-speci®c endogenous retroviruses(ERVs) appear to have been active in the mammaliangenome. Mammalian retroviruses fall into three classes (I±III),each comprising many families with independent origins. Most(85%) of the LTR retroposon-derived `fossils' consist only of anisolated LTR, with the internal sequence having been lost byhomologous recombination between the ¯anking LTRs.DNA transposons resemble bacterial transposons, having terminalinverted repeats and encoding a transposase that binds near theinverted repeats and mediates mobility through a `cut-and-paste'mechanism. The human genome contains at least seven majorclasses of DNA transposon, which can be subdivided into manyfamilies with independent origins 148 (see RepBase, http://www.girinst.org/,server/repbase.html). DNA transposons tend to haveshort life spans within a species. This can be explained by contrastingthe modes of transposition of DNA transposons and LINEelements. LINE transposition tends to involve only functionalelements, owing to the cis-preference by which LINE proteinsassemble with the RNA from which they were translated. Bycontrast, DNA transposons cannot exercise a cis-preference: theencoded transposase is produced in the cytoplasm and, when itreturns to the nucleus, it cannot distinguish active from inactiveelements. As inactive copies accumulate in the genome, transpositionbecomes less ef®cient. This checks the expansion of any DNAtransposon family and in due course causes it to die out. To survive,DNA transposons must eventually move by horizontal transferto virgin genomes, and there is considerable evidence for suchtransfer 149±153 .Transposable elements employ different strategies to ensure theirevolutionary survival. LINEs and SINEs rely almost exclusively onvertical transmission within the host genome 154 (but see refs 148,155). DNA transposons are more promiscuous, requiring relativelyfrequent horizontal transfer. LTR retroposons use both strategies,with some being long-term active residents of the human genome(such as members of the ERVL family) and others having only shortresidence times.Table 11 Number of copies and fraction of genome for classes of interspersedrepeatNumber ofcopies (´ 1,000)Total number ofbases in the draftgenomesequence (Mb)Fraction of thedraft genomesequence (%)Number offamilies(subfamilies)SINEs 1,558 359.6 13.14Alu 1,090 290.1 10.60 1 (,20)MIR 393 60.1 2.20 1 (1)MIR3 75 9.3 0.34 1 (1)LINEs 868 558.8 20.42LINE1 516 462.1 16.89 1 (,55)LINE2 315 88.2 3.22 1 (2)LINE3 37 8.4 0.31 1 (2)LTR elements 443 227.0 8.29ERV-class I 112 79.2 2.89 72 (132)ERV(K)-class II 8 8.5 0.31 10 (20)ERV (L)-class III 83 39.5 1.44 21 (42)MaLR 240 99.8 3.65 1 (31)DNA elements 294 77.6 2.84hAT groupMER1-Charlie 182 38.1 1.39 25 (50)Zaphod 13 4.3 0.16 4 (10)Tc-1 groupMER2-Tigger 57 28.0 1.02 12 (28)Tc2 4 0.9 0.03 1 (5)Mariner 14 2.6 0.10 4 (5)PiggyBac-like 2 0.5 0.02 10 (20)Unclassi®ed 22 3.2 0.12 7 (7)Unclassi®ed 3 3.8 0.14 3 (4)Total interspersed1,226.8 44.83repeats.............................................................................................................................................................................The number of copies and base pair contributions of the major classes and subclasses oftransposable elements in the human genome. Data extracted from a RepeatMasker analysis ofthe draft genome sequence (RepeatMasker version 09092000, sensitive settings, using RepBaseUpdate 5.08). In calculating percentages, RepeatMasker excluded the runs of Ns linking the contigsin the draft genome sequence. In the last column, separate consensus sequences in the repeatdatabases are considered subfamilies, rather than families, when the sequences are closely relatedor related through intermediate subfamilies.880 © 2001 Macmillan Magazines Ltd NATURE | VOL 409 | 15 FEBRUARY 2001 | www.nature.com
Page 2 and 3: articlesGenome Sequencing Centres (
Page 4 and 5: articleswell as targeted regions of
Page 6 and 7: articlesultimate goal of a complete
Page 8 and 9: articlesthat were used to `seed' th
Page 11 and 12: articlesFingerprint clone contigPic
Page 13 and 14: articlesof the end of the contig, s
Page 15 and 16: articles®ngerprint map. However, m
Page 17 and 18: articlesnucleotides to collections
Page 19: articlesTable 10 Number of CpG isla
Page 23 and 24: articlesposable elements in categor
Page 25 and 26: articlesProportion of genome compri
Page 27 and 28: articlesgenomes 184±187 . By study
Page 29 and 30: like termini show the typical diver
Page 31 and 32: articlesvelocardiofacial/DiGeorge a
Page 33 and 34: articlesaSum of aligned bases (kb)c
Page 35 and 36: articlesthe IAC anticodon for a Val
Page 37 and 38: articlesTable 21 Characteristics of
Page 39 and 40: articles30% to 50%. The correlation
Page 41 and 42: articlesTable 22 Properties of the
Page 43 and 44: articlesTable 23 Properties of geno
Page 45 and 46: articlesmultifunctional proteins an
Page 47 and 48: articlesaYW, F, VBr Br Ba Br Br Br
Page 49 and 50: articlesSegmental history of the hu
Page 51 and 52: articlesThe most extreme mechanism
Page 53 and 54: articlesinclude the DiGeorge/veloca
Page 55 and 56: articlesbecause of the availability
Page 57 and 58: articlesNature 380, 152±154 (1996)
Page 59 and 60: articles265. Sorensen, P. D. & Fred
Page 61 and 62: articles443. Roth, F. P., Hughes, J

Initial sequencing and analysis of the human genome - Vitagenes

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?