Molecular biology of the cell, 5th ed

gso r

l0tt

Z60L

L LOL

LgOL

090 r

?s0 r

HIMOUDII]f CNV NOISIAICrtl) Jo toulNol

stslNl)ol^)

SISO1IW

]SVHd S

wlls^s tourNofltf^)-Itf IHI

llf^) Il)lHl ro MTAU]AO

raldeq) slql ul

eql sreSSlrl ll ^|oq upldxa pup urals^s loJluo3 elc.{J-lleJ aqt eqlJ3sep ueql eM'elJltJ

IIaJ aqt Jo ̂ ar^reô Jalrq B ql.Irv\ u€aq eM'paleulpJooJ puu pallorluoJeru.{.eql ̂\oq puE alf,l{J IIoc eqlJo slueê snoIJEA aql saqlrcsep reldBqJ sIqJ

'uorsr^rp naql qll1!\ (ss€ru IIef, ur esuercul JIeq] 'sl ]BI{}) q}Mor8Jraql eleulpJooJ lsnru silac Surpr^rp 'azrs Jreql urPluIEIu oJ'uolsplp qcPe qlyvlJellErus le8 plnoM sllef, ratqSnsp 'esMJeqlo lselnJelotuoJletu pue selleueSJoJaqto rreqt aluJlldnp oqe sllaf, lsoru 'euloue8 rlaqt Supucrtdnp o1 uoyllppe uI

'Jecuuc q llnseJueJ suorsr^rp IIac e^rssesxe 'suorlJuruluu uralsrts eql uel{lv\ dpoq aqr 1o sensspaql ul sJeqrunu [ec Surlup8ar ul eloJ IeJlueJ p suq eJoJeJeql uralsds lortuoc alcdc-lla3 eql 'lou aJE ,{aql ueq^| ll 3u14co1q puu pepeeu aJE slleJ eroru ueq a uols-FIp IIaJ Supeprups 'slleJ reqlo uo4 spu8ts ot arusuodsar,(1q8q st uralsds aql'leurrue JplnllacrlFru p uI 'ilec eql eplslno suolllpuoJ srolluoru osp rualsrts 1or1-uoo eqJ 'a1a1duoc s1 uorlecqdal VNC lpun

'aydurexe JoJ 'saluosoruoJqt paleo-gdnp 3o uone8ar8as eql JoJ suoqerudard lnuad tou saop 11 'palalduoJ ueeqeAEq sluala JerlJpa Ipun sluele ralel sdulep pue alcrb IIaf, eql q8norql uotsser8-ord srolruoru rualsrts IoJluoJ aql 'llal eql eplsul 'lleJ eql eplstno pue eplsu qloquro4 spu31s snolJpl o1 puodsar ol rualsds IoJluoJ eql ,lrollu pue uoIsIAIp IIaJyo '{rqapg eq} acupqua uoqeln8ar;o srar{.e1 puolllppe 'sllef, }sotu uI 'uoue8ar8as

pup uorlporTdnp aurosoruorqc Surpnlcur 'alcztc eql Jo stuaâ u1eru eql elelllul

lEqt saqJtrMs lBf,ruaq]orq Jo selJas perapJo ue s1 rualsrfu slq] Jo aroc aql 'a1cdc

IIaJ eql q8norqt uorssarSord surano8 ferTt'utalsts ToJluoJ ayctc-11ac aq] sE u ôu{'suralord ,fuo1ep8ar Jo {Jomlau xayduroc e peAIoAe eêq slleo cqorfuucng

'(1-2,1 arntlgl auroua8 erque eqlgo,tdor E selreJal qf,Ee lpql os 'sllal rarq8nep oMl aql o1 6palnBa8ag petnqulslpdlalerncce eq uaql lsnrx seruosoruoJqc paleclldar eql pue 'sardoc alaldruoc orrnlacnpord o1 paleclldar IIIryqrleJ eq lsJg tsnur eruosotuorqo qcea ul VNC eqt 'sllao

ralq8nep leJpuepr ,(lpcpaua8 oa4 acnpord oI 'slleo Jo uopureue8 xau eql ol

uoppruroJul cpeua8 slrJo uo Surssed eqt :{sul lplueuepury tsoru sll qsqdurocceol lr ^\olle lpql esoql are uro;rad o] seq ilec e lpql sassaoord Jo las IunIuIuIueqJ'lesre^run eru talemoq 'sJIlslJe]JBJEqJ uIPuaC 'eJII slrrspe8ro ue uI setuptuaraJJrp lE pue ursrue8ro ot rusrue8ro uro4 fuen a1c,tc 1ac eqlJo sllutep aqJ

's[ep rrag E ulqllm erp plnom arvr-aldruexe ro3 'sz{er-xgo asop a8re1I-razr e ol arnsodxa fq-paddols aJa^ uorsrllp IIaJ IIe JI :aAIAIns o1 dldurrs puo-cas drala sllao Jo suollnu ,(ueur arnlce;nueru lsnru sn Jo qcea 'IJBJ uI 'alp leq]s11ac aceldar ol pepeeu,'tlpnsn sr uorsrnrp 1ac

'r(poq l1npe eql uI uelg 'rustue8ro

3u1uo11cung u ecnpord o1 parmbar eJe suolsrnlp IIaJ Jo sacuanbas xaldruocpue Suoy 'sarcads JulnlleJplnru u1 'rusrue8ro a,rau alalduroo p seJnpoJd uotsvrtp

IIac qJBa 'stsead pue BrJatf,uq se qJns 'salcads JEInlleJIun u1 'acnpordar s8uqt3ut rl 11e qclqm rQ usrueqf,eru prluasse aql s1 'a1cfc

ilac eqt sE trmou{ 'uotstrttp

pue uorluJrldnp 1o alcdc srql 'or\^l q sepplp ueql pue slueluor qt salecqdnp

ll qJIq a ur sluene go acuanbas ,{lrapro ue Sunuro;rad dq sacnpordar 1ac y'o8e srear( uoIIIq eeJql JeAo quug uo aJrI

;o s8uruu€aq eqt o] etuu ul lceq Eulpua[e uols1lrlp pue qunor8 IIer Jo spunorpaleadar;o slcnpord aJp 'leurru€ur Jelnllacr]Fru eql o] runlJeloeq JulnllaJrunaqt uroq 'srusrue8ro 3upil 11V

'aJII Jo ,fignupuoc eqt JoJ a8essaur puno;ord e 1t

qllm sarJJeJ 'r{-rntuac qluaa}eup aqlJo alppnu aql ul paqslqelse }sJrJ '1ceg aldurssr{I'stsxa.{pearp teq} IIeo u elecgdnp ol $ IIaJ rvrau e e{Eru o},,(e,rn dpo aql

el)/() Ile) eql

'(srsaur)ol^))

oMl olut sapr^rp Jlaslr lla) eql q)tqMroue '(srsollul ut) rol)nu lalq6nep Jo

lred e olul paleoejbas ale saulosoulolrl)petP)rldnp aql uaqM,aseLld t^l

pue 'pale)tldnp alp sauosotuolLl)aql uaqM'aseqd s ut rn))o al)I) lla) aqlJo slua^a leuosoLuorq) Joreul aql .al)^)

lla) aql ]o slua^o rolpu atll z-l I arn6tl

pate8efas aq] pue 'palquassesrp ueq] sr alpurds eql 'epurds aql Jo salodalrsoddo ol palpd aJe qcrqm 'sppeurorqc Jalsrs eql saleredas asaqdnun 1o yelseql lp uorseqoo prleruoJqr-Jalsrs Jo uorlonJlsep aql 'asaqdaput pelleJ a8els eur rolenba alpurds aql lu u8rp srelsrs 1e

'rtlenluale 'pue 'alpurds aql;o salodaltsoddo ol peqJBpE aJE sprteruoJqJ Jelsrs '(gt raldeql ur pessnJsrp) saFqn]-oJf,IIu go Lerre relodrq luer8 e 'a1purds Jqollut aql ol peqop11e aurocaq srrcdpDp{uoJqc Jelsrs aql 'srso}nu ur Ja}el selquasspsrp adolazrua J€el3nu aql uer{M'uo&aqn p!ruu,toJqc-Ja7s1s ,tq raqlaSo] pa{ull ureruar qJIq&\ 'spllBruoJqJ Jel-sls paller spor lcedruoc pue pr8rr;o srmd otur pesuepuoo pue papueluasrp z(1pn-per8 are selnrelolu VNC o1\^l eq] 'asatldotd palpc a8els e lp srsolrru ur ,(Feg 'sa8e-1uq uralord pazqercads ,(q raqtaSor fpq8p pleq pue peurm]relur erp seruosoru-oJqJ palerrldnp yo rrcd qcea ur selncelotu VNCI eqt 'aseqd

S Jo pue eqt ]V'17-21 arnt;g) omt ur sapwp Jleslr IIeJ eql ueq.^ 'srs

-auryo1tc ro 'uorsnrp cnuseldo/.c pue lrelJnu ralq8nep;o rmd e olur petnqrnspaJB seruosoruorqc pardoc eql qJrqm Suunp 'srsolrut Jo 'uorsrlrp JeelJnu :slualerofeur om] sasrrdruoc aseqd 6

'(llef, uBIIBururEru B ur Jnoq uu ueql ssel) eruq sselqcnur sarrnbeJ qJrqm '(srsoln JoJ W) asuqd y1 ur JnJJo uorsrlrp IIaJ pue uone8-ar8as aurosoruoJqJ 'aseqd

S JaUV'lleo uerprurueu pcrddl e ur arurt a1c.,b-1acaqtJoJIeq lnoqe sardncco pue srnoq 71-91 se.rrnber qcrq a'(srsaqtuis VNCI roJ S)asoqd g Surrnp srnrro uo4eclldnp euosouoJq3 'a1c,{c

fiac eq] Jo saseqd roferuoml eql aurJap sassacord eseqJ 'sllal rarq8nup IpJrluepr ,tlucllaua8 o^ l olur.,{lasrcard sardoc aql a}e8arSas ueq} pue saruosoruoJqc aqr ul VNC Jo lunoruelsEA aq] ,{lalerncce alecrldnp o1 s alcd: IIaJ eq} Jo uorlf,unJ f,rseq }soru eql

saseqd rnol olu! pap!^!c sl all^) lle) )rlo^re)nl eql

'a1c.,b 1ac aql dprys o1

pasn suralsds IIaJ Iapour puu spoqlau aq] Jo eruos JaprsuoJ uer{l aM 'a1c[c 1ac

cqo,,fuecna arlt p sasaqd rnoJ aqt Jo uoqdrrcsap ;arJq E qlrm uorloas slql urBaq all

tt)A) ttr) lHl_ Jo ltAUtAo

'peluurpJooJ eJB sasseoord or4 asaql ,rroq pue uorsrnlp puu qlr,ror8 IIaJ Jo seleJaq] uralo8 spu8rs JEInllacEJue ̂aoq Japrsuoc a,u.'dleurg 's1ac relq8npp oml eqlolur pale8ar8as uaqt pue palecrldnp eJe seuosoruoJqJ eqt qJrqm ur 'a1c[c

11acaql;o sa8els roleu eql 1elep ur Japrsuol xeu aM'ayc[c aqt go s]uele ]ueJeJJIp

slslNt)o1A)

o6

uorle)rld npa|.xosourorq)

'sa;>{t ;;a: leuorlrppe q6norqr 6urob Iq

apr^rp ot enuttuo) uauo lltM s;;at ralqbnepaqr Jo q)el 'a;r,(r q>ea ur parnpotd

ate s;;ar ralqbnep le)rluapt {lletrleue6 oan}MOq alerlsnllr ol uMoqs st sot.uosorloJtlf

omt Ll]rM ;;et rrlol.rerna lerlteqrod,(qe Jo uolsr^rp eq1 'a;r{r

lla) aql 1-11 arn6r1

Ioo

E

NOrrvglu9lsSWOSOI OUHf

\

\

NOr-LV)r'rd3U]tAOSOIAOUHf CNV

Ht_l ou9'l'lt) L

a1:(1 1;a1 eqt:/ t rardeq3 t5g t

OVERVIEW OF THE CELL CYCLE 1055

cytokinesismitosis

metaphase-to-anaphase transition

INTERPHASE

DNA repl ic

chromosomes are packaged into separate nuclei at telophase. Cltokinesis thencleaves the cell in two, so that each daughter cell inherits one of the two nuclei(Figure l7-3). <TACT> <TCAA>

Most cells require much more time to grow and double their mass of pro-

teins and organelles than they require to duplicate their chromosomes and

divide. Partly to allow more time for growth, most cell cycles have exrra gap

phases-a G1 phase between M phase and S phase and a Gz phase between Sphase and mitosis. Thus, the eucaryotic cell cycle is traditionally divided intofour sequential phases: Gr, S, G2, and M. Gr, S, and G2 together are called inter-phase (Figure l7-4, and see Figure 17-3). In a typical human cell proliferating in

culture, interphase might occupy 23 hours of a 24-hour cycle, with I hour for Mphase. Cell growth occurs throughout the cell cycle, except during mitosis.

The two gap phases are more than simple time delays to allow cell growth.

They also provide time for the cell to monitor the internal and external environ-ment to ensure that conditions are suitable and preparations are complete

before the cell commits itself to the major upheavals of S phase and mitosis. The

G1 phase is especially important in this respect. Its length can vary greatly

depending on external conditions and extracellular signals from other cells. If

extracellular conditions are unfavorable, for example, cells delay progress

through G1 and may even enter a specialized resting state known as Go (G zero),

in which they can remain for days, weeks, or even years before resuming prolif-

eration. Indeed, many cells remain permanently in Ge until they or the organismdies. If extracellular conditions are favorable and signals to grow and divide arepresent, cells in early G1 or G0 progress through a commitment point near the

end of G1 knorvn as Start (in yeasts) or the restriction point (in mammaliancells). We will use the term Start for both yeast and animal cells. After passing

this point, cells are committed to DNA replication, even if the extracellular sig-

nals that stimulate cell growth and division are removed.

M PHASE

mitosis(nucleardiv is ion)

G2 PHASE

5 PHASE

Figure 17-3 The events ofeucaryoticcel l division as seen under amicroscope, The easily visible processes

of nuclear division (mitosis) and cel ldivision (cytokinesis), col lect ively cal ledM phase, typical ly occupy only a smallfraction of the cell cycle. The other, muchlonger, part of the cycle is known asinterphase, which includes S phase andthe gap phases (discussed in text). Thefive stages of mitosis are shown: anabrupt change in the biochemical stateof the cell occurs at the transition frommetaphase to anaphase. A cel l can pause

in metaphase before this transition point,

but once i t passes this point, the cel lcarr ies on to the end of mitosis andthrough cytokinesis into interphase.

Figure 1 7-4 The four phases of the cellcycle. In most cells, gap phases separatethe major events of 5 phase and M phase'

Gr is the gap between M Phase and5 phase, while G2 is the gaP betweenS phase and M phase.

cytokinesis(cytoplasmic

(DNA repl icat ion)

G1 PHASE

1056 Chapter 17:The Cell Cycle

Cell-Cycle Control ls Similar in All Eucaryotes

Some features of the cell cycle, including the time required to complete certainevents, vary greatly from one cell type to another, even in the same organism.The basic organization of the cycle, however, is essentially the same in alleucaryotic cells, and all eucaryotes appear to use similar machinery and controlmechanisms to drive and regulate cell-cycle events. The proteins of the cell-cycle control system, for example, first appeared over a billion years ago.Remarkably, they have been so well conserved over the course of evolution thatmany of them function perfectly when transferred from a human cell to a yeastcell. we can therefore study the cell cycle and its regulation in a variety of organ-isms and use the findings from all of them to assemble a unified picture of howeucaryotic cells divide. In the rest of this section, we briefly review the threeeucaryotic systems most commonly used to study cell-cycle organization andcontrol: yeasts, animal embryos, and cultured mammalian cells.

Cell-Cycle Control Can Be Dissected Genetically by Analysis ofYeast Mutants

Yeasts are tiny, single-celled fungi, with a cell-cycle control system remarkablysimilar to our own. TWo species are generally used in studies of the cell cycle. Thefission yeast schizosaccharomyces pombe is named after the African beer it isused to produce. It is a rod-shaped cell that grows by elongation at its ends. Divi-sion occurs when a septum, or cell plate, forms midway along the rod (Figurer7-5A). The budding yeast Saccharomyces cereuisiae is used by both brewersand bakers. It is an oval cell that divides by forming a bud, which first appearsduring G1 and grows steadily until it separates from the mother cell aftei mito-sis (Figure l7-58).

gene, because we avoid the complication of having a second copy of the gene inthe cell.

Many important discoveries about cell-cycle control have come from sys-tematic searches for mutations in yeasts that inactivate genes encoding essen-tial components of the cell-cycle control system. The genes affected by iome of

YEAST (Schhosa cch a ro myces po m be)

Gr

ISTART

YEAST (5accharo myces cerevisiae)

Figure 17-5 A comparison of the cellcycles of fission yeasts and buddingyeasts. (A) The fission yeast has a typicaleucaryotic cel l cycle with Gr, 5, G2, andM phases. The nuclear envelope of theyeast cel l , unl ike that of a highereucaryotic cell, does not break downduring M phase. The microtubules of themitotic spindle (light green)form insidethe nucleus and are attached to spindlepole bodies (darkgreen) at its periphery.The cell divides by forming a partition(known as the cel l plate) and spl i t t ing intwo. (B)The budding yeast has normal G1and S phases but does not have a normalG2 phase. Instead, a microtubule-basedspindle begins to form late in S phase; asin fission yeasts, the nuclear enveloperemains intact during mitosis, and thespindle forms within the nucleus. lncontrast with a fission yeast cell, the celldivides by budding.

G r

@

('sllrw ̂uof Jo ̂sauno)) uorlernleu66a;o ssarord aql 6uunp uruop

ua)iorq seq aoolaûa real)nu aqt'alnlltoaqt ur uaas aq louue) slql q6noqtly

'use;doy(r 66a aqt 1o ra{e; ate;rns aq1 urlueubrd u/Aotq aqt pareldsrp spq q)rqM

'snal)nu aql Jo alrs aql smoqs dol aqlreau ]ods a;ed aq1 'uo11ezrp1.ra;.ro; Ipear

'66asndouayarnleur V g-11 arn611

urur s 0

'(uMor,|s lou) lno unJ sa^rasar

lp)ruaq)orq raqt uaqM uo 6urpuadap-a;rl{r eq1;o e6e1s Iue le ,{lprezeqdeq

]lpq'(uortlnpord 61y se qrns) qlrvror6pue srsaqlu{sorq ro; apl(r aq} }noq6nolq}

{ressarau sr leql ssaro.rd e ur }uar)UapJr'siuelnur )pJ-uou'lseltuo) {g a6re;

{;;erurouqe aLuolaq (aql anor6 o} anur}uo)

lllls sluPlnur rp) aql asnp)ag '(aldurpxa

srqi ur 'aseqd s Jo uorler]rur) alaldLuo)

o] alqeun are {aq} 1eq1 dals rrlreds eq} olaiuor {aql ;r1un a;rb aql q6norql sser6ord

ol anurluol slla) luelnLu aqi'Illeurouqesuorl)unJ lrnpord aua6 luelnr.u eql

q)!qM le 'arnleradura] (q6!q) arrlplrrsar aqlol 6urLurerrir uO (B) '(rolo) s1r ,(q pale>rpur

sr lla) aqlJo eseqd aqt) apl{l eqllo saseqd

lle ur puno} are pue,{1;eu.rrou apr^rp slla)aql'arnleradural (Mol) anrssrurad aql lV (V)

'luelnul rp) aArl!suos-srnlereduele Jo ,rorêqsq eq19-11 arn6r1

LSOL

IAZ9S

( xesraqn ga;;o,(sa1lnol)'aseqd W alel Jo )llsllal)ereq) aJe q)lq^^'spnq

a6rel qllm {;uro;tun lsa.ue {aq}'}lnsal e sy'srsautloy(l pue slsolltLl ulol} }lxa aq} alalduro)

]ouue) ]nq aseqdeue alelduor s;;a>'ernleredua] a^rllrJlsar aql le ua,rotb luPrnLu 9t)pf e ul (B)'a6e1s a;r(r-;14) aql ol 6urptotte azts ut Ilen spnq 'slla) lseaI 6utleta;r1otd;o uorle;ndod ;eulou

e ul (V) 'uoltetn w )p) e Iq palsarre s;1al lseel 6ulppnq lo I6oloqdrou aq1 ;-1 1 atn6t1

r.uri 0Z

Surureluoc o^Jqua ue aleJaue8 ot 'Sut,r,roJ8 lnoqlu 'sepIAIp IIaJ luEIS elSuIS

aql qcrqm ur'suorstt'trp aSanaalJ pallel 'suoISIAIp IIeJ Jo asuenbas prder ,r{1$urqsr

-uolsE ue sraSSlrt BBa sndouay aql Jo uoUPZIpraC '(8-ZI arnBIJ) ,r(poq ueutnq

aql ul IIaJ a8pJaê uE ueq] urspldoldJ eJour sarull 000'00I suleluoJ pue Jala-ruerp ur ruru I JaAo sr 'aldruexa ro!'sndouax 3or; aqrlo 33a aq1 'uoISIAIp

IIaJ roJpapaau suraloJd aqt Jo salld{Jo}s a8Jel .{rrer qcrqm 'sletulue ̂'(ueru;o s33a pazr-llueJ luBI8 aql uI paz.{IeuB Lllpeer aJe alJ.{f, IIaJ eql Jo saJn}EaJ leJlruaqcolq aqJ

soltqul

leuruv ur ,(lle)lutoq)o!8 paz^leuv a8 ue) lotluo) al)^)-lla)

'(z-zI arnSlc) palsarresr tuptntu aql qtrqâ ]e ap,{t aq} ul lulod aql aleJlpul 'ezIS pnq pue 'pnq e JoaJuasqe ro asuasald eql :s[a] aql lE SuDIool ]sn[ ̂q ad,{1 srql;o lseJJe alJ-'{J-lleJ

ruroJrun e trelep uer a^\ 's1sea.{ Surppnq uI '(9-zI arnSlt) lpq -{aq] lulod slql

tE pue 'ssaJSoJd raqunJ JoJ paJlnbar sI eua8 luelnlu aql Jo uollJunJ aql aJaq^

lutod aql qoeal êql l!]un alc,{c IIaJ aql q8norql anulluoJ sllao 'ern}eJadrue}

raq8rq aql lV 'aua8 tuelntu aql Jo uorlJunJ er{l JJo qJ}I ^s ol (uourpuoJ anuJulsa.t

aqt) aJnteJedural raq8rq E ot pasIPJ uaql pue (uoutpuoJ ansslruJacl eql) aJnl-eradual l ol e lP pelEBEdord aq uec luelnut JpJ aAIlISuas-arnleJedlual V

'seJnl

-eJadual llrol ]E uorsrnrp IIas .rrrolle o1 qSnoua IIe^ suorlJunJ lnq saJnlpJedrual

q8rq fe uollrunJ ot sllEJ uralord ]uplnlu aql qcrqM uI 'suoltDinLu anqrcuas-aJnl-DJadraal ere suollelntu all^{J-llal PuolllpuoJ lsolN 'suoplpuol llJlsads UIEUaJ

ur .,{1tro uortJunJ ol slleJ }JnpoJd aua8 lue}nu aql JI 'sI wql-IDuolllpuoc s ad^l

-oueqd rlaq] JI .{1uo paurclulelu puB pellalas aq uBc s}uE}ntu ,pJ '.snqJ 'pa}EB

-edord aq louuec laâmoq 'alJ^3 IIaJ aql elelduo) louuel leql luelnru V

'1urod s1q] tsed IIac aq] la8 ol pallnber sI lJnpoJd aue8 leurJou aql lBI{1 3ul-tsa38ns 'elct(t

IIaJ aqt q lulod JrJrceds e te lsaJJe ol sllao asneJ suollPlnlu aseql

;o .rfueyq 'saua3 cp3 Jo 'saua8 alcrb-uolspqp-Ilac se uÂoDI erE suollelnlu aseql

(8)

L9

run-LvutdFlsr (Mo'I) 1^l55ll/!U3d (v)

l1)A) lll) lHllo l llAUlAo

tunrvu3dtAtl (H9rH) l^lr)lul-slu (8)

'l)ejlxa )tuseldol^) aaJJ-lla) stqt ul6ullerado sr ulalsls lolluo) al)l)-lla) aql

leql 6urle>rpur 'stsoltLU pue uorlerrldery116 1o sa;:{t paleadal qbnorql

o6 uaql pue asuapuo)op re;tnu r.uradsaql dlv qll/vl raqta6ot'tr ol poppe ale

ra;>nu utiads pue'pal)allol sr r-use;do{rpalnlrpun aqf 'sluauoduto)

lla) raqloruor; urseldoy(> aql etetedas pue s66e

6ol;1o q>req e6re; e uado leelq ol pasnsr uorle6nplua) altuag .urelsls aa.tJ-lle)

e u1 a1>{> lla) aql 6ur{pn15 g 1-1 1 ern6r3

ulur 09-0t :apib rrlolrur aer+-lla)

'suoprpuor alqellorluoJ puE pa$ldl.uls dlqSlq rapun sluaâalJ.,tr-llal alElndruEru pue a^Jasqo uEJ a^\'slJEJlxe IIaJ qJns uI '(0I-ZI eJngIC)eqru lsal P ur alr^J IIaJ eql Jo sluaâ dueu alruUsuoceJ puE sBBa sndouay ]UJ04ruseldol,b eJnd lsorup aredeJd ol alqrssod osle sr ]I 'uorsseJSord alcdc-1ac uo]caJJa Jreql aurruJalep ol 33a ue olur seJuBlsqns ]sa] lcefur ol ,,(sea dlanrlelal sl ]I'azrs e8Jpl Jraql sl srs^lpup a1c,,b-1ac ro; solJqua dlJea eseqtJo aBEluB^pp Jaqlouv's11ac ralq8nep oml otur uorle8eJ8es slr pue etuoue8 aql Jo uorlecqdnp aq1-sluarueJrnbar pluaurepury lsoru aq] aârqf,E ol papeeu unurrurru eql ol perJrld-rurs pue urrop paddrrls uals.{.s IoJluoJ alcdc-1ac eql Jo sBuDIro ̂ eql IBaAeJ eJoJ-e;eqt saIJ[J 71ac cruofuquta fpaa asaqJ 'selJ.,(c

IIaJ xaldruoc eJoru ur a]EJedo leql(JalEI pessnf,srp) surstueqlaru IoJluoJ eql Jo ̂ueur Jo qllr,roJ8 Jaqlre Jo aJuesqeaqt rn uorsrlrp prder ,{lSurpaarxe Jo alqpdec snq} erB 'apqdosotg,{g 1pr3 aqrpuealnsrdg ruplJ eqtJo asoql se [eâ se 'sndouay

1o s11ac cuod;qura i{yrea aql'saseqd zg

ro Ig alqelcarap lnoqllm'qcea salnurlu gI ]noqeJo saseqd ntr pue S olur papwpst a1c-,b qJEg 'sJnoq

2 ulr{tlm sllao (zrZ) g60t }noqp Suronpord ,slplra}ur a}nur{u-69 1e 'dlsnouorqcu,{s ssal Jo aJoru tnJf,o suorsrlrp I I lxeu eql ,salnur{u

06 }noqese{et tpq} uorsrlrp }srrJ E raUV <JJJV> '16-21 arn81g) s11ac raleurs Jo spupsnoq}

'(lou st itito;aq 6orJ aql lnq) ale)s aures aq] uo lle are,rnor dol aql ur s6u;anerp aql'6urpea; sur6aq a;odpelaql uaqM {;uo s1re1s qlMorg'azts aues aq} sureurer oftqLua aLl} pue'uotst^tp q)ea q}!M re1;eus,(;anrssat6ord ta6 s;;at

aqf 'oMl to {ep e ulqtlm alodpel Jelnlla)rllnu e 6urr.uro1-salnurur 0€ {rane a;>I> uotst^tp auo }o atel e te ,(1;erlrur-I;plderIlan sanea;> 66e eql'uo!tez!llue1 uodn'(lZ ratdeql ur passn)srp) 66a ue olur salnleu I;;eur; pue 6o.11 taqlou aq];o,{reno

aql ul sqluoul ,(ueu.r .ro; 6ulprnrp lnoqllM sanorb a/roo aq1'sndouay ur e6eaea;> 66e pue qlMoJ6 ey(rog 6-4 1 arn6l1

rI6oJJ llnpe ue

seuo)aq pue'stsoqdlouJelautsao6rapun 'snnot6 'spea; e;odpe1

'. - :,r,r:.+i.:iiirrii;////////

uruj I

(sqluour)6urprnrp lnoqlrnl saaol6 ep(roo

epl3 11a1 aqJ_:1.I raldeq) BS0 t

(srnoq)6urnirol6 lnoqltM saprnrp 66a paztltpa+NOIIVZt'lU_Ull

('ralqang-lq)arqlVratuanD Jo ̂satrno)) 'slselqorqu

ler are qder6ot)rur uol])ala 6uruuetssltlt u! slla) aqf 'ernlln) ur 6urlere;r;o,rd

slle) uelleu.rureyg 1 1-41 arnOrl

uiri 0 I

('rarusor) olr)g) Jo IseUno)) 'aIp lua)salonUpar e qllM peurets are slla) aql lv

'pail6) serpoqrlue npi€-llue lualselonuqllM 6ulleqel lo, patedald pue paxu seM anssll eql q)!qM laue 'nprg

ol pesodxe seM qsu aql'ln6 ilsuelqaz aqr Jo slle) le!laqtlda peleqel-npl8 JoqdeJ6oJ)lru a)ue)seronUounLuur uV'slla) eseqd-S 6u;1aqe1 ZI-LL etn69

'S 'IC Jo sqlSuel aql eulruJelep o] dJleruoltc rlog asn osp uec eM'(8I-ZI aln

-t;g) s1aclo srequnu e8rplJo srs,(1euu Jlleruotnu pue pldeJ eql smolle lqlrqr.,'taj-auoilb mo[ B pup se^p Surpurq-ygq ]uarsarongJo esn el{lfq palelllrcugdpearBsr qceordde sql 'eseqd

S Suunp selqnop qJIq ^ 'lueluoJ OtO sll SuFnseaur

dq sr alcdc ilec eq] "' peqceer s'q rrer " *uijj"?;:uj,:lTff;1j"#i"'ul,l1X""o

pue 'IC olur aseqd ry q8norqr 'aseqd htr olul z9 q8norqr ssarSord o1 lac aseqd

-S uE JoJ selel ]r 3uo1 moq eurruJalep uBJ e a'elullJo sqfuel peJnseeu ro3 alcri.ceqt punor€ enurluoo ol slleJ aql 8u!vro[e puu npJg go aqnd e 3un€ Lq 'uo1]

-lppe uI 'aseqd htr Jo uorlernp eq] aleu4lse uet at't''(xapu! Jlioryu eql) slsotlru ul

sllao Jo uoprodord eql uorJ ,(yrupurrg 'uoll€rnp alrdc-1ac eloqm aqt Jo uollcerJu se aseqd S Jo uorlernp aq] ateurltsa uEf, ar'l''(xapu! Suqaqay eql) pelaqel aJE

teqt uopepdod e qcns ur slleJ Jo uoqrodord aq] ruorc 'npJg Jo aslnd;auq e ,tq

paleqel eruooeq pue luelsul due le aseqd g ul eq ill \ o/o1V-OE lnoqe 'dlsnouorqc

-u,,(se 1nq fprder Supera;rtord ile arp leq] s[a];o uorlulndod e ur ,{1pctd,,(;'Qt-Lt a.rnt;g)sar

-poqrlue npJg-rlue r{tyn Sururels r{.q pazqunsll ueql ale llpJg palerodrocut anuq

teqt Ielrnu IIaO '(nprg) aurprrndxoap-oruorq Sopue aupnudqt [elsgluu eq] sE

qJns 'VNC pazrsaqtur(s fpu.au olut pelerodrocul eJe ]eql sepcelotu alqEZIlBnsIqtrâ rueq] Surdlddns,{.q adorsorcl{u eqt uI palJlluepl eq uec slac aseqd-5 dpep-urrg '(alpurds Jrlolnu aq] Sulearral) sepqruoJJlru aql su qJns sluauodruoc repl-1ac cgrcads azruSocar ]eq] salpoquue qllm Jo (slsollru uI seluosoruoJqc Jo uou-Esuepuoc eql lealar qcqnlr) sari.p luarsarong Surpurq-VNq tlllm s11ac SutumlsLq uoqrsod alcdc-1ac lnoqu senlo Ipuop1ppp utu8 uec aM'slseuDloltc 3o ssacordaqt ur pelJesqo aq upc sJeqlo '(II-ZI arn8rg ees) slsolltu uI eJE pue dn papunoraêq slleJ eql Jo uollJu4 E leql slealer eJnllnJ ut Suqura;qord sgac uEIIBtutuEru

;o uorlelndod e 1e acue13 y 'adocsorcrru p qlp s11ac 3urn1 ]e {ool o1 ,tldruts st,i.urrn au6 ;a1crb 1ac aqt ur peqJEeJ suq IIaJ IeruIuP ue a8els leq ^ ile] ao\ uec ,rroH

s{ery1snolren u! palpnls eg ue) uolssal6old el)I)-lle)

'(97 raldeq3 uI pessnJslp) JaJueo uI sloJluoJ asel{l Jo ssolaq1 SurpuelsJepun JoJ osle lnq sensslt uI sJeqlunu IIaJ Jo sloJluoo IEIuJou eqlSurpuelsrapun JoJ Lluo 1ou lueuodrut aJe salpnls qJnS 'suspe8ro replacrlpuur uoqereJrTord 1ac Jo loJluoc aq] Suurano8 srustueqcalu Julncalou aql Surur-ruexe JoJ InJesn ̂,(lercadsa uaeq alpq sllal uelprurueru pemlpJ Jo salpnls

'loJluof, alcri.c-11ac uI peAIoAuI suralord aq] Jo sls-d1eue pcruaqf,orq ol alqeuerue aru ,(aql pue 'sluela a1c[c-1ac Jo suopelJesqopcrSolo$.c pelrelep ^ olle 01 a8rel ,tlluagelJJns are sllaf, eseql 'uolllppu uI 's[ac

snoaua3oruoq ,(lecrlaua8 Jo eoJnos pellru[un ue apnord ,bql asnecaq-rtne-raua8 dSolorq [ac roJ pue-selpnts alcrb-1ac ro3 pasn dlapu ere saull IIaJ qsns'leturou lou are daql q8noqtff 'sauq

IIaJ ,.pazqeuouul,, sP eJnJInc ur ,{laltugapuraleragqord uraql dlaq reqr suollelntu o8rapundleuolsef,Jo slleJ u€IIEIUTUPIN

'ralpl ssncsrp eM se 'aJuaJsauas IIaJ an!ruJlldal pelleo sseloJd e 'suo1s

-1rr1p 0?-gZ ra4e Sutpnrp aseec dlluaueurrad 'aldruuxa JoJ 'slsEIqoJqIJ ueunH'salczb uorsr^rp Jo Jaqrunu palltuI E JaUP 8upplp dols uago daq] 'suo1]1puoc

pJepuets ur paJnllnc aJB sanssll uelPruruBur leruJou ruo4 slles uel{ \'Jeâll/Ioq'uorlerrldurol B sr eJeqJ '(II-ZI a.rn8;g) sJopeJ Jeqlo pue sluelJlnu lelluesse;o acuesard eql ut saqsp eJnllnc cpsuld ur uvrorS pue sJorun] Jo senssu IEruJouuro4 palelosr ueeq aêq lpql sllef, esn eJoJaJaql IoJluoJ alcdr-11ar uelprurueruuo serpnls lsohtr 'lPruruPru lf,Blul ue uI sllal lPnpmlpul a^Jasqo ol r{.sua lou sl }I

slla) uerletxurew pelnllnJ ul palpnls o8 ue) lorluo] ol)^)-lle)

650[llf^) Ilf lHl- lo M3lAUlAo

(strun ̂reilrqre)

lla) rao vNo +o lunoure e^tlelaJ

L'

tuels,{s IoJluoJ eql 'tuaê alJ^J-lla3 qf,Ee Jo uoFaldtuoJ aql JoJ aturl Jo }unourppaxrJ e saprloJd ]eql Jarurt pauueJSoJd ,{lpr8rr p a{ll sr ruals.{s loJ}uoc aql-rarlrea passnJsrp sa1c,b 1ac cruodrqura umop-padduls aql ur uaas se-ruJoJlsaldrurs stl uI '(1I-ZI arn31g) acuanbas las E ur a1c-,b 1ac eql Jo sluela aqt sra8-3rl teql JotellrJso Jo rarurl e a{rl qJnru salerado rualsrb loJluoc alcfc-11ac aq1

al)I) lla)aql ro stua^l tofew ar{} sta66!rl urals{s lolluo) al)^)-lla) aql

'ap,{r 11ar aql

Jo sluaâ aq] eleurpJooJ pup eurl ol raqlaSol 4roan.,{aql moq pue rua}sLs IoJ}uoJaq] Jo slueuodruoc uratord aqt ssnJsrp uaq] aM'salerado rualsds lorluoc alodc-[ec eql qJIqM uodn saldrcurrd crseq ar{} JaprsuoJ }sJrJ ellt 'uol}Jes sq} uI

'uo os pue 'uope8ar8as aruosoru-oJqJ 'uorleJrldar y111q;o sassacord aql uroyrad lBql suralord aq] ruo4 lJurlslpare .{aql }eq} uotezrleal aq} qryrrr Suop 'rua}s,{.s

IoJluof, aql Jo suralord La1 aql youortecrJrtuapr aql qlu!\ s086I elpl aq] ur eruef, q8norqtlearq roferu V'sallasruaqlpalloJluoJ llroqatuos srsauqo{c pup 'srsoutu 'srsaqluf.s

VNCI Jo sassacord aqlJaqlaqm ro 'ure1s,{s

IoJluoJ aleredas e sem aJaql Jaq}aqm JealJ uale }ou spl }I'lleJ eql aprsu xoq )Je1q e,(1durs se^,r,t uals,,(s IoJluof, alc,rtc-1ac eql 'stuala asaqlSuryyorluoc ureunJ aqt purqaq .{e1 leq.u;o papr ou peq lnq srsauqo{c pue 'srs-olrlu 'srseqlu,r(s ygq;o noqs laddnd aql paqcle^ slsrSolorq 1ac srea.{ ,tuelu Jo{

wltSAS toutNo) ltf^f-tt]) IHt_

'IoouoJ a7c[t-71ac ulofuacna lo mam patltun a oi palanoq-atnqnJ ur slp) uvtlalutuDra puo'sofuqua lvlaruD'slsoaf.Sutpnput-stualststoaBuat aptm o ut satpnls puD 'uounlona Suunp panrasuoJ [7r.13tt1 uaaq anaq lor]uor puvuo4aztua8to a1c,(t-17a3'uorssatSotd a\ctc-yat ala\nBat qauBp r\lnnar2r#a puD ralnl-Iarortul snouon ua14m 'z) puD It pa1pc sasaqd doB [q paryndas [11ansn atD asotld ygpua asaqd g'(usaut4o1[c1 oml ut s1t1ds uaqi llal aqi puv'(srcoilut) npnu pnprnrput ojulpala&at&as an sataosotuottlr pa1o41dat atll 'awt1d 779 Suunq 'a1c[c aql moqBnotql [1sno-tluquor palauldnp atu siuauodutoJ IIar raLlp lsola soaraqm 'a1c[.c

77ac aql to asaqd gSuunp sJnJJo uouuJr1dnp awosowoJqJ 'snal nlqBnop oml olur slualuoJ asotp to uorl-nqu1s1p ,tq pamollol 's1ua1uor sJIaJ aql lo uo4act\dnp qum su8aq [1\onsn uosrnlp IpJ

{reuun5

'a1c.rtc aql q8norql sserSord slleJ aql^ oq IeaAaJ sllaf, Jo uorlelndod pazruorqcufs p qJns uo slueuraJnspeur ]ua]uocy111q :ap.,{c IIaJ aq};o aseqd relncrlred auo ur aq o} pa}Jalesard uaaq aêq }eq}sllal palaqel-VNq Jo uorlelndod e arup rano 8ul,r,ro1o;,r(q 'sasuqd

IN + zC pue

'uo!lelndod

srq] u! W 1 z9 ue{l rabuo; sr L91eq1 6urmoqs 'eseqd lA + zg ut upq} Lgur slla)jo sraqunu;aleer6 are araq] ]eql sate)tput polellsnllt asp) aqt ul

slla) Jo uorlnqulsrp oqf 'aseqo s ur are pue vNc Jo ]unoue aletpaulalut ue

aêq ]eql asoql pup'aspqd W ro z9 u! are pup (lualuo) VNO LD eql o)tMl)VNC Jo luauraldulot pale>r;dar I11n; e aêq leq] asoql'lD ut atoJaraql alp

pup VNC;o iuaua;duo: paletr;datun up a^pq ]pq] osoq] :sarrobalet aalqlotur llpj slla) aql lla) q)pa ul VNC Jo lunourp aq] o] ;euolt.rodotd I;l>atrp

sr 3)ua)saronlJJo lunouJe aq] ]pql os'vNc ol sputq lt uaqM lua)salonlJsau,ro)aq leqr a,{p e q}!M pau!e}s araM aloLl pazl;eue slla) aql'(z-g

arnbr3 aes-erua)salonlJ ltaql ol 6urpro>>e slla) tlos ol pasn aq osle up)'SfVl ro 'rauos

lla) pale^tl)e-a)ua)salonU e pallp) osle'lataLuol^) MolJ V) 'Jalauol^) MolJ e ut pautuilalap st slla) lenpt^tput slt Jo tualuo) VNCaql uaqM uorle;ndod ;;at 6urlera;r;o.rd e ro; paurelqo stlnsor ;errdIl smoqsqderb srql'.ra1eruo1fu ltog p qllm tualuot VNO ,o srs(;euy g 1-2 1 aln6r1

JC

35q

o

a1r{3 11a1 aql:/ [ raldeLl) 090 ]

THE CELL-CYCLE CONTROL SYSTEM

ls a l l DNA repl icated?

ls environment favorable?

G2lM CHECKPOINT

Are all chromosomesattached to the spindle?

METAPHASE-TO-ANAPHASETRANSITION

1 061

Figure'17-14The control of the cellcycle. A cell-cycle control system triggersthe essential processes of the cel l cycle-such as DNA repl icat ion, mitosis, andcytokinesis.The control system isrepresented here as a central arm-thecontroller-that rotates clockwise,tr iggering essential processes when i treaches specif ic checkpoints on the outerdial. Information about the completion ofcel l-cycle events, as well as signals fromthe environment, can cause the controlsystem to arrest the cycle at thesecheckpoints. The most ProminentcheckDoints occur at locations markedwith vellow boxes.

START CHECKPOINT

ls environment favorable?

in these cells is independent of the events it controls, so that its timing mecha-nisms continue to operate even if those events fail. In most cells, however, thecontrol system does respond to information received back from the processes itcontrols. Sensors, for example, detect the completion of DNA synthesis, and ifsome malfunction prevents the successful completion of this process, signalsare sent to the control system to delay progression to M phase. Such delays pro-vide time for the machinery to be repaired and also prevent the disaster thatmight result if the cycle progressed prematurely to the next stage-and segre-gated incompletely replicated chromosomes, for example.

The cell-cycle control system is based on a connected series of biochemicalswitches, each of which initiates a specific cell-cycle event. This system ofswitches possesses many important engineering features that increase the accu-racy and reliability of cell-cycle progression. First, the switches are generallybinary (on/off) and launch events in a complete, irreversible fashion. It wouldclearly be disastrous, for example, if events like chromosome condensation ornuclear envelope breakdor.trn were only partially initiated or started but notcompleted. Second, the cell-cycle control system is remarkably robust and reli-able, partly because backup mechanisms and other features allow the system tooperate effectively under a variety of conditions and even if some componentsfail. Finally, the control system is highly adaptable and can be modified to suitspecific cell types or to respond to specific intracellular or extracellular signals.

In most eucaryotic cells, the cell-cycle control system triggers cell-cycle pro-gression at three major regulatory transitions, or checkpoints (see FigureI7-I4).The first checkpoint is Start (or the restriction point) in late Gr, where thecell commits to cell-cycle entry and chromosome duplication, as mentionedearlier. The second is the GzlM checkpoint, where the control system triggersthe early mitotic events that lead to chromosome alignment on the spindle inmetaphase. The third is the metaphase-to-anaphase transition, where the con-trol system stimulates sister-chromatid separation, leading to the completion ofmitosis and cytokinesis. The control system blocks progression through each ofthese checkpoints if it detects problems inside or outside the cell. If the controlsystem senses problems in the completion of DNA replication, for example, itwill hold the cell at the G2i M checkpoint until those problems are solved. Simi-larly, if extracellular conditions are not appropriate for cell proliferation, thecontrol system blocks progression through Start, thereby preventing cell divi-sion until conditions become favorable.

TRIGGER ANAPHASE ANDPROCEED TO CYTOKINESIS

ENTER CELL CYCLE AND PROCEED TO S PHASE


The Cell-Cycle Control System Depends on Cyclically ActivatedCyclin-Dependent Protein Kinases (Cdks)

Central components of the cell-cycle control system are members of a family ofprotein kinases knor,rm as cyclin-dependent kinases (Cdks). The activities ofthese kinases rise and fall as the cell progresses through the cycle, leading tocyclical changes in the phosphorylation of intracellular proteins that initiate orregulate the major events of the cell cycle. An increase in Cdk activity at theG2lM checkpoint, for example, increases the phosphorylation of proteins thatcontrol chromosome condensation, nuclear envelope breakdown, spindleassembly, and other events that occur at the onset of mitosis.

Cyclical changes in Cdk activity are controlled by a complex array ofenzymes and other proteins that regulate these kinases. The most important ofthese Cdk regulators are proteins known as cyclins. Cdks, as their name implies,are dependent on cyclins for their activity: unless they are tightly bound to acyclin, they have no protein kinase activity (Figure f7-f 5). Cyclins were origi-nally named because they undergo a cycle of syrthesis and degradation in eachcell cycle. The levels of the cdk proteins, by contrast, are constant, at least in thesimplest cell cycles. Cyclical changes in cyclin protein levels result in the cyclicassembly and activation of the cyclin-cdk complexes; this activation in turntriggers cell-cycle events.

There are four classes ofcyclins, each defined by the stage ofthe cell cycle atwhich they bind cdks and function. All eucaryotic cells require three of theseclasses (Figure l7-f 6):

l. G1/S-cyclins activate Cdks in late Gr and thereby help trigger progressionthrough Start, resulting in a commitment to cell-cycle entry. Their levelsfall in S phase.

2. S-cyclins bind Cdks soon after progression through Start and help stimu-late chromosome duplication. S-cyclin levels remain elevated until mito-sis, and these cyclins also contribute to the control of some early mitoticevents.

3. M-cyclins activate Cdks that stimulate entry into mitosis at the G2lMcheckpoint. Mechanisms that we discuss later destroy M-cyclins in mid-mitosis.

In most cells, a fourth class of cyclins, the Gl-cyclins, helps govern the activitiesof the Gr/S cyclins, which control progression through Start in late G1.

In yeast cells, a single cdk protein binds all classes of cyclins and triggers dif-ferent cell-cycle events by changing cyclin partners at different stages of thecycle. In vertebrate cells, by contrast, there are four cdks. TWo interact with Gr-cyclins, one with G1/S- and S-cyclins, and onewith M-cyclins. In this chapter, wesimply refer to the different cyclin-Cdk complexes as G1-Cdk, Gr/S-Cdk, S-Cdk,and M-Cdk. Thble l7-l lists the names of the individual Cdks and cyclins.

How do different cyclin-cdk complexes trigger different cell-cycle events?The answer, at least in part, seems to be that the cyclin protein does not simplyactivate its cdk partner but also directs it to specific target proteins. As a result,

' , , G r t Mi_- metaphase-anaphase

i M G 1

APC/C

cycl i n-dependentkinase (Cdk)

Figure 17-15 Two key components ofthe cell-cycle control system. Whencycl in forms a complex with Cdk, theprotein kinase is activated to triggerspecific cell-cycle events. Without cyclin,Cdk is inactive.

Figure 1 7-16 Cyclin-Cdk complexes ofthe cell-cycle control system. Theconcentrations of the three major cyclintypes osci l late during the cel l cycle, whilethe concentrations of Cdks (not shown)do not change and exceed the amountsof cycl ins. In late G1, r ising G1lS-cycl inlevels lead to the formation of G1lS-Cdkcomplexes that tr igger progressionthrough the Start checkpoint. S-Cdkcomplexes form at the start of S phaseand tr igger DNA repl icat ion, as well assome early mitotic events. M-Cdkcomplexes form during G2 but are held inan inactive state by mechanisms wedescribe later. These complexes areactivated at the end of G2 and tr igger theearly events of mitosis. A separateregulatory protein, the APC/C, which wediscuss later, ini t iates the metaphase-to-anaphase transit ion.

G,iS-cycl in

Gr/s-Cdk S-Cdk

THE CELL.CYCLE CONTROL SYSTEM

Table 17-1 The Major Cyclins and Cdks of Vertebrates and Budding Yeast

G1-CdkGrlS-Cdk5-CdkM-Cdk

cyclin D* Cdk4 Cdk6cyclin E Cdkzcyclin A Cdk2, Cdkl**cyclin B Cdkl

Cln3Cln1,2c lbs, 6c lb1 ,2 ,3 ,4

cdkl "*cdklcdklcdkl

" There are three D cycl ins in mammals (cycl ins D1, D2, and D3)**The original name of Cdkl was Cdc2 in both vertebrates and f lssion yeast, and Cdc2B inbudding yeast

each cyclin-Cdk complex phosphorylates a different set of substrate proteins.The same cyclin-Cdk complex can also induce different effects at different timesin the cycle, probably because the accessibility of some Cdk substrates changesduring the cell cycle. Certain proteins that function in mitosis, for example, maybecome available for phosphorylation only in G2.

Studies of the three-dimensional structures of Cdk and cyclin proteins haverevealed that, in the absence of cyclin, the active site in the Cdk protein is partlyobscured by a slab of protein, like a stone blocking the entrance to a cave (Figurel7-L7A). Cyclin binding causes the slab to move away from the active site, result-ing in partial activation of the Cdk enz)ryne (Figure l7-l7B). Full activation of thecyclin-Cdk complex then occurs when a separate kinase, the Cdk-activatingkinase (CAK), phosphorylates an amino acid near the entrance of the Cdk activesite. This causes a small conformational change that further increases the activ-ity of the Cdk, allowing the kinase to phosphorylate its target proteins effectivelyand thereby induce specific cell-cycle events (Figure I7-I7C). <TAGA>

Inhibitory Phosphorylation and Cdk Inhibitory Proteins (CKls) CanSuppress Cdk Activity

The rise and fall of cyclin levels is the primary determinant of Cdk activity dur-ing the cell cycle. Several additional mechanisms, however, fine-tune Cdk activ-ity at specific stages ofthe cycle.

Phosphorylation at a pair of amino acids in the roof of the kinase active siteinhibits the activity of a cyclin-Cdk complex. Phosphorylation of these sites by aprotein kinase knor,vn as Weel inhibits Cdk activity, while dephosphorylation ofthese sites by a phosphatase knor,rm as Cdc25 increases Cdk activity (Figure

17-18). We will see later that this regulatory mechanism is particularly impor-tant in the control of M-Cdk activity at the onset of mitosis.

Binding of Cdk inhibitor proteins (CKIs) also regulates cyclin-Cdk com-plexes. The three-dimensional structure of a cyclin-Cdk-CKl complex reveals

activating phosphate

(c)

Cdk-activating kinase (CAK)cyclin

1 063

Figure 17-17 The structural basis of Cdkactivation. These drawings are based onthree-dimensional structures of humanCdk2, as determined by x-raycrystal lography.The location of the boundATP is indicated.The enzyme is shown inthree states. (A) In the inactive state, withoutcyclin bound, the active site is blocked by aregion of the protein called the T-loop (red).(B) The binding of cycl in causes the T-loop tomove out of the active site, resulting inpartial activation of the Cdk2. (C)Phosphorylation of Cdk2 (by CAK) at athreonine residue in the T-loop furtheractivates the enzyme by changing the shapeof the T-loop, improving the abi l i ty of theenzyme to bind its protein substrates.

active site

(A) TNACTTVE (B) PARTLY ACTIVE


that CKI binding stimulates a large rearrangement in the structure of the Cdkactive site, rendering it inactive (Figure f 7-fg). Cells use CKIs primarily to helpgovern the activities of Gr/S- and S-Cdks early in the cell cycle.

The Cell-Cycle Control System Depends on Cyclical Proteolysis

\fhereas activation of specific cyclin-Cdk complexes drives progression throughthe Start and G2lM checkpoints (see Figure 17-16), progression through themetaphase-to-anaphase transition is triggered not by protein phosphorylationbut by protein destruction, leading to the final stages of cell division.

The key regulator of the metaphase-to-anaphase transition is the anaphase-promoting complex, or cyclosome (APC/C), a member of the ubiquitin ligasefamily of enzymes. As discussed in Chapter 3, many of these enzyrnes are usedin numerous cell processes to stimulate the proteolytic destruction of specificregulatory proteins. They transfer multiple copies of the small protein ubiquitinto specific target proteins, resulting in their proteolytic destruction by the pro-teasomes. Other ubiquitin ligases mark proteins for purposes other thandestruction.

The APC/C catalyzes the ubiquitylation and destruction of two major pro-teins. The first is securln, which normally protects the protein linkages that holdsister chromatid pairs together in early mitosis. Destruction of securin at themetaphase-to-anaphase transition activates a protease that separates the sistersand unleashes anaphase. The S- and M-cyclins are the second major targets ofthe APCi c. Destroying these cyclins inactivates most cdks in the cell (see Figure17-16). As a result, the many proteins phosphorylated by Cdks from S phase toearly mitosis are dephosphorylated by various phosphatases that are present inthe anaphase cell. This dephosphorylation of Cdk targets is required for thecompletion of M phase, including the final steps in mitosis and the process ofcytokinesis. Following its activation in mid-mitosis, the APC/c remains active inG1, thereby providing a stable period of Cdk inactivity. \Mhen G1/S-Cdks are acti-vated in late Gr, the APC/G is turned off, thereby allowing cyclin accumulationto start the next cell cycle.

The cell-cycle control system also uses another ubiquitin ligase called SCF(after the names of its three subunits). It ubiquitylates certain cKI proteins inlate G1 and thereby helps control the activation of S-cdks and DNA replication.

The APC/C and SCF are both large, multisubunit complexes with somerelated components, but they are regulated differently. APC/C activity changesduring the cell cycle, primarily as a result of changes in its association with anactivating subunit-either cdc20 during anaphase or cdhl from late mitosisthrough early G1. These subunits help the APC/G recognize its target proteins(Figure l7-2oL). SCF activity also depends on subunits called F-box proteins,which help the complex recognize its target proteins. unlike Apc/c activity,however, scF activity is constant during the cell cycle. ubiquitylation by scF iscontrolled instead by changes in the phosphorylation state of its target proteins,as F-box subunits recognize only specifically phosphorylated proteins (FigureI7-20H.

actrvecycl in-Cdkcomplex

inactivep27-cyclin-Cdk

comprex

Cdk activatingphosphate

INACTIVE

Figure 17-18 The regulat ion of Cdkactivity by inhibitory phosphorylation.The active cycl in-Cdk complex is turnedoff when the kinase Weel phosphorylatestwo closely spaced sites above the activesite. Removal of these phosphates by thephosphatase Cdc25 activates thecycl in-Cdk complex. For simplici ty, onlyone inhibitory phosphate is shown. CAKadds the activating phosphate, as shownin F igure 17-17.

Figure 17-19The inhibit ion of acyclin-Cdk complex by a CKl. Thisdrawing is based on the three-dimensional structure of the humancycl in A-Cdk2 complex bound to theCKI p27, as determined by x-raycrystallography.The p27 binds to boththe cycl in and Cdk in the complex,distorting the active site of the Cdk. ltalso inserts into the ATP-binding site,further inhibit ing the enzyme activi ty.

']uaê elcô-lleJ JUrJads E sJaSSul qclq^\Jo qcpe (seqUrMs Ipcr

-ureqJorq Jo selJas P elPArlJE o1 dlsnoluouolnE ̂llp4uessa saleJedo qcrq ^ ({JoM-lau lsnqoJ e ruroJ ol JeqlaSol pe{ul ,r(leuorlcurg are suralord asaq; 'rualsrts

IoJluoJ alcdc-gac eqlJo slueuoduroc roferu eqlJo aruos sezrJer.uruns Z-Llelqe;

seq)lrMs le)!ueq)otg,o lro/vuaN e sp suolpunl u,lels/(s lolluoJ al){)-lle) eql

'rrl\/\ou)lun aJE sJeqlo ,tueur;o suorlJury aql lnq 'suon-rury a1cl.c-1leJ rrlr,roDl qlyv\ sureloJd apocua saua8 asaql;o eruos 'a1r",b

1ac aqlSurrnp elellrJso slelel esoqm sVNUur epoJua saua8 aql Jo %0I lnoqe ,aldruexa

ro; ']sear( Suppnq u1 'Sursrrdrns aJp serpnls esar{l Jo s}FseJ aql 'a1cdc ilac eql

q8norqt sassarSord IIaJ aql se auoua8 aqt u1 saua8 eql Jo IIe go uorssardxa aq]ur sa8ueqc azf1eue ot (B Ja]deql ut pessncsrp) sderreorcrur VNC esn ueJ aAA

'slleJ lsoru ut sleêl uqcdc yorluoc dlaq 'aldruexa ro; 'uorldrrcsuerl

aua8 uilcrb ur sa8ueq3 'uorlep8ar;o Ialal leuorlrppp ue sapnord IoJluo3 Ipuou

-drrcsuerl :alamoq 'sadft 1ac tsoru Jo salcrb 1ac xaldruoc aJotu eql u1 'suratord

la8rer 4aql puu sase8q uppblqn pue qp3 3o uopeln8ar eqt eAIoAul teql sursru-EqJeru puorldrrcsuerl-lsod uo f1arusn1cxa spuadap loJluoc alcdc-1a3 rnccolou saop uopdlrcsuerl auaB terlJea pessnosrp a1c[c lac cruortrqura 3o4 aql uI

uorteln6au leuotldulsuell uo spuadac oslv lotluo) al)/()-lle)

1v{osvtJ.oudtrtt tX: lO F

NOt-LVCVU9tC

seuAzuauorle;r$rnbrqn

m (r))) ( nbrqn utalold rolrqrqur 1P1

+ *CJutlrnbrqn{;od

'ltunqns xoq-; rr1>adse Iq pazruboral aq o1 1a6le1 aql smolleiumoqs

ly) aqt se q)ns ,ura1o.rd 1a6le1e ;o uor1e1{roqdsoqd aql.sad,{1 luaragrp

{ueu ate ataql q)tr.lM Jo ,sutalold

xoq-l palle) sltunqns 6urpurq-elellsqnsuo spuadep 115 ase611 urlrnbrqn

aqr 1o,(r;n;lre aql (g) 'eulosealoid

e ur paper6ap pue pazrubotatuaql s! tabrel perelr(lrnbrqn{;od

aq1'uralotd 1a6le1 aq1 oluo saln)aloururlrnbrqn aldlrlnu sle;suerl f/)dv

aql'zl pue I j palle) suralotd leuorltppeomt Jo dlaq oqr qll6'sulalord 1ebre1

raqlo pue ur;r{t-y1 uo sa:uanbas prreourue lrpads sezru6olal q)tqrvr,gz)p)lrunqns 6utlenrlre aql qllM uotlet)osseIq srsoilu ut pale^r])e sl l/)dv aqJ_ (V)

'ep{r ;1er aql 6urrnp J)S pue )/)dV r(q

sgs(;oalord 1o lorluo) aq1 g7-4 1 arn611

s90t

315 Aq srs{;oalord 1o lot1uo: 191

seu{zuauorle;Ipnbrqn

.t 1f; urlrnbrqn

t)/)dv a^rpe

1\l/1tu,)^r-^

\ )l)d lleul

)-'{/t

(02)p)) lrunqns6urlenrpe

)/)dV ^q srs{;oalord +o loJ}uot (V)

wlr_s^S tourNo) rt)^)-ttl) lHl_

alouexalo]'sall^) lla) )tuoi(lqula Iuea ul

6urssrr.u ere ,{ueu :sad{t ;;ar ;;e ur luasaldlou are susrueq)alu asaqf'fdo, sluaâ

apAr-;;a> alaldurorur pue'a6euep

lla)'luauruorAUa Jplnlla)ellxa aqt tnoqpuorteuroJur aprnord q)rqM'sursrupq)aLu

^rollqlqul snorren Iq pa)uanllulos;e sr xa;duor qtea;o ,{1rnr1:e

aql latPl lrelap alour ur passn)srp sv'(/Aoila() saxa;duor 1p1-urp,(> 1o seuas

e Jo slsrsuo) uals{s ;oiluot a;r,{r-;;ataql jo aro) aql 'ua1s{s

;o,rluor a;r{>-;;a>aqt Jo rvra!^ralo uy 17-11 arnbrg

lP)-S - lp)-slrD )p)-t9

rg

lp)-l l-

TT

\\a6euep VNC

VNC pale:rlda:un

,{}!nllreslql ro; palrnber {1;ensn ura}ord 1a6let;o uor}el^ror.ldsoqd i(sleurueu u LZd'$ee( Eulppnq

u! t)lS) sl)) oLuos 6urpn1>ur 'lojluor Lg ur pa^loûr suralord {ro1e;nbar Jo uoltep0lnblqn saz^lele)rernrlre lp) ^q pallqlqu!

:Lg lnoLl6norql pue eseqdeue rayp ̂lr^rl)e J/)dV surelureu leqt l!unqns 6utle^tl)e-)/)dv{}tnr}re lp)-ti! ,(q palelnurl}s luol}lsueJ}

aseqdeue-ol-aseqdplau te )/lldv jo uolle^ll)e lelllu! sra66u] lsllal lle ut ltunqns 6ulle^lpe-f/)dvs]runqns 6ullenllre qllM uollellosse {q pa1e1n6et lsurll^l-y\1 pue -S pue ulln)as

6ulpn;rur 'srsolrLu urorj lrxa ur {;ueurud panlonur sura}ord ,(roteln6ar;o uorlel,tlnblqn sazllele)

rafue) ur pele^rl)eur r{;}uenbet;:t9 ul ̂}l^l})e )pf-tD sassalddns

obeuep y56 6urano;;o; serlrnrl)e )pf-S pue )pf-S/tD sassarddns

315 {q uorle;ftrnblqn s}l sre66rr} Z)pf Iq uoltellloqdsot{d jo}etluarejJtp I;;eululal(aqt uaqnn ep{r glar uorJ MerpqllM s1;ar sd;aq :Lg ur sar}r^r})e lp)-S pue Ip)-S/Lg sassarddns

uorl)nrlsap stt sta66rrt LD,o pua aqi te [)p]) ,{q uorlel,(roqdsoqd :19 ut ,{ltnl}le 11p1 sesselddns

stsolttrr Jo tesuo otlt lP uotle^tl)e tlpf 6ur;;orluol ul pa^loûl I;tteuttdisleLuueur ul () 'g 'VSZrp)) srequaLU {qruie; ealql 1qp) LUorJ sateqdsoqd ,{toltqtqu; saôuar

slsol!Lllero;aq ,{lrnr1re 11p) 6ursserddns ur po^loûr I;ueuud lslpf ur satrs fuollqlqur salel{loqdsotld

$lp) ul alts 6urlentl:e ue sa1e1&oqdsoqd

luauuoJrAuarelnlla)erlxa

elq eroê]

(sleLUueu) 9[d(sleuueul) tZd

(sleuLueul) 1Zd(rsea^ 6urPPnq) [)lS

(sDl)} suFlord roUqlqul Ip)

aseleqdsoqd g7rpl

aseur) leaM()V)) aseul) 6u tlentlre-1P1

s1p1 {1potu leq} sase}eqdsoqd utelord Pue saseu!l ula}old

'aseqd S qllr,r Surlrels 'llelep arotu uI se8els a1c,{c-1ac

esaql ssnJsrp ol ,{peal ,{i\ou are a A'Ja}EI ssnJSIp a^ se 'polrad Ic elqBls E uI 8uI-llnsar 'srsol[u rage .,!r,,r.r1ce 1p3 ssarddns o] aleJoqelloJ slusluBqcaru alduptu'a1a1duoc sr srsolnu uaqr\\ ' (

I Z-Z 1 arnS;g) slsolltu Jo uopalduroc aql pue uoue8-aJBas prlpruoJqJ-Jalsrs Sutqsealun,{qaraqt 'uolJlsuerl aseqdeue-01-aseqdelauaql le surlJ^c puE urJnf,as Jo uollcnJlsep aql sJeSSul '\zJpJ JolPAIlsBstr qtr^ raqlaSol '3734y aqr -{1eurg 'alpurds rl}o}Iu aqt;o rolenba aq} }e splleru-oJqc Jalsrs ;o luaruuSrle aql o] Surpeal 'slsolllu .{Fua ;o sluala aql pue lutod-{3eqJ htr/zC aql q8norqt uorssarSord sraSStr} uaq} uol}PAIlcE {pl-N

'slso}Ilu Jo

sluaâ .{Fea aruos o} salnqlJ}uoo osle pue aseqd 5 uI uol}eJlldnp eluosoluoJq3salerlrul qrlq \ ',{tnr}re

)pl-S Jo aAP^ B qseapn qpl-S/i9 'Ja}el ssnJSIp allr

srusrueqJeur.,(g 'lurodlcaqJ uels eqt q8norqt uolsserSord seAIJp uaqt {pl-S/15)

Jo uorlelrlce Suqpsar aq; 'sur1c,r{c-S pue -S/I9 Sutpocua sauaS;o uolsseJdxeaql selBlntups uJnl ur qJrq ^ 'Ipl-I3

Jo uoIleAIlJB aql alBlntulls spuSts pu-Jalur pue Ieurelxa snoIJeA 'lqBIJ ere uouera;t1ord IIeJ JoJ suoplpuoo ueq \

lfs

tr-lp)

0z)p)

)DdVsrolP^!l)e rraql pue sese611 utl1nb1q1

suralord ltuolelnEeg aP{3-11a3 ro[e61 aq1 1o {leuutn5 Z-l I alqel

al)^) lla) aql:/ [ ra]deq) 990 t

5 PHASE

Summary

The cell-cycle control system triggers the euents of the cell cycle and ensures that theseeuents are properly timed and occur in the correct order The control system respondsto uarious intracellular and extracellular signals and arrests the cycle when the celleither fails to complete an essential cell-cycle process or encounters unfauorable enui-ronmental or intracellular conditions.

Central components of the cell-cycle control system are cyclin-dependent proteinkinases (Cdks), which depend on cyclin subunits for their actiuity. Oscillations in theactiuities of uarious cyclin-Cdk complexes control uarious cell-cycle euents. Thus, acti-uation of S-phase cyclin-Cdk complexes (S-Cdk) initiates S phase, while actiuation ofM-phase cyclin-Cdk complexes (M-Cdk) triggers mitosis. The mechanisms that con-trol the actiuities of cyclin-Cdk complexes include phosphorylation of the Cdk sub-unit, binding of Cdk inhibitor proteins (CIQs), proteolysis of cyclins, and changes in thetranscription of genes encoding Cdk regulators. The cell-cycle control system alsodepends crucially on two additional enzyme complexes, the APC|C and SCF ubiquitinligases, which catalyze the ubiquitylation and consequent destruction of specific regu-latory proteins that control critical euents in the cVcle.

S PHASEThe linear chromosomes of eucaryotic cells are vast and dyramic assemblies ofDNA and protein, and their duplication is a complex process that takes up amajor fraction of the cell cycle. Not only must the long DNA molecule of eachchromosome be duplicated accurately-a remarkable feat in itself-but the pro-tein packaging surrounding each region of that DNA must also be reproduced,ensuring that the daughter cells inherit all features of chromosome structure.

The central event of chromosome duplication is replication of the DNA. Acell must solve two problems when initiating and completing DNA replication.First, replication must occur with extreme accuracy to minimize the risk ofmutations in the next cell generation. Second, every nucleotide in the genomemust be copied once, and only once, to prevent the damaging effects of geneamplification. In Chapter 5, we discuss the sophisticated protein machinery thatperforms DNA replication with astonishing speed and accuracy. In this section,we consider the elegant mechanisms by which the cell-cycle control system ini-tiates the replication process and, at the same time, prevents it from happeningmore than once per cycle.

S-Cdk Init iates DNA Replication Once Per Cycle

DNA replication begins at origins of replication, which are scattered at numer-ous locations in every chromosome. During S phase, the initiation of DNA repli-cation occurs at these origins when specialized protein machines (sometimes

called initiator proteins) unwind the double helix at the origin and load DNAreplication enzymes onto the two single-stranded templates. This leads to theelongationphase of replication, when the replication machinery moves outwardfrom the origin at tuvo replicationforks (discussed in Chapter 5).

To ensure that chromosome duplication occurs only once per cell cycle,the initiation phase of DNA replication is divided into two distinct steps thatoccur at different times in the cell cycle. The first step occurs in late mitosisand early Gr, when a large complex of initiator proteins, called the prereplica-tive complex, or pre-RC, assembles at origins of replication. This step is some-times called licensing of replication origins because initiation of DNA synthe-sis is permitted only at origins containing a pre-RC. The second step occurs atthe onset of S phase, when components of the pre-RC nucleate the formationof a larger protein complex called the preinitiation complex. This complexthen unwinds the DNA helix and loads DNA polymerases and other replicationenzymes onto the DNA strands, thereby initiating DNA synthesis, as describedin Chapter 5. Once the replication origin has been activated in this way, the

1067


prerepl icat ive complexes at repl icat ion or ig ins

repl icat ionelongat ion forks

M-Cdk activation |

.hrotoro." segregation

,.4^

I

G l

pre-Rc is dismantled and cannot be reassembled at that origin until the follow-ing G1. As a result, origins can be activated only once per cell cycle.

The cell-cycle control system governs both assembly of the pre-RC andassembly of the pre-initiation complex (Figure lZ-22). Assembly of the pre-RCis inhibited by Cdk activiry and, in most cells, is stimulated by the ApC/C. pre-RC assembly therefore occurs only in late mitosis and early G1, when cdk activ-ity is low and APC/C activity is high. At the onset of S phase, activation of S-Cdkthen triggers the formation of a preinitiation complex, which initiates DNA syn-thesis. In addition, the pre-RC is partly dismantled. Because S-Cdk and M-Cdkactivities remain high (and APC/C activity remains low) until late mitosis, newpre-RCs cannot be assembled at fired origins until the cell cycle is complete.

Figure 17-23 illustrates some of the proteins involved in the initiation ofDNA replication. A key player is a large, multiprotein complex called the originrecognition complex (oRc), which binds to replication origins throughout thecell cycle. In late mitosis and early Gr, the proteins cdc6 and cdtl bind to theORC at origins and help load a group of six related proteins called the Mcm pro-teins. The resulting large complex is the pre-RC, and the origin is now licensedfor replication.

The six Mcm proteins of the pre-RC form a ring around the DNA that isthought to serve as the major DNA helicase that unwinds the origin DNA whenDNA synthesis begins and as the replication forks move out from the origin.Thus, the central purpose of the pre-RC is to load the helicase that will play acentral part in the subsequent DNA replication process.

Once the pre-RC has assembled in Gr, the replication origin is ready to fire.The activation of s-cdk in late G1 triggers the assembly of several additional pro-tein complexes at the origin, leading to the formation of a giant preinitiationcomplex that unwinds the helix and begins DNA synthesis.

At the same time as it initiates DNA replication, S-Cdk triggers the disas-sembly of some pre-RC components at the origin. cdks phosphorylate both theoRC and cdc6, resulting in their inhibition by various mechanisms. Further-more, inactivation of the APC/G in late Gr also helps turn off pre-RC assembly.In late mitosis and early G1, the APC/C triggers the destruction of a protein,

Figure 17-22 Control of chromosomeduplication. Preparations for DNAreplication begin in Gr with the assemblyof prereplicative complexes (pre-RCs) atrepl icat ion origins. S-Cdk activation leadsto the formation of multiproteinpreinit iat ion complexes that unwind theDNA at origins and begin the process ofDNA replication. Two replication forksmove out from each origin unti l theentire chromosome is dupl icated.Duplicated chromosomes are thensegregated in M phase. The activation ofrepl icat ion origins in S phase also causesdisassembly of the prerepl icat ivecomplex, which does not reform at theorigin unti l the fol lowing G1-therebyensuring that each origin is act ivatedonly once in each cel l cycle.

S PHASE

u 1

COMPLETION OFDNA REPLICATION

prerepl icat ive complex (pre-RC)

ORC (or ig in recogni t ion complex)

t61M

geminin, that binds and inhibits the pre-RC component Cdtl. Thus, when theAPC/C is turned off in late G1, geminin accumulates and inhibits Cdtl.In thesevarious ways, S- and M-Cdk activities, combined with lowAPC/C activity, blockpre-RC formation during S phase and thereafter. How then, is the cell-cycle con-trol system reset to allow replication to occur in the next cell cycle? The answeris simple. At the end of mitosis, APC/C activation leads to the inactivation ofCdks and the destruction of geminin. Pre-RC components are dephosphory-lated and Cdtl is activated, allowing pre-RC assembly to prepare the cell for thenext S phase.

Chromosome Duplication Requires Duplication of ChromatinStructure

The DNA of the chromosomes is extensively packaged in a variety of protein

components, including histones and various regulatory proteins involved inthe control ofgene expression (discussed in Chapter 4). Thus, duplication ofa

1Mg

Figure 17 -23 Control of the initiation ofDNA replication. The ORC remainsassociated with a replication originthroughout the cel l cycle. In early G1,Cdc6 and Cdtl associate with the ORC.The resulting protein complex thenassembles Mcm ring complexes on theadjacent DNA, resulting in the formationof the prereplicative complex (pre-RC).S-Cdk (with assistance from anotherprotein kinase, not shown) thenstimulates the assembly of severaladdit ional proteins at the origin to formthe preinit iat ion complex. DNApolymerase and other replicationproteins are recruited to the origin, theMcm protein rings are activated as DNAhelicases, and DNA unwinding al lowsDNA repl icat ion to begin. S-Cdk alsoblocks rereplication by triggering thedestruction of Cdc6 and the inactivationofthe ORC. Cdtl is inactivated by theprotein geminin. Geminin is an APC/Ctarget and its levels therefore increase in5 and M phases, when APC/C is inactive.Thus, the components ofthe pre-RC(Cdc6, Cdt1, Mcm) cannot form a newpre-RC at the origins unti l M-Cdk isinactivated and the APC/C is activated atthe end of mitosis (see text).


chromosome is not simply a matter of duplicating the DNA at its core but alsorequires the duplication of these chromatin proteins and their proper assemblyon the DNA.

The production of chromatin proteins increases during S phase to providethe raw materials needed to package the newly synthesized DNA. Most impor-tantly, S-Cdks stimulate a large increase in the synthesis of the four histone sub-units that form the histone octamers at the core of each nucleosome. These sub-units are assembled into nucleosomes on the DNA by nucleosome assembly fac-tors, which typically associate with the replication fork and distribute nucleo-somes on both strands of the DNA as they emerge from the DNA synthesismachinery.

Chromatin packaging helps to control gene expression. In some parts of thechromosome, the chromatin is highly condensed and is called heterochromatin,whereas in other regions it has a more open structure and is called euchromatin.These differences in chromatin structure depend on a variety of mechanisms,including modification of histone tails and the presence of non-histone proteins(discussed in Chapter 4). Because these differences are important in gene regu-lation, it is crucial that chromatin structure, like the DNA within, is reproducedaccurately during S phase. How chromatin structure is duplicated is not wellunderstood, however. During DNA synthesis, histone-modifying enz).rynes andvarious non-histone proteins are probably deposited onto the two new DNAstrands as they emerge from the replication fork, and these proteins are thoughtto help reproduce the local chromatin structure of the parent chromosome.

Cohesins Help Hold 5ister Chromatids Together

At the end of S phase, each replicated chromosome consists of a pair of identi-cal sister chromatids glued together along their length. This sister-chromatidcohesion sets the stage for a successful mitosis because it greatly facilitates theattachment of the two sister chromatids in a pair to opposite poles of the mitoticspindle. Imagine how difficult it would be to achieve this bipolar attachment ifsister chromatids were allowed to drift apart after S phase. Indeed, defects in sis-ter-chromatid cohesion-in yeast mutants, for example-lead inevitably tomajor errors in chromosome segregation.

Sister-chromatid cohesion depends on a large protein complex calledcohesin, which is deposited at many locations along the length of each sisterchromatid as the DNA is replicated in s phase. TWo of the subunits of cohesin aremembers of a large family of proteins called sMC proteins (for Structural Main-tenance of chromosomes). cohesin forms giant ring-like structures, and it hasbeen proposed that these might form rings that surround the two sister chro-matids (Figure 17-24).

5mc molecule

y'--'-} .r

h ingeC N

ATPasedomain

Figure 17-24 Cohesin, Cohesin is aprotein complex with four subunits. Twosubunits, Smcl and Smc3, are coi led-coi lproteins with an ATPase domain at oneend; together, they form a largeV-shaped structure as shown. Twoaddit ional subunits, Sccl and Scc3,connect the ATPase head domainsforming a r ing structure that may encirclethe sister chromatids as shown.

20 " t

(A)

(c)

Jo slueueSuBJJeeJ pue edolaûe rEelf,nu aql Jo u^\op{Balq aq} sa}oruoJd oslE

{pl-W 'slla3

IEruruE uI 'saJn}3nJls e{ll-poJ '}JEdrrros olul spllPruoJqs JelsrspaurMualur eql Jo uorlBZruBSJoeJ elef,s-e8JEl a\l 'luo!rysuapuoJ awosoutoJqJsre88u] osp tI 'alpuds aqt Jo alod elrsoddo eq] ol peqopnp sr rrcd p ur prleru-oJqr Jelsrs qcpe luql ernsue pue alpurds Jllollur aqt go ,tlqurasse aq] eJnpur]snur Ipf,-W

'urnrurunu e lV 'srsolnu go sa8els [1rea aql ur rnoco ]eql slueru-a8ue.rrear 1ac xalduroc pue asJelrp aql Jo IIE lnoqe s8urrq '{pO-I

tr 'eseur{ ura}

-ord ay8urs p leq] sl IoJluoJ alcdc-gac Jo saJnleal alqe{Jeual tsotu eql Jo euo

srsolrW olutr(rluf seÛO Ip)-W

'uaq] etErtseqrro c/ldv aql pue {pl-W,lt.or{ pue srsolrru Jo sluale IuJrueqoaru ,{a1 aql equosap a,rr 'uorlJes sq} uI

'srsauqog,b ̂,(q 1ac aq]Jo uorsrllp aq] pup 'alpurds cpotlx aqlyo l.1q-Iuessuslp aql 'aseqdeue

3o uorlalduroo eql Surpnlcur 'aseqd t{ elBI Jo sluela IIB

ro; parmbar sr qf,rr{,t,r 's1a8re1 1p3 go uoqeldroqdsoqdap eql pue uorlelrlcpur {pJ

ot speal qJIq ^ 'sur1c,b Jo uorlJnrtsap aq1 sra33rrl oslp 3/ldv eqJ 'spueluoJqc

Jalsrs eqlJo uoperedas sal€qrur ̂qaJeql pue urseqoJ seleelc ]eql asealord e 8ur-leJeqII 'uunJas

Jo uorlJnJlsap aql sraSSrr] l/JdV aql uaqm 'uorlrsue4 asuqdeue-o1-aseqdelau eql lB su€aq srsolru;o ged roleu puocas eq; 'srrcd pl]EruoJr{Jralsrs eq] ot tueruqoptlp slr pup alpurds ollot1ru aqr go .{lqurasse eqt ot Surpeal'suralord;o rilarJel E alef i-roqdsoqd saseuq uralord crlollu reqlo [BJeAas pue {pO-y41 'porrad s1q] Surrng '(asuqdelaur puu 'aseqdeleruord 'eseqdord) srsolrur dlrea

Jo sluane aq] sraSSrrl lurod4caqc W/zC aqr le.,br.qce {pl-hl ur eseeJJur ldnrqeue 'lsJIC 'ruals.{s

IoJluor a1c[c-1ac aq] Jo s]ueuodtuoc ]curlsrp dq paurano8 qcea'suud roferu o.tt] o]ur pep4lp eq UBJ srsolnu 'r,rar,r.go 1urod,,{rolep8ar e ruorg

'aseqd W Jo stuale rofeur aq] sezrJeru(uns I-ZI larred

'snelf,nu

Ief,rluapr ue qtlm qoea 'sellpq oml olur IIeo aql saprlrp-srseuqo{c-aseqd 61Jo luala rofeu puocas aq] 'pa]aldruoc sr srsolrru sV <WJJ> <JIVJ> 'adocsorcrur

e uI uees sE Jorleqaq aruosoruoJql Jo srseq eql uo ,{preurrd peugep---?su2t4do7a1pue'asnqdnua 'asnqdnlatu 'asoqdnlauotd'asnqdotd-saBels ang olur paprnrp ,(1e-uonrpeJ] sr srsolrhl 'aruoua8 aql;o .{doc umo s}r q}lm qJEe 'ralJnu ralqBnup pcll-uepr Jo rrcd u ot {pary&a8as) pa}nqrJtsrp pue paleredes erp sprtpruoJr.IJ Jelsrs eqlqclqm Suunp 'srsolrur qlyrt sur8aq srq; 'aseqd yq;o pneaqdn cBprueJp aql sao8-Jepun IIaf, aq] '.zg q8noJql uonrsupJl pue aseqd g;o uorlaldurof, aql 3ura,ro11og

srsolrw'a1c[c

77ac ryau aqi Urun pasnaraq rcuuoJ puo asnr"ld S qJaa u! aJuo [1uo pua acuo patyf st utBuo qcaa 'snq1 'uru&u

uotpct\datvNcl aruuru! oJ u!8tro ruqj monD o1 patlnbat arq jotlj sutajotd vlqlqu!osID 4pJ-S

'asoqd g Suunp pawnlpo sr utBrto uo4an1dat a a?uo 'sur&tto uotl.ac-tPaJ panoc VNe aqt ut salls iD uouDJrldat st! alDlltut puo VNe aLIt purmun ruLH sulaj-otd saJnnuca ryrum'4p)-Sto uotlantlca aql tq pata33u1 st uotlau\dnp awosotuoJ.qJ'uotl.cunt atuosotaotqc lo stcadso snounn utanoB pun y111q aW rylm ajonosst) !.oq| sunj-otd uryawottlc aql lo uoryattldnp a4j sD lpm sD'alaosoraoJqJ qcoa w alnralow VNeat\ua aq1 to Surtdoc aruJnJOo aql sanlollur asnt4d g ur sawosoluolt\c aql lo uollvcl\dnq

{reuun5

'Jel€l aqrJJsep alr,r se 'saxalduroc aseql;o uoqdnrsrp uo dpreurud spuadap eJoJalaql uorlrsueJl aseqdeue-o1-aseqduleruaqt ]e uorseqoo Jelsrs Jo ssol aqJ 'saxaldruoc urseqoJ uo dyreruud spuadep uors-eqo3 prlEruoJrlJ-Jelsrs 'paôruer ueeq seq uoueualec eql ecuo '(tz-g aln8r{ ees)VNO ]nc aq] Suqeasar uaql pue '{perq eq} q8norqt reqlo eql Surssed ,ayncaloru

VNC euo 8uq1nc dq srsolrur,r(1rua pue asuqd S ueemlaq sVNCI Jelsrs pelpueluJ eqlsalSueluesrp dlpnper8 II asBJaurosrodol aruufuue aq;.srsaqluds y1qq Suunp laaur$lJoJ uorlecrydar om1 ueq a sJnJco leql selnJalour VNC Jelsrs Jo Suruunlralur aql'uorlaualal y IO ruo4 'lred ur lseel le 'sllnseJ osle uorsaqoo pqeuorqo-Jalsrs

LLOLstsoltt

adolaûa reallnu

+o sluau6Pr+

-1

, ' alodalpulds le

a u osotlu al

]SVHdVl]IAOUd Z

alpuldsaqi ]o salod alrsoddo

ol sprle|.r]orLls .latsrs qf, e]]P

solnqnlorJrLU oioqlolaur)aql saloa alpur0s aq]

uaaMlaq AeMplrr iilnurds

aql ]o rolenba aql ]epau6rlP ale sarrosoLLrorq3

aql ]v

uorloir .l^r])P u auosouloJq)

lUdLIlaAOrL/ JAll)P

obrapun pue saroq)olaur)raq] erA sal nq nlor)t tt.l

alpulds ol q)PllP /ôu UP)

saurosourorqJ Jdolaûareapnu aq] +o irMop)eojq

aqt qlrM AllonrqP

su e]s

uaat 6 a )e salnqnlol)lulpue a6ue)o pJUrPls are

saLUosoLUorq)'qd Pr60r)il-uoloqo aql ul ]uasalo aLl-ros

-oLUOrq) q)Pa +o sardo) oA ]aq plnoM aiaql'slla) proldrp

ul uMoqs ale saLUosoLl.lolqlaarq] ̂luo A|rrldLuts ro3

Uede poôur pue patertldar

aAeq q)rqM'sat]-]osorlua)

o^^l aq] uaaMlaq solqu.lassPalpurds lrlo]rul Jql'snalrnu

aql aprslno asuapuol'sprleuorq) rolsrs paleDosse

,41aso1r onnl +o 6urlsrsuolq)ea'saL!osoLUorq)

pelerrlder eq]' ]V

o ln q nlorc il..Iroro Llcolau r)

a;od a;purds

]e aLUosolluac

]SVHdVT]IA T

q16ua1 rraqt 6uo1e raqla6o] plaq sprlpurorq) lalsls oM]

1o 6urlsrsuor'aL!osouorq) palettldar 6utsuapuol

alnqnlorlruaroq)olaur)

/ aroLllolaurl

.,t"' adolaûeleallnu

,,:, ]lelul

]SVHdOUd L

a lpu rds ,l

)rlollu '- .t

burrurol ./",.,,,,..,......... ./

au.losollua)

; !..

ANAPHASE

s n o r l e n n g

k i n e t o c h o r e

m i c r o t u b L r l e

5 TELOPHASE

o v e r l a p

n t I c r o r L t D L t I e s

6 C Y T O K I N E S I S

cof t rac i l l€ ] i l ng

c r e a t l n g c l e a v a g e

f r r r row

N-s)\C\\-\)

-

d a L l q n t e f C n r O m O S O m e S n r" h L t h e s r s t e r

c h r o m a t i d s s v n c h r o n o u s l Y

sePa ra te t o t o rn l two' daugh te r ch ron rosomes ,

a n d e a c h i s t r r u l l e d s l o w l Y

towa rd t he sp tnd le Po le i t

f aces The k r l e t ocho rem ic ro tubu les ge t sho r t e r ,

. a n d t h e s p i n d l e p o l e s a l s orr ' move apart ; both

processes cont f lbute to' ch romosome se l l r ega t t on

s p i n d l e p o l emov rng ou twa ro

D u r i n g . t he two

se t s o f daugh le r ch romo-

somes a r r i ve a t t he po les o f

t h e s p i n d l e a n d d e c o n d e n s e

A n e w n u c l e a r e n v e l o p e

r e a s s e m b l e s a r o u n d e a c h

se t , comp le t i ' r , J l l r e [ o rma t i on

o f t w o n u c l e r a n d r n a r k i n g

t h e e n d o f n r i t o s i s T h e

d i v i s i o n o f t h e c y t o p l a s m

b e g i n s w i t h ( o i l l r a c t i o n o r

t he con t rac t i l e r i ng

c e n t r o s o r n e

n u c l e a r e n v e l o p e r e a s s e m b l i n ga r o u n d i n d i v i c i u a l c h r o m o s o m e s

, : . . , . ,

: : :1

-1..,.:

R. r } J

GiS

c o n t r a c t r l e n n gs tar t rng toC O N I T A C I

, : : . : t : : : . ' .

:..1 '::;, ...

' :,;:

ta- r.

, - t , : ' "

D u r i n g , t h e

cy top lasm i s d i v i ded i n two

by a con t rac t r l e r i r r g o f

a c t r n a n d m y o s r nf i l a m e n t s , w h r c h p i n c h e s

the ce l l i n two t o c rea te

two daugh tc r s . each w i t h

o n e n u c l e u s

r e - f o r m a t i o D o i n l e r p h a s e

a r r a y o f m r c r o t r , l ) u l e s t l L t c l e a t e c l

l l v the cent rosonre

rl.l:

r-.----:\q.-\,

C\\a/

s e l n ' d a U g h t e ' u l t r , , t n o s o m e s

a t s p i n d l e p o l e

-S)- iC2 ot

c o m p l e t e d n u c l e a r e n v e l o p e

s u r r o u n d s d e c o n d e n s i n g

c h r o n t o s o m e s

(M i c rog raphs cou r t esy o f Ju l i e Canman and Ted Sa lmon )

'IaaM lrqtqut ol lpf_wJo IIlrqe aq] Iq pe)uequa st lleqpeaJ

srql'))eqpaaj a^rlrsod ut 6utllnsai'lp)-W e^tt)e Iq pele;nutls raqunl

s! SZ)p) '97rp1 asereqdsoqd aql ̂q zD

Jo pua aql le pelenrt)e uaql sr xa;dtuorlpf-W a^lr)Pu! 6ulllnsar aqf 'aseutl

14e11 aql {q salrs r(rolrqrqur ;o lrede uo pue ()v)) aseull 6ullp^ll)e-Ip) aql,(q alrs 6ur1en;1)e ue uo pale;fioqdsoqd

sr xa;duot )pf-W 6ultlnseraqf 'asu I;;enpet6 urp{t-y11 1o s;ena;aql se urp{>-y1 qllm sa}e!)osse tlpf

'Ip)-W Jo uolle^ll)e aq1 97-11 arn6r3

)p)-l laArpeul

oseurl lp)-lA^rolrqrqur-lpf a^rpeulDp)

eseurl6urlenrpe-1p;r

ur;:{:-61

eseleqdsoqdeArl)eu I

{pl-W Jo uor}Blndodqns B Jo uor}?Arlre lel}red eq} ot spPel OIpf,-S dq sdeqrad)gzJpJ Jo uortE^rlJE lellJed eql 'lepolu e^rlcerltP srql o1 Suproccy '(92-LI em-3r4 aasl sdool {ceqpaal el4rsod sallolur srsolllu uI uollplllcp {pC-hl tuqt s}se8-3ns 11aa111 rotrqrqur uâo slr llqlqul puE (gzcpf,) roteÎlJe u^ o sll eleÎlse ol

Ip3-ht Jo ̂rIIqE eql 'IeaM eseuDl frolrqrqur aql llqlqq osle deur {p]-N )pC-yq '1a3ru1 sll ^q 'ued ul lseal ]e rpelelllJe aq osp ueo gzopl 'd18uqsara1u1

'gzJpJ alBlnruus esEqdoJd dlrua puE zc uI aAIlsE are teq]s{pl-S aqt }eq} sI ItilqISSod euo 'poo}srepun

IIa^\ tou are stsolnu d.lJea uI (IeeAsseJddns pue) Ilrûcp gZJpJ qseaFn lpql susFeqJeru eqJ 'sespeJ3ul &yrlfce

{pC-hl lEqt Suunsua JeqUnJ'passarddns sI IeeMesBurn eqlJo IIIÎIJE drollqqulaql 'erlrp eluPs eql lM'Gz-Ll arn81g; {p3-hl ulerlsar lBql selEqdsoqd,r{Joltqquteql selorueJ r{JrqÂ 'gZJpJ ese}Eqdsoqd uta}ord aql Jo uoIlEAI}Je aq} sI

tuale IETJnJJ aql;elrdlcots {pl-^ eq}Jo uorlplrlop aql sra38u} 'ueql 'tBtI71\'esPuD[ eql Jo a]rs a^rl3e aq] Itolq lPql selEqdsoqd,(q passarddns sI lnq ]JP ol

Lpear pue parurJd sr ]eq] {pO-IAI Jo a161co1s tuepunqe uP supluoc tI 'zt Jo pua

eql seqJeer IIec eql etuu eql ,{q 'snql '(8I-zI arnStg ees) se}IS SurroqqSrau o,\/\l }uuopel,fuoqdsoqd.,fuorrqrqur .,tq a1e1s e^llJEul uE ul ll sploq Iae A esEuDI ulalordeql terllPe pessncsrp sp '$Vl) aseuDl Supenpce->1pf, eql ^q elIS SuIlPAIlce uP

1e pa1e1froqdsoqd sr sexaldruo3 eseqt uI {p] eql q8noqflV'slsollru saqceordde

ilet eqt sP (urr3l.r-t{ puB IIpl Jo xaldluof, aql) Ipc-N Jo uollBlnrunsceSurpuodsarroJ e o] speal uralord uITJIJ-I I uI espeJJul aq1'uoqduJsueJl eue8u17ctc-1t11uI esperJul ue o1 ,tpreupd Suulo 'hl pue z5 Surrnp seseaJlul stsaqlu.{surlcdc-y41 leêmoq 'sadd1

1ac tsoru uI 'aseqdralut ut utalord eqt Jo Irllqels qBHaql uorJ sllnser uorlelnun3oe uqc^,b-141 pue 'e1czb

IIaJ eql lnoq8norqt luelsuoJsr uqcrb-6;o srsaqlurts aql 'sa1c,b

1ac cruo.{rqrua uI '(I-ZI elqEl ees :slle3 elEJq-euel ur g unc^J) u11c,b-61 Jo uopelnurncop eql qlpn sur8aq uoltelllJe {pl-N

srsotrw Jo tasuo aql le lp)-w sale^ll)v uollel/(loqdsoqdec

'JeelJ lou eru srusrueqceu uollelllJu asIJaJd eql lnq ,trplrce >lp3-4 uo spuadapseseuDl erornv pue sasEuDI el1-olod Jo uoIlEAIlcV relul ssnf,slp ertl sP 'elpulds

aq] ol spleuorqJ relsls Jo tuauqJellp sloJluoo g-EJornV seaJeq^\ 'alpurds aql

;o,fi1rqe1s pue ,tlqurasse aqt ura,ro8 luql sulalord Ionuoo sdlaq osp V-EJoJnVaseuDl BJoJnV aqJ 'srsotltu ut f.pea salod aypurds aq] Jo uoperedas uI pellonulsuralord salul,fuoqdsoqd 1r asnecaq tred ut 'ayputds JItotIu reyodtq e;o dlquesse

Ieturou eql JoJ parmbar sr 'aldurexe JoJ '{ld eseuDl e)I-olod eqJ'stuala cltol1ru,(1rea ;o loJluoc aq] 01 suollnqlJluoJ ]ueuodrut eIeru osp 'sasou!4 aJoJnVaql pue sasau!4 aql-olod aql 'sesBupl uralordgo serlrruBJ lEuolllppP o^ J

'sISolIu

.,tpea ur pe^loûl surelord,{a4 ate$roqdsoqd ol auop IJP tou saop {pf,-IAI'pegpuepl uaaq ]art lou aêq surelord asaql Jo lsoru qSnoqlp 'ssacoJd eql uI

pallolur suralord cryrcads saleldroqdsoqd ryC-lt ueq.r,r paraSSln eq 01 rq8noqlsr sassacord esaql Jo qceg 'snleJeddu r31og eql puu uolele{soldJ uI}Je aq}

af )^) lle) aql:/ [ raldeq3 vLOL

MITOStS

:'fl ;;".'"';'*'#:il:?:il[::ffi ffi :?ili:,T::''.",:fr i:ffl :flH'along their length. The constricted regions are the centromeres. (Courtesyof Terry D. Allen.)

complexes, which then phosphorylate more Cdc25 and Weel molecules. Thisleads to more M-Cdk activation, and so on. Such a mechanism would quicklypromote the complete activation of all the M-Cdk complexes in the cell. Asmentioned earlier, similar molecular switches operate at various points in thecell cycle to promote the abrupt and complete transition from one cell-cyclestate to the next.

Condensin Helps Configure Duplicated Chromosomes forSeparation

At the end of S phase, the immensely long DNA molecules of the sister chro-matids are tangled in a mass of partially catenated DNA and proteins. Anyattempt to pull the sisters apart in this state would undoubtedly lead to breaksin the chromosomes. To avoid this disaster, the cell devotes a great deal of energyin early mitosis to gradually reorganizing the sister chromatids into relativelyshort, distinct structures that can be pulled apart more easily in anaphase. Thesechromosomal changes involve two processes: chromosome condensation, inwhich the chromatids are dramatically compacted; and sister-chromatid resolu-tion, whereby the two sisters are resolved into distinct, separable units (Figure

17-26). Resolution results from the decatenation of the sister DNAs, accompa-nied by the partial removal of cohesin molecules along the chromosome arms.As a result, when the cell reaches metaphase, the sister chromatids appear in themicroscope as compact, rod-like structures that are joined tightly at their cen-tromeric regions and only loosely along their arms.

The condensation and resolution of sister chromatids depends, at least inpart, on a five-subunit protein complex called condensin. Condensin structureis related to that of the cohesin complex that holds sister chromatids together(see Figure 17-24).It contains two SMC subunits like those of cohesin, plus threenon-SMC subunits (Figure 17-27). Condensin may form a ring-like structurethat somehow uses the energy provided byATP hydrolysis to promote the com-paction and resolution of sister chromatids. Condensin is able to change thecoiling of DNA molecules in a test tube, and this coiling activity is thought to beimportant for chromosome condensation during mitosis. Interestingly, phos-phorylation of condensin subunits by M-Cdk stimulates this coiling activity,providing one mechanism by which M-Cdk may promote chromosome restruc-turing in early mitosis.

The Mitotic Spindle ls a Microtubule-Based Machine

The central event of mitosis-chromosome segregation-depends in all eucary-otes on a complex and beautiful machine called the mitotic spindle. The spin-dle is a bipolar array of microtubules, which pulls sister chromatids apart in

anaphase, thereby segregating the two sets of chromosomes to opposite ends ofthe cell, where they are packaged into daughter nuclei. M-Cdk triSgers theassembly of the spindle early in mitosis, in parallel with the chromosomerestructuring just described. Before we consider how the spindle assembles andhow its microtubules attach to sister chromatids, we briefly review the basic fea-

tures of spindle structure.As discussed in Chapter 16, the core of the mitotic spindle is a bipolar array

of microtubules, the minus ends of which are focused at the two spindle poles,

and the plus ends of which radiate outward from the poles (Figure 17-28).<GTCT> The plus ends of some microtubules-called the interpolar micro-tubules-interact with the plus ends of microtubules from the other pole,

resulting in an antiparallel array in the spindle midzone. The plus ends of other

1 075

1 *

DNA

Figure 17-27 Condensin. Condensin is af ive-subunit protein complex thatresembles cohesin (see Figure 17-24).The head domains of its two majorsubunits, 5mc2 and Smc4, are heldtogether by three addit ional subunits. l tis not clear how condensin catalyzes therestructuring and comPaction ofchromosome DNA, but i t may form a r ingstructure that encircles loops of DNA asshown; it can hydrolyze ATP and coil DNAmolecules in a test tube.

ATPase domain

sp ind lepo le r ep l i ca ted

1076 Chapter 17:The Cel l Cycle

chromosome kinetochore(sister chromatids) ,/

motor+ protern

astral microtubules kinetochore microtubules interoolar microtubules

microtubules-the kinetochore microtubules-are attached to sister chro-matid pairs at large protein structures called kinetochores, which are located atthe centromere of each sister chromatid. Finally, many spindles also containastral microtubules that radiate outward from the poles and contact the cellcortex, helping to position the spindle in the cell.

In most somatic animal cells, each spindle pole is focused at a proteinorganelle called the centrosome (discussed in chapter l6). Each centrosomeconsists of a cloud of amorphous material (called the pericentriolar matrix) thatsurrounds a pair of centrioles (Figure lz-29). The pericentriolar matrix nucle-ates a radial array of microtubules, with their fast-growing plus ends projectingoutward and their minus ends associated with the centrosome. The matrix con-tains a variety of proteins, including microtubule-dependent motor proteins,

l p m

Figure | 7-29 The centrosome. (A) Electron micrograph of an S-phase mammalian cel l in culture,showing a dupl icated centrosome. Each centrosome contains a pair of centr ioles; although thecentr ioles have duplicated, they remain together in a single complex, as shown in the drawing of themicrograph in (B). One centr iole of each centr iole pair has been cut in cross section, while the otheris cut in longitudinal section, indicating that the two members of each pair are al igned at r ightangles to each other. The two halves of the repl icated centrosome, each consist ing of a centr iole pairsurrounded by pericentr iolar matrix, wi l l spl i t and migrate apart to ini t iate the formation of the twopoles of the mitot ic spindle when the cel l enters M phase. (C) Electron micrograph of a centr iole pairthat has been isolated from a cel l .The two centr ioles have part ly separated during the isolat ionprocedure but remain tethered together by f ine f ibers, which keep the centr iole pair together unti l i tis t ime for them to separate. Both centr ioles are cut longitudinal ly, and i t can now be seen that thetwo have dif ferent structures: the mother centr iole is larger and more complex than the daughtercentr iole, and only the mother centr iole is associated with pericentr iolar matrix that nucleatesmicrotubules. Each daughter centr iole wil l mature during the next cel l cycle, when i t wi l l repl icate togive r ise to i ts own daughter centr iole. (A, from M. McGil l , D.p. Highfield, T.M. Monahan andB.R. Brinkley, J. ultrastruct. Res. 57:43-53,1976. with permission from Academic press; C, fromM. Paintrand et al., J. Struct. Biol. 108107-128, 1992. with permission from Elsevier.)

cent ro50me

Figure 1 7-28 The three classes ofmicrotubules of the mitot ic spindle inan animal cel l . The plus ends of themicrotubules project away from thecentrosomes, while the minus ends areanchored at the spindle poles, which inthis example are organized bycentrosomes. Kinetochore microtu bulesconnect the spindle poles with thekinetochores of sister chromatids, whileinterpolar microtubules from the twopoles interdigitate at the spindle equator.Astral microtubules radiate out from thepoles into the cytoplasm and usuallyinteract with the cel l cortex, helping toposit ion the spindle in the cel l .

200 nm

microtubule per icentr io lar matr ix pair of centr io les

MlroSlS 1477

coiled-coil proteins that link the motors to the centrosome, structural proteins,and components of the cell-cycle control system. Most important, it containsthe y-tubulin ring complex, which is the component mainly responsible fornucleating microtubules (discussed in Chapter 16).

Some cells-notably the cells of higher plants and the ooc)'tes of many ver-tebrates-do not have centrosomes, and microtubule-dependent motor pro-teins and other proteins associated with microtubule minus ends organize andfocus the spindle poles.

Microtubule-Dependent Motor Proteins Govern SpindleAssembly and Function

The assembly and function of the mitotic spindle depend on numerous micro-tubule-dependent motor proteins. As discussed in Chapter 16, these proteinsbelong to two families-the kinesin-related proteins, which usually movetoward the plus end of microtubules, and dyneins, which move toward theminus end. In the mitotic spindle, these motor proteins generally operate at ornear the ends of the microtubules. Four major types of motor proteins-kinesin-5, kinesin-14, kinesins-4 and 10, and dynein-are particularly important in spin-dle assembly and function (Figure f 7-30).

Kinesin-S proteins contain two motor domains that interact with the plusends of antiparallel microtubules in the spindle midzone. Because the twomotor domains move toward the plus ends of the microtubules, they slide thetwo antiparallel microtubules past each other toward the spindle poles, forcingthe poles apart. Kinesin- l4 proteins, by contrast, are minus-end directed motorswith a single motor domain and other domains that can interact with a differentmicrotubule. They can cross-link antiparallel interpolar microtubules at thespindle midzone and tend to pull the poles together. Kinesin-4 and kinesin-10proteins, also called chromokinesins, are plus-end directed motors that associ-ate with chromosome arms and push the attached chromosome away from thepole (or the pole away from the chromosome). Finally, dyneins are minus-enddirected motors that, together with associated proteins, organize microtubulesat various cellular locations. They link the plus ends of astral microtubules tocomponents of the actin cytoskeleton at the cell cortex, for example; by movingtoward the minus end of the microtubules, the dgrein motors pull the spindlepoles toward the cell cortex and away from each other.

Two Mechanisms Collaborate in the Assembly of a Bipolar MitoticSpindle

The mitotic spindle must have two poles if it is to pull the two sets of sister chro-matids to opposite ends of the cell in anaphase. In animal cells, the primary

focus of this chapter, two mechanisms collaborate to ensure the bipolarity of the

spindle. One depends on the ability of mitotic chromosomes to nucleate andstabilize microtubules and on the ability of the various motor proteins just

described to organize microtubules into a bipolar array, with minus ends

spindle microtubule

kinesin-14

ki nesi n-5

Figure 17-30 Major motor proteins ofthe spindle. Four major classes ofmicrotubule-dependent motor proteins(yel/ow boxes) contribute to spindleassembly and function (see text). Thecolored arrows indicate the direct ion ofmotor movement along a microtubule-b/ue toward the minus end, and redtoward the plus end.

kinesin-4,1 0 /

s ister chromat ids

centrosome

'ralse ue palle) salnqnlol)tt! Jo ̂eJelPrpPl uMo slr saleal)nu Mou alllosoJluaf

q)el ateredas ol ut6aq sa^leq o/V\laql pue oM] u! sl!lds xalduo) oql uaLlM

'aseqd W lo 6uruurbaq ogl lllun xa;durot

leurosorluol a;6urs e ul taqla6o1 aso;tureLuar sred oloulua) onrl aq1 z9 i(q

palaldtuo) {;;ensn sr olou}ua) ralq6nepaq];o uorle6uola aql'tl o1 e;6ue

]q6u p te pue alotJluo) raqtou q)paJo aseq aql reau anot6 o1 sur6aq oloulua)relq6nep e'eseqd 5 6urrn6 slalaLuol)tr.u

ana; e Iq aleledas rred eqi;o salouluo)oMl aq]'Lg u! tulod utetla) p lv'uaar6)

xuleur relouluatrrad paleoosse puerPo aloulua) e Jo Slstsuo) aLuosollua)

aq1 'uo11e>1;dar alotrlua] 1g-2 1 arn613

lririiir!t,:', iir'. 1ti{iliiiixii,g

JealJnu eql Suop uede aôtu seurosoJlual o^ l aql '{sllal IEturue u1 z(lqruasse

alpurds saterlrur -,tu,rtce )pl-W ur asrJ uappns aq] ,srsolnu Jo SuruurBaq eqt lV

aseqdord ur ̂lqurassv elpulds saletltul{p)-W

'a1c,{c 1at Jad aJuo o} uor}eorldnp euosoJ}uac }1urll sllao qcns ̂\oq umouT lou

sr lI 'palJolq sr uorleJrldnp auosotuoJqJ Jr sanupuoJ uorlecrldnp aruosorluacpuE rusruPqJaur e qcns urEluol lou op 's3or; pue 'surqJJn Ees ,saru

Jo so^Jqrua,{pea aqt ur asoq} Surpnlcur lalamoq 'sed^l

IIaJ raq}O 'IJaqJ ur lda{ sr Jeq-runu aruosorluac qcrq,,rn,{q usrueqJeru auo SurprôJd ,uorlecrldnp auosoJlua3sTJolq srsaqlu.{s VNC Jo uoqrqrqur p}ueurJadxa 'sedd}

IIaJ ̂ueru uI 'ureueJun

are a1c,{c lac.rad aJuo ot uoqecrldnp aruosoJlueJ lltull leql susrueqcau eqJ'uoup8eJ8as auosoruoJqc ur sJoJJa snq]

pue ,tlquasse alpurds ur slf,aJap ol peel plnoJ saruosoJluac Jo Jaqunu ltalroJurue :sardoc orq dpo q4nr srsollu sJalua IIaf, aq1 ]Eql arnsue ol ,alJ.{J

1ac radacuo ,{1uo puB acuo a}ecrldar }snru 'saurosoruoJqc elrl .sauosoJlue3 'JIeq ̂\eue Jo uorlJnJlsuo3 roJ sa1e1dual se elJas pue aleredas sa^pq oml aq] qorq A ur'uouecrldnp

Jo tusrueqlaru anrlelJesuoo-rures e asn qlog .uopecqdnp eruosotu-orqJ pue uorlecqdnp eruosoJlual uee^Naq slalered Surlsaralur are areqJ

'srsolllu sra]ue IIaJ aq] Flun snalJnu aql Joaprs auo uo reqtaSol sureruar rred aurosoJlual slqJ'(Ig-ZI arn8lfl xrJletu JBIorJl-uacrrad pa8relua up urqlrl srred alorJluac oA\l ur Surlpsar ,elorJluaf, ̂\au a18urse Jo uorlerxJoJ eql saleelJnu qJea pue 'aleredas etuosoJlual aql ur salorJluaJo1\{l aqJ'uonectldnp aluosorlual salelllul osle ̂Jlua alrdc 1ac sraSSrrl teqt (I-ZIalqeJ aes :sllar Ieurrue ul alp3 pue g uqc^,tc;o xaldruoc E) {pl-S/IC aq1 'aseqd

S sralua flac aq] sE erur] arups eq] ]noqe ]e sur8aq uorlecqdnp aruosor]ua3'saruosorlual o,\/\l aJp aJaql srsol[u saqceer IIaJ aq] auE aq1 .{q leql os ,a1c.{c

11acaq] srelua [ac eq] uaqm saleJrldnp aurosor]uao eqJ 'salnqnlolJrru crurseldoi-,bs,lleJ aql Jo lsotu satealcnu leq] euosoJluac a13urs e ureluoJ sllal Ierurue lsolN

al)^) lla) oqf ur {ltel sln))O uorle)tldnc auosol}ua)

'umop{eeJq adolanua Jealcnu JaUe lup}-rodrur,r(lrepcrlred saruocaq pue saruosoJlual arrnbar lou saop leqt rusrueqcauuorlezrue8ro-Jlas aql reprsuoJ uaqt a A'srsolrur z(pea ur dlquasse luapuadap-aurosoJlual qtl,tlr Suruu€aq 'dlqruasse alpurds;o sdals aqt aqrrtsep llrou aM

'aruosoJtuac aqt po,rtorlsep seq ueeq Jasel e qclq ,t, ur sllaf, palnllnf, ur puesauosoJluac 1ce1 ,,(leurrou leql sllal ur sruro3 aypurds puorlJunJ E aturs lala-moq 'alpurds relodrq e go ̂,{yqruasse aql JoJ lertuasse }ou aJe saruosorlua3 .sa1od

alpurds peleorrqeJard go rred e Surpmord ,r{q ,{lqruasse alpurds relodrq a}p}rIrJEJsatuosoJlual oÂl aqJ 'salnqnloJs[u

3o ,r(erre prpeJ e sa]ealcnu qJIqM Jo qsEe'seurosorluecao rred e qtr.{\ srsolru sratua IIar leurue pcrddl y'sa1od alpurds aq}uro; dlaq oJ seruosonuac;o rftryqe aq] uo spuadap raqto aqJ .auozprru alpurdseq] uI Jaqlo qcea qll,tr Surlceratur spua snld pue salod alpurds oml le pasnJoJ

al),() lle) aql:/ [ ra]deq) 9LOL

MITOSIS 1079

(B) (c)

2 p m

Figure 17-32 The influence of opposing motor proteins on spindle length in budding yeast. (A) A differential-interference-contrast micrograph of a mitotic yeast cell.The spindle is highlighted in green, and the position of the spindlepoles is indicat ed by red arrows. The nuclear envelope does not break down during mitosis in yeasts, and the spindle formsinside the nucleus. In (B-D), the mitotic spindles have been stained with fluorescent anti-tubulin antibodies. (B) Normalyeasr cells. (C) Overexpression of the minus-end-directed motor protein Kar3 (a kinesin-14 protein) leads to abnormallyshort spindles. (D) Overexpression of the plus-end-directed motor protein CinS (a kinesin-5 protein) leads to abnormallylong spindles. Thus, it seems that a balance between opposing motor proteins determines spindle length in these cells.(A, courtesy of Kerry Bloom; B-D, from W. Saunders, V. Lengyel and M.A. Hoyt, Mol. Biol. CellS:1025-1033, 1997. Withpermission from American Society for Cell Biology.)

envelope, and the plus ends of the microtubules between them interdigitate toform the interpolar microtubules of the developing spindle. At the same time,the amount of y-tubulin ring complexes in each centrosome increases greatly,increasing the ability of the centrosomes to nucleate new microtubules, a pro-cess called centrosome maturation.

Multiple motor proteins drive the separation of centrosomes in early mito-sis. In prophase, minus-end directed dlnein motor proteins at the plus ends ofastral microtubules provide the major force. These motors are anchored at thecell cortex or on the nuclear envelope, and their movement toward the micro-tubule minus end pulls the centrosomes apart (see Figure 17-30). Followingnuclear envelope breakdown at the end of prophase, interactions between thecentrosomal microtubules and the cell cortex allow actin-myosin bundles in thecortex to pull the centrosomes further apart. Finally, kinesin-5 motors cross-linkthe overlapping, antiparallel ends of interpolar microtubules and push the poles

apart (see Figure 17-30).The balance of opposing forces generated by different types of motor pro-

teins determines the final length of the spindle. Dynein and kinesin-s motorsgenerally promote centrosome separation and increase spindle length. Kinesin-14 proteins do the opposite: they are minus-end directed motors and interactwith a microtubule from one pole while traveling toward the minus end of anantiparallel microtubule from the other pole; as a result, they tend to pull thepoles together. It is not clear how the cell regulates the balance of opposingforces to generate the appropriate spindle length (Figure 17-32).

M-Cdk and other mitotic protein kinases are required for centrosome sepa-ration and maturation. M-Cdk and aurora-A phosphorylate kinesin-S motorsand stimulate them to drive centrosome separation. Aurora-A and PIk also phos-phorylate components of the centrosome and thereby promote its maturation.

The Completion of Spindle Assembly in Animal Cells RequiresN uclear Envelope Breakdown

The centrosomes and microtubules of animal cells are located in the cytoplasm,

separated from the chromosomes by the double membrane barrier of thenuclear envelope (discussed in Chapter 12). Clearly, the attachment of sisterchromatids to the spindle requires the removal of this barrier. In addition, many

of the motor proteins and microtubule regulators that promote spindle assem-bly are associated with the chromosomes inside the nucleus. Nuclear envelope

breakdor.rm allows these proteins to carry out their important functions in spin-

dle assemblv.

overexpression ofCinSp

(D)

overexpression ofKa13p

normal spindles


Nuclear envelope breakdown is a complex, multi-step process that isthought to begin when M-cdk phosphorylates several subunits of the giantnuclear pore complexes in the nuclear envelope. This initiates the disassemblyof nuclear pore complexes and their dissociation from the envelope. M-Cdkalso phosphorylates components of the nuclear lamina, the structural frame-work that lies beneath the envelope. The phosphorylation of these lamina com-ponents and of several inner nuclear envelope proteins leads to disassembly ofthe nuclear lamina and the breakdown of the envelope membranes into smallvesicles.

Microtubule Instabil ity lncreases Greatly in Mitosis

Most animal cells in interphase contain a cytoplasmic array of microtubulesradiating out from the single centrosome. As discussed in chapter 16, the micro-tubules of this interphase array are in a state of dynamic instability, in whichindividual microtubules are either growing or shrinking and stochasticallyswitch between the two states. The switch from growth to shrinkage is called acatastrophe, and the switch from shrinkage to growth is called a rescue (see Fig-ure l6-16). New microtubules are continually being created to balance the lossof those that disappear completely by depolymerization.

Entry into mitosis signals an abrupt change in the cell's microtubules. Theinterphase array of few, long microtubules is converted to a larger number ofshorter and more dynamic microtubules surrounding each centrosome. Duringprophase, and particularly in prometaphase and metaphase (see panel l7-l), thehalf-life of microtubules decreases dramatically. This increase in microtubuleinstability, coupled with the increased ability of centrosomes to nucleate micro-tubules as mentioned earlier, results in remarkably dense and dlmamic arrays ofspindle microtubules that are ideally suited for capturing sister chromatids.

M-Cdk initiates these changes in microtubule behavior, at least in part, byphosphorylating two classes of proteins that control microtubule dynamics (dis-cussed in chapter 16). These include microtubule-dependent motor proteinsand microtubule-associated proteins (MAPs). Experiments using cell-freeXenopus egg extracts, which reproduce many of the changes that occur in intactcells during the cell cycle, have revealed the roles of these regulators in control-ling microtubule dyrramics. If centrosomes and fluorescent tubulin are added tothese extracts, fluorescent microtubules nucleate from the centrosomes, and wecan observe the behavior of individual microtubules by time-lapse fluorescencevideo microscopy. The microtubules in mitotic extracts differ from those ininterphase extracts primarily by the increased rate of catastrophes, in which themicrotubules switch abruptly from slow growth to rapid shortening.

TWo classes of proteins govern microtubule dlrramics in mitosis. proteinscalled catastrophe factors destabilize microtubule arrays by increasing the fre-quency of catastrophes (see Figure 16-16). one of these proteins is a kinesin-related protein that does not function as a motor. MAps, by contrast, have theopposite effect, stabilizing microtubules in various ways: they can increase the fre-quency of rescues, in which microtubules switch from shrinkage to growth, or theycan either increase the growth rate or decrease the shrinkage rate of microtubules.Thus, in principle, changes in catastrophe factors and MAps can make micro-tubules much more dFramic in M phase by increasing total microtubule depoly-merization rates, decreasing total microtubule polymerization rates, or both.

constant throughout the cell cycle, the balance between the two opposing activ-ities of the MAP and catastrophe factor would shift, increasing the dynamicinstability of the microtubules.

eql azrue8Jo dlaq saurosouloJqJ aql 'seluosoJlual ululuoc ,{lerurou }Eql sllaJ

ur ua1g '(gt-ZI arnglg) saluosoJlue3 lnoqllv\ dola,rap so^rqlua clueSouaqUgd

aseq] ur selpurds JItolIu eqt '(IZ Jalduq3 uI passnJslp) 33a ue sezITIUeJ

tl ueq^ errrosoJlual eql saplôJd ri.lprurou ulJeds etqlsc-:(/ttlDJ!rcuaSouaquad'sr

teqt) uopezlllpeJ lnor{tlm s33a uro4 dolanap ol pacnpu ueeq aAPq }eql sodJque

]Jasur ureuec ur selpurds alquasse ol pasn ssacoJd eql oslE s-r 1I 'sapulds IUJOJ

ol ssaooJd uorlgzruefiJo-JIas peseq-euosoluolqo slql esn 'sal.{coo plulue i(ueru

puB slueld raq8lq Jo esoql sE qJns 'setuosorlual IJBI [llPluJou ]uq] slle3

'(?t-ZI arn8lf,) selod apulds olrt] eql luroJ 01

sepqnloJtrru aql Jo spua snul1u eql sncoJ pue Iuq-ssoJf, uel{} sule}old Surputq

pue-snuru snorJpl pup ,7I -ursauDl ,uleu,{c 'saruosoruoJqJ aq} uorJ LeÂE spue

snuru eql qsnd dlaq osp surre aluosoruoJqJ eql uo 0I pue ?-suISeuI) 'lJede

spua snulu Jreql qsnd puB uolleluelJo IalluJEdUuP ue uI saFqnloJJIIu IUII-ssoJJueql '(gt-ZI aJnBrC ees) /i.lilueJ g-urseuDl aqlJo srequaur dlrclnolued'sulaloJd

Jotontr 'seruosoluoJqc eq] punoJe sepqnlorclu Jo uopez[qe]s puE uopEalsnu

aql qtl^/\ urSeq o] lqSnoql sr flqulasse epurds puosorlueJv 'JeIIJee pessns-srp sE ,seurosorlueJ

Jo aJuasqe eql q salpulds JBIodIq IUJoJ o1 sllec salqeue

]eql saFqnloJJru azruesJo pue ez$qels o] seruosotuoJqf, Jo 'iruqu sHl sI 1I'saluosotuoJqc punoJe seFqnloJJIIU

Jo uol}ezllqBls puE uopEalcnu Iusol aq1 Supuprups fqaraqt '1oso1dc aql ul

saxalduoc ulaloJd luo4 sulaloJd 8urz1qe1s-aFqnloJf,Ilu seseeleJ '(71 raldeq3

ur pessnssrp) ]rodsuerl JeelJnu uI peAIoAuI oslE sl qlHlv\ alD-ueu pelE^lpe

eql aCIO Jo ereld ul dJD pulq ol uua pe1cr Iosot{r eql uI asEdIC lptus E se]EI-nrurls Cgg aqt :ullBtuorqc ol punoq sI ]eq] (CgD) JolsPJ e8ueqcxa eppoallnu-auruEn8 e uo 'UEd uI lseal lP 'puedap ol sruaes seruosouroJqc eql Jo ,'bradord

sql .ezrJau^lodap uorlrsod raruroJ s,aluosoruoJqt aq1 ]E selnqnloJJlu elpulds

eql alrqM ,aurosoruorqJ peuousod dpvrau aq] punoJE sJBeddP flptder saFqnl-oJlrru a1p111ds l au Jo SSpIu e 'luaurufirp

Jo lno pe33n] sI eluosoluoJqc alSuIS P

Jr 'aseqdplelu uI sllaf, etuos JoC 'peuJoJ sBq alpulds aql JaUB uraql uoplsodeJ o]

elpeau ssEIS eurJ u 3ursn.{q palerlsuouap eq up3 saruosoruoJqs aq}Jo aruanuul

eqJ'uoueuro; alpurds ur ued aAIlcE ue /{eld.{aqt 'uoFezIIIqEls epqnlorcllu pue

uopealJnu alnqn]orrrtu qloq sro^BJ l€q] lueluuoJlûe lesol e SullEaJJ ,(S ,(tq-tuessp epulds;o ssacord aql ut sra8uassed ellssed lsnf ]ou eJB SaIuoSoIuoJqO

{1q ruassy al pu !dS r elod ! g e}oulord seuosotllol t{) )llorlUl

ro1:e1 eqdorlsele:pue dvn

1o palaldap dvt^ +o palaldap l)erlxa'peJlxa )rlolrur '])eJua )llolll.ll )llolltx

fplt sraqs!lqnduellrLu)ew urorJ uorssrrj.uoo qllM'0002

'61-EL:Z'lo!8 ila) ?pN "le ]a azrqournol'u

ruorl) 'salnqnlor)ru aqt ratloqs aql'sa1er eqdo.rlseler aqt raq6rq aqt teql alou

lslaued do1 aql ur uMoqs are suotttpuo)

leluauilaoxa luarajJrp aql u! pauroJsralsp aql ro sqde.r6o.trtt-u afua)saJonll

]o])ej aqoorlsele) aql pue dvwaq] uoaMlaq a)ueleq aql uo spuaoap elel

aqdortsele> aql 'snqf'sl)erlxe lrlolrul ulsaqdorlseler 6ur1e;nut1s ro; aqqtsuodsal strol)ej srql ]eql 6urlertput 'sl)pllxa )ttoltul

palaloap-dvt^ aql ur aler aqoollsele)aql sa)npar {1teat6 (1eryy utalotd

palelar-ursaur) aql) Jol)eJ eqdorlsPle))uDaos e Jo uorllqrqul 'sl)erlxe )llolltu

ut saqoorlsele) stlq!Llu! dvw srq] lpql6urlerrpur 'a1er aqdotlsele) aql saspal)ul

sl)prlxa )rlo]rur aql urolJ (S !Zdeux pallP))

6yy11 rrpads e;o uorla;dap aql'sl)erlxaaseqdralur ur ueql frlo]rur ut taqbtq st a1e.taqdorlseler aql'palredxa sy'{do:sot>ttu

oapr^ a)uo)sorong {q paano1lo1seM sat!osoJlua) aql LUor] paleal)nu

salnqnlol)rru lPnpr^rpur ro rorêqaq aLllpue'urlnqnt lua)saronlJ pue sauosorlua)qllm paleqn)u! oraM sl)eJlxe 66a sndouay)llollur ro aseqdralu;'r,ll6ual alnqnlor)!ul

pue seqdorlsele) olnqnlor)!trt

;o {ruanberl sql se)uanlru! sdVW puPs.ro1>e; eqdorgsele) uaa/nlsq a)ueleq aqlleql a)uoprâ ;e1uaulradx3 gg-41 arnbtl

t80 [

]ferlxaaseqdralu t

T

s'0

o

n'r 3

5o

s't .a

307 a --

co

sz

r.uri 0 t

stsollt

(ssar6 {lrsrenrun rallala))ou aql rrrorl uorssrr.ulad q}lM .966 t ,t6E [-€g€ I: tt t 'lolg ila) f 'ue^rllnS 'M pue olleqd lules ap .B Luoll) .saulosourorr.,l)

palerr;dar oql ale6al6as o] olqe aip sa;purds 1o sed^1 qlog .16usaop saurosorlua) ]noqltM peLuloJ elputds aql seaJeqM ,a;purds aqr;o a;od

q)ea ]e Jalsp ue spq sauosollua) qlt/vl alputds aql leql eloN .uotleztltuoJ

lnoqtlM luaurdo;anap paletltut leLll ollqula ue ut saurosollua) lnoqltmpauro; a;purds e sMoqs qde.r6olrru woyoq a'41'ofuqwa DJnDS, paztltuaj

{;leurrou e ur saurosol}ua) qltm peulJo1 a;purds ;eulou e smoqs qder6olrrr"ua)ua)seionU clol aql'par sauJosoulolrl) ar4l'uaatb pautels alp salnqnlol)tru

aq1 '(1eu6 snbunl ro) D,rDIrS: l)asul aql 1o sol(lqura >lleuabouaqlredu! seuosorluo) lnoqllM ̂lquesse a1pulds.relod;g gg-41 arn6;1

ruo4 SurMoJB salnqnlorc[u 'eurll erues aql lV.luaur{Jupe uo-pue ue o] pagal-uoc dlprde.r sr eruosoruoJqJ eq] o] lualuqculle leJelpl aqJ .eruosoJluec aqlprelv\ol 1l Suop z(prder epIIS ot ueqt pup eFqntorsrru aql Jo eprs eqt ol purq ollsJrJ uaas sr eJoqtolaupl aql 'edoJsoJorru e ur luala eJnldeo Ierlrq eq] elJesqoue3 a^ eJaq ̂ '.slleJ Sunl lmeu uI 'pppuroJq3 Jelsrs auo Jo eJoqcoleuDl aqlernldpc dlenluala pup sauosoJluec aq] uorJ pJeMlno elerpEJ selnqnloJf,rruJo spua snld cnueu.,i.p aql 'elpurds eql ot seuosotuoJql Jrlolru Jreql qf,elleol rusruBqJaru ,,eJnldp3 puB qcJEas,, u doydrua saruosoJlueJ 8ururc1uoc s11a3

'relel ssnJsrp al sE'alpurds eq] uo ]uetualoru euosoruoJqJ Jo IoJluoJ eql JoJ IBJTITJO sr eJoqooleuDleq] ]e uo4ezuaur,(yodap pue uorlezrrarudlod pua snld Jo uoue1n8ag .et-Ll

arnS;g) pua slq] ]B slrunqns u{nqnt Jo IBôruer Jo uortrppE eq} 8urâolle ilrlselrqâ aroqoolauDl eql o1 dpq8q aFqntoror{u eqt Suploq fqaraql ,pua s]r rpauepqnloJcrru aql spunoJJns leql Jelloc uraloJd e surpluoJ elrs ]uetuqtelle qceg'auo ]sn[ ure]uoc saJoqooleuDl ]spe^ seaJaqM ,sa]rs ]ueruqcE]]e esaql Jo 0t-0IurBluoc saJoqJolauDl IIac puruv 'aJoqsolauDl aql ulqlll\ir selrs lueruqJellp-apqnloJorru pazquroads ut uo-peeq peppeqrue ale sepqruoJorru eJoqooleuDlJo spua snld aql'(gS-ZI arnt;g) euosoruorr{J eq}Jo uor8er crJeruor}ue3 eq} lestuJoJ leql urleruoJqJoJeleq aql uo llmq sr leq] aJnlJnJls uralord paradellpru'luer8 e 'aloqiolauDl aql ]e ppeuoJqt Jalsrs qlee ol paqcellu eJe selnqnloJorrualpurdg 'seruosoruoJr{J aq} ol ,r(eup eql Jo luatuqcp11e eq} sr uorleruJoJ alpurdsur dals Jofeur puooes aq] ,(eJJp alnqnloJctu JBIodrq E Jo dlquasse eq] Buu oiloC

elputds aql ol sptletuolq) lalsrs q)ellv satoq)otou!)

'srsauqoilc ur sarllpruJouqE urSuqpsar 'pauoqlsodsltu ueuo st alpurds eql 'llnseJ e sp isllec purue ur alpurdsaql Suruoursod roJ alqrsuodsar aJp qJrqm ,selnqnlorJrr.u

IeDSe $lf,el ll fptu-Jou satuosoluoJqJ ale8ar8as uuc alpurds leruosoJluece Suqpsar eql q8noql1v'paôlueJ aJB saruosoJlual eql y alputds Jllolru JBIodrq e 3o .,(lqruasse eqlaloruord uEJ 'suralold Jolour snoIJeAJo dlaq aqf qllm'pue salnqnloJJrru alpurds

lled e olut spua snutul asoql sn)oJ'suralord jaqlo snoraurnu ql;m leqtabol'sroloru tl-ursourl or, ,,."nj-"'l:1:5il::i:aql uot; {eane spuo snuluJ qsnd pue sture aurosoLuorq) ol salnqntor)rul aq} lurl 0[ pue t-sursau!l pol)arp pua-snlo

allqM'salpunq lalleledllue olut salnqntol)tt,r't asaql eztue6lo (0E-1[ arnbrl aos) sutalold Jotoru S-utseut) .soulosotuorq) aq]1o Ilrut>tn aql ul salnqntot)rr.li Jo uorleuro; aql alouroro pue aleal)nu lpq] suralord selelrl)e q)rqM,(umoqs rou) dfgueU

Jo uoll)npold le)ol aql alelnulls saurosor.rrorq) )rlolrl l'suralord rolour iq uoglezrue6ro-1;es alpurdg t€-ll aln6rl

uri O!

g1'7-ursaurl {qqsnd preMlno

g-ursaurl {q6ur4ur;-sso.o lelleledtlueuotleel)nu

salod alputds

tt-ursaut) pue urau{p{q salod 1o bulsntol

el)r() lle) aql:lt raldeq) ZgOt

'aloqlotaurl aql o] paq)Plle sureuarolnqnlor)lul aql allqM pua snld pasodxo

aqt le rn))o ol uollezuau.l^lodappue uorlezuau,(;od 6urmolle

'pua sn;d alnqnlor)ru, aqt spunornslq-t4l (tôilat)arnl)nrls rello) e uteluot ol

tq6noql sr alls q)el'aroq)olaull e u! allsluauiq)e11e alnqnlor)!ur V /€-lI atn6ll

f aJoq)olaurl Iur peppequla Isalnqnlor)rLU

L

luauraôurprleuiolql

+o uorl)ejrp

alnqnlorltrllaroqrolau rl

+o pua snurur

'ueql loeJJo3 ol peAIoAe eAEq sluslueqcaur drol-e1n3ar luefiala pup tnJJo op sluaruqJelle ]JeJJof,uI 'ssalequaleN 'alod alputds

aluusaqleJeJuusSeJoqso}auDlqloqlsqlpooqna{IleqtSeJnpeJleqluopuluelJo

{3uq-o}-)ceq B ur palsnrlsuoJ alp seJoqcoleuDl Je}sls lEqt sI Ja1v\suB eql Jo uEd

;sa1od apurds qloq ol aroqsoleuDl euo Jo lueuqsullE aql ro alod alputds arues

aql ol seJoqsoleuDl qloq Jo lualuqceile aql slualard IEI{ \ ipaêlqlu 'uollulua

-IJo-Iq pellef, ,lueurqJeue Jo epou slql sl ̂ oH 'aseqdeue ur aleredas daql uaqru

1"r "ql-;o.pua alrsoddo ot elorrr ,{aqr reqr os 'alpurds cpotgr eq} Jo salod elIS

-oddo o] qcEllp rred e ur spDprrroJqJ Jalsrs teq] spuBuep sISolIIu Jo sseJ3ns eqJ

rorlf pue lelrl ̂q peârq)v sl uollelueuo-!8

'fty-Ll arn8tg aas) alod alpurds eql 1B sulelord -rolour

Lq pasnco; pu€ spua snulur Jeqlo ol pe{uq-ssoJf, eluooeq AIIEn}ueê ppo^,salpurds eerJ-atuosorluec ur spue snuru Jeqlo aIII 'selnqnlorlllu eJoqsoleupl

asaqlJo spue snurru eqJ'aroqooleuDl eql uroq ^,i.ea,re sepq$oJolru aqt;o qyvror8

ur llnsal uaql ppo.ry\ spua snld esaql le uopezgraruflod 'aloqso]auDl eql Jo SJPI-1oc Surpurq-pua-sn1d eq] q pappaque euoceq pu? saloqsoleupl qlIM ]rurelul

seuosoruoJqi "qt

go ,tprcn aql III sepqnloJcr{u Uoqs tuq} sr dlqrqlssod aug

iseluosoJlueJ Jo acuesqB eql ur JnJlo lualuqcellE aruosoruoJqf, seop lAoH'(8t-ZI arnt1$ luaruqoeile relodrq u Suturro; 'eluosotuoJqo

aqlJo eprs alrsoddo eqt uo eroqootaur{ aql o} qoet}e alod alpurds alrsoddo aql

('99151 ruorl uorssrutao qllM'0/6 t'26-LLiEZ'pS'lo!8 'f 'isny'a)Mol 'f'l pue sdeag-11apr6 'O'f uor; '1 :r(a11uug 'g'g;o ,(salrno> 'y) 'sta'{e1 leuolrlppe q}!M

alnl)nJls xa;dr,rjo: {1;ensnun ue seq (e6le uae.r6 e Luorr) araq uMorls auo aq}'arn})nlls JeululellJl e aêq saloq)olaull }sotualrqM'aroLl)oleurl slr ol paq)eue salnqnlor)r,u qtlM plleuloJLl) aseqdeue ue;o qde.rbolrlLu uorl)alf (f) 'slla) uelleululeul

autos ut 0' ol lsee^ 6urppnq ut t ruolJ satje^ aloq)olaurl eseqdelar-u e ol punoq salnqnlol)lul Jo laqunu aql'aleuollua)aql ro a)eJJns aqt uo enbeld p suloJ aloqrolautl qrel 'selnqnlol)ru aroq)olautl Jo spua snld eql o] paq)elle splleuolq)

lalsts o11l s1r 6urmoqs aulosotuolq) aseqdelau e;o 6utmetp V G)'pai paulels alP'plleuioJq) q)ea qllM palelfosse

auo ,saloq)olaut) oMl aq1'surato.rd aioq)otautl rr;Deds qllM l)per leqt satpoqtlueolne ueulnq qtlM pue a^p lua)salonu6urpurq-yy6 r qrlr* p"rlrtt arrrosotuolq) aseqdelatu e;o qder6otrtu a)uarsaronlj V (V) 'aroq)olau!1 aq19g-41 eln613

(8) (v)prleuo.Ll)

aurosoulo.lq)pale)r loeJ

aJoq)oleur) rauul

auosol.!olql +o

€80t

aloq)olau r)

SISOIIW

'a)eld ur pel)ol ̂qalaqtsr tuauq)eup t)ailo) stql.^lturte

6urpurq elnqntol)tul ut aseal)ut uesra66u1 q)tqM /solts 6urpurq-a;nqn1o.orr.u

roql ssol)e uotsuol asuas ol rq6noqrare saroq)oleut) lalsts aql /atoq)olaut)

puo)as aql o] sputq a;od alrsoddoaqt urolj alnqnlor)trrJ puo)as e uaqMalet)osstp ol spuel salnqnlol)tur oMl

aql Jo auo ]eql os laâMoq ,olqetsun etesluauJq)ellp l)alro)ut asaql.aloq)olautl

auo ol rl)ellp ue> sa;od alpulds qroquorj salnqnlol)tut lo ,aloq)olautl lalsts

reqlo aqt ol q)ellp ue: e;od a;purds aulesaql ulol; alnqnlol)tul y'slem snouenur auosoLuolq) oql o] putq uaql ue)

salnqnlorlil,.U leuotltppV I ted ptleuorq)Jalsrs e ut atoq)olaut) auo ol sputq

alod e;purds e LUoIJ alnqnlortru a;6urse'{;;ertrul'luaruq)eltp auosouroJtl)

Jo sruro] a^lleural;y 69-1 1 atn6r1

'outptls auosouolq)Jo uott)altp aql sale)tput (l) ul

uouo trotb aql oltqM,qlmol6 elnqntol)rulJo uoll)altp aql salp)tput (V) ut MdrD

par aqI 'soJoq)olau!I {q sa;nqn1o.rtlueuosorlua),o arnlder aq1 gg-4 1 etn6r3

ltSvl_s

'alrs lueuqJellp aql Jo [1ru4;e eql SursparJur pue uor]B1d-roqd-soqd aroqcolaur4 SurcnpeJ dqaJaql 'sJnJJo uorlBluarJo-rq ueq ^ petE^rlJEur sr g-EJornv'pua snld epqnloJJrru e JoJ &rugJu s,elrs aql SurspaJf,ep ,alrs luaruqcE]lBalnqnloJf,ru aq] Jo slueuodluos IeJeês saleldroqdsoqd 11 'uorsual

Jo eJuasqeaql ur lualuqJenB alnqnloJcrru Jo qlSuans aql seJnpar ]eq] IEuSrs d-rolrqrqureql aleJeua8 o] lq8noql sr g-eJoJnV 'aroq3oleur) aql qly!\ pelerf,ossE sr qorqlv\'g-proJne aspuDl uralord aq] uo spuadap rusrueqteur Bursuas-uorsua] eql

'salnqnlororru a1d41nu;o pesodruoJ Ja q{ aJoqropqry {JIq} p Jo uor}eruroJaq] ur slFSeJ srqJ 'aJoqJoleuDl aq] 01 saFqnloJJrru puollrppe Jo ]ueluqcBneaq] o] spBel oslP ]nq elrs ]ueluqJene aq] Jo ̂lrurJJp aq] sesEaJf,ur ,{luo tou uors-uel 'slleJ

IEruruE uI ']uaruqf,EDe alnqnloJsrru SuruaqlSuarls ,lpuSrs .,trolrqrqureqt JJo stnqs aloqf,oleur{ aq} lE uorsual q8rq aqf ,sJnoJo uor}e}uerJo_rq

-auDI eq] saop /v\oq '1ng 'ace1d ur pa)Jol arp sluaruqf,Ellp lJaJJoJ alrr{lv\ r}sel }ouop pue elqelsun duStq aJE slueruqJeltp lJeJJoJur :aldrcurrd aldurrs e uo paseqsI leql rolJa pue IBrJ] Jo rua1s,{s e .,(q palcarroJ aJe slueuqceup IJaJJoJUI

l'l8vr-sNn

&

luaut qlellelelodtq ol ielodtun

liaûo) ol alod alpurdsalrsoddo txol+ salnqnlol)tul

salnlde) aloq)oleutl eaJ+

elnqnlo])tujoloq)oleut) pelle)

MOU alnqnloJ)trrj:luaulqleDp lelodtun

'uo-pua o] sueAUo)

G) ]ueLuq)elle lerotel (c)a;od a;purds pleanolseprls aulosorrJolqr ())

aseqdelatlo.rdle eurosouloJq)

peq)el]eun (v)

rrSvisNn

a I nq nlol)t ullerlse ue o1 {11eta}e1

spurq aroLllolaut) auo (8)

\a;od a;purds

lrsvl_sNn

el)^) lle) aql:/ [ roldeqf t8O I

'alod alpurds aql le pua snurulalnqnlor)ilJr aql preMol aloq)otautl

aql aôur llrM slq]'eldr)uud ul 'pua snldalnqnlol)rru aql spunollns leLll aJnl)nlls

rello) aqt lsurebe qsnd pue (g t-g tarn6rl oas) preMtno Unr alnqnlor)il.u

aqt Jo sluaureluolord aql 'srn))o

uollezuaulllodap uoqM'alod elpurdseql PleMol aioq)olau!{ oq} llnd r(euruoltpzlraurllodap ,vloH ot-l t arn6rJ

eroq)olaur)

'lzv-Llern8ld) alEld esEqdEleu ar{} tB srlud ppeuoJqc Je}srs peluelJo-Iq eql UBIIEsdleq ll ueqm 'eseqdpletu pue eseqdelauord uI lupuodlul dlJelnJlued sI eJJoJsrql'sayod alpurds aql uor; derrne sauosoruoJqc aql lrodsuuJl pup selnqnlorolrrrrelodralur qll,lr lf,eJelut srure eruosouloJqf, uo srololu 0I puu t-usauDl pelJeJlp-pue-snld 'auol uotlca[a n1od et4 sr seruosoruoJqo uo Supce acroJ prlql V

'aseqdeuu ur aleredas .{aqt rage splle{uoJt{J Je}sIS Jo }uau-eôru prerr,ralod eq] puE aloqcoleupl eql le uorsualJo uorlsreuaS eql ol salnqlJl-uoo qJlqa 'ecJoJ pJemalod e sacuarradxe xng qcns Suto8rapun eFqnloJclrue o] paqrepp sr leql aJoqooleuDl duy 'salnqnlorcrtu relodretu pue eJoqJolaul{qloq uo salod aql pJemo] Ie Erl lpql saplcads ]ueoseJong ,(ur13o acuureaddu aqlur stlnseJ slql'(I1-ZI arnt;g) s11ac 3urn1 olut palcalul aJe uIFqru ]uacsarong

Jo slunoue 1eurs.{ral qJIqM ul poq}eu snorua8ur ue dq uaas eq uEf, salputdsaseqdelaru ul xng e1nqnloroll l 'a1od alpurds aql premol sepqntoJ3lru Jo tueu-aloru aql atldsap ]uelsuoJ surprueJ r{13ua1 epqnloJf,Itu }Eql os 'pue snulur aql]e u1nqnl Jo ssol aqt roJ salesuadruoo elnqnloJJrru e Jo pua snld eql tu ulFqnl,,lreuJo uorlrppp aql 'aseqdeuuJo lasuo eq] Ulun

'spua snuru Jreql ]E palluerusppue salod alpurds eql pJumol peloru eJp sellesueq] saFqnloJJlru aql ,ri.qararylt'xnfl alnqnlo.rc;ur Lq sadril 1ac auos ul papr,rord sI ecJoJ prerralod puoces V

'(gt ;aldeql ur passnJsrp) eFqnlororru e Jo pua eql o] sppe lrun-qns urnqn] e reue srnf,Jo teqt dJC;o srs,(1orp,tq eqt (uoq saruoJ it1aleu4p 11lsazrarudlodep apqntorJrru eqt ueqm paspaler sl puu epqntoJJllu aqt ul perolssr luerueloru eql selrJp leql .{3raua eql 'eloru ,{qaraql pue sepqnlorcrur 8ur-zrraur.{.1odap o} paqf,E}le urprueJ uec 'luasard

dJV ou q}lm'aqn} }sa} E ur seJoqJ-oleuDl pagrrnd lpql rrmoqs ueeq spq tl tnq'lsJg ]e alqrsneldurr ruaes rq81ru srq;

6;y armbar ]ou saop acJoJ pJp^\elod palerauaS-aroqcolaupl sql ',{.13uqsara1u1'Jalel ssnf,srp aal se 'asuqdeue ur alurudas daql rage salod eql pJEMot sppeluoJqcratsrs Surnotu ro; luuuodrur ,i.lreprqred sl pu€ aseqdularu pue aseqdelaruordSurrnp sauosoruoJqc uo slnd ecJoJ slql''p'V-Zt arn8gg) pre,nelod eJoqcotauplaql slpd teql eJroJ e sateraua8 ,lroqeruos eFqnlorcrru eqlJo pue snld aql te uol]-ezuaru^,t1odep 'usrueqJeru ureuef,un ue dg Jlestr aJoqcoteupl eql lB suratord,(q pecnpord q iI 'alod alpurds aq] pre^ ol aFqn]ororru eroqcoleupl aql SuopeuosoruoJqJ paleroosse slr pue eJoqcoleuDl aqt slpd acro; roleru ]sJrJ er{I

'tueilodrur dpulncqred aq o1 lq8noqleJe seJJoJ roferu aarq; 'alpurds JI]olIu aql Jo seFqnloJJrru eql uo saruos-oruoJqo aloru lpql secroJ eql aleraua8 srusrueqceru Jeqlo pue suralord Jolontr

alpulds eqt uo sourosoruoil4) aôw salrol oldlrlnw

'Jelel ssnJ-slp ellt sB 'seurosoruoJqc aql Jo lspl eql Jo luaruqcu1p ruyodrq eqt JeUe 'aurq 8EIalqercpard p qllm 'pacnpord sr pu8rs aq1 'aleredas o] spupuorqJ ralsrs eq] JoJpu8rs aqt Suqpare 'a1e1d aseqdetau aq] le ,tltua8 alelllf,so ueql saruosoruorqJaql 'sIIeJ alerqeuel u1 'e1eyd asuqduleru eqr pellec uolllsod e 'se1od elpulds oanleql ueemleq luelspmba uorlrsod e Sununsse rtlpnlue,ra 'quog pue 4cuq pa33n1eJe saruosoruoJqr aq] 'sa1od alpurds o^N aql ol lueruqJelp Jleql 3urarro11o4

salnqnloJ)ru.r eroq)oleurl+o pue snlo ]o

uo rlez u a ur{1 od a p

980rsrsolrw

'aloo eqt preMol saulosoulolr.l)llnd ol lqbnoql ale'xnU olnqnlol)rul

ql!M raqla6ol'aloq)olautl aLllle uortezuaullodap Iq paleloue6 so)ro,

premelod '(O€-/ t aln6rl aas) .rolenba

alpulds eql pJemol saulosoulolL.l)saqsnd q>rqnn'ariol uorlrafa re;od eq1elereua6 ol salnqnlol)l.u qltm l)elalul

ol lqOnoql ele sulle auosoruolq) aql uo(0 t-ulsaull pup t-utsoutl) suralo.rd .rolor.u

pal)arp-pua-sn;6'a1e1d aseqdeleu aq1ol saurosomolLlf anoul ol ateledoo> {elusarro; 6ursoddo olvrl Moq Jo lapou V (g)

'uotslndar pespoJ)op e 6urlraget'elod aql plemol sonoul oJoq)olautl

aql qllM lleo oql seoreqM'alod aql uolJ,{eme r(;prder paqsnd st eloLl)olaut)i e

lnoqllM aurosoutoJLlf paJaâs aql Jo uedaqf 'salnqnlol)rul aroq)oleurl {q e10d

a;6urs e o] paq)eup st leql euiosotuorrl)aseqdelauo.rd e slanos t!eoq Jasel

e'lueuuadxa s!Lll ul .Jolenbe elpurdseql pleMol selod alpurds aql ulor,

(earre sauosoluolqt saqsnd lpql o)roJuoll)afa rel0d e to; e)uapr^] (v) .aleld

eseqdegaur aql ol sauosoutolq) aÛp(eur sarrol Eursoddo mog 79-41 e.rn6r3

a;od u.rol1 ,{elaeseôur eroq)oleu!l

lnoqllM trjle

suteloro rolor.!0 ['t-ursaur)

pepal rp-pue-sn lo

el nqnlor)r urlerlse lo

JelooJalu I

eql lseJeau eJoqJoleupl aql ur uorsual selPJeueS ecJoJ uopJefe Jelod SurseeJsurue 'elod elpurds aq] pJpMot aôru seruosoruoJqf, se lpq] 'alduruxa roJ 'pesod

-otd ueeq seq ]I 'eJoqooteupl aql ur uolsuel Jo eeJ8ep eq] uo puadap deur satels

o^tl eql ueeMlaq q3ll.\/\s eqJ'elod aq] ruo4 I?âE ser.uosoruoJqJ eq] seqsnd eJJoJ

uorlJefe Jelod eqt puE JJo peuJnl erE seJJoJ pJeâelod aql uaq,\/\ 'elPls 'lsJlneu

Jo '(dV) alod-eql-uo4-itu^^p ue pup 'elod aql pJe^\ot pellnd eJe saluosoruoJqJeq] ueql\i\ 'elels (d) pJB^\elod e-salels o^ 1 uaeMleq qsllr\ s 01 uaes eJE sluelu-aôur eql 'slleJ 3un1 luau ur ^doosoJJllx oepl^ dq parpnls uel{ A

'esEqdEleru

puP aseqdeleruoJd ur saruosoruoJqc eql Jo luelxeôru ,{roleltcso sno-nurluos eql sr sllal alerqeue^ ul srsolrru Jo slcedse 3uoF1s lsou eql Jo euo

('Pl'l slaqs!lqnd uelllul)ewurolj uotssilIlad qllM ' looz 'Lz-ll1:E'tolg

ilD ?DN 'uor.ules 'o'l pueuosrq)llw 'r1 uor,') pue g) 'puo snuru aql uioJ] peôuer eJe slrunqns

urlnqnl se ater aurps aql ]e pua snlo olnqnloj)rur aq] le pappe ere slrunqnsurlnqn] ̂ au asne)aq ,(1lue>;;1u6;s e6ueq) tou saop alpulds eseqdeleu

aql u! qlbual elnqnlol)lW (O) preMalod 6utnou alP salnqnlor)rr,!aql leql 6ulterlpul 'uruTruri S/'0 lnoqelo aler e le salod aql ple^ ol

onour ol uaas aq ue) sall)ods lenprnrpul 'aurll Joô uor6ar aql;o abetuotu

p oleur ol aprs (q eprs palsed pue seurl ;erluanbes le ualel aJaM (g)ul (/Au)D) uor6ar paxoq lelnOuel)ar 'urql'6uo; eqlJo saoeLul '{do>sot>ttu

oaprn esde;-aur;1 r(q palro;1o1 eq uer sa;1rads lenpt^tpulJo luaulaôulaql (f) par are sal))ads urlnqnl aqt pue'ulAorq parolo) ale saulosoulolq)

aql'lla) 1e;laql;da 6un; vvrau 6urnr; e ur alpurds lrlolru e Jo sqdel60l)lr.lla)uo)saronll (g) ',(do>sor>ru.r aruarsaron;; r{q peMetA uoqm arueieadde

pallrads e eêq salnqnlor)il.u q)nS 'utlnqnl luo)salonU jo uotuodotd

lleuis ̂le^ e qllM uiloJ salnqnlor)tr! lenpl^!pul lPql os slla) 6ul^llolur palrafur sr urlnqnl lua)saronpJo lunoue lleuls fuaA e'xnU alnqnloJ)rtu

a^lasqo o1 (y) 'alpu1ds aseqdelaur aql u! xnu elnqnlor)lw 1y-4 1 aln6g

auJ] <-. (v),selllaos,

6 \t8 c tt

oo8 8ô*t

tH. f

el)^) lle) aql:/ t ratdeq)980 r

'uolleÎ]f,Eur u,\/\o slr snq] pup uorlJnrlsap uqcfs o1 speel ]Eq] ssasoJd ̂JolPFSeJe uorloru ur slas {pl-W :dool Treqpaal elr}e8au e sa}earr,ri.11,t11ce l/ldV-gZJp:)atoruord ot {pl-htr go ,,h11qe aq1 'aseqdeue o}ur uorssarSord rog a8els aq} stesosp lr 1nq 'aseqdelaru o] dn Surpeal sluela ollolFu,,(pea aqt s;a88ul dpo 1ou

{pl-W 'snqJ

)pO-I I sI l/JdV aqt arEAItrE snq} pue ale/.roqdsoqd }eq} saseuqaq] Suoruv 'xaldruoc enrlJe ue atparc ot Surdlaq dqaraqt 'llldv aqt ot pqqgzcp3 sdlaq 3734y aqt;o uor1u1.{roqdsoqd 'puoces 'aua8 slt;o uotldrrcsuerl aq1ur esearcur ue ol 3uu,ro'srsolnu saqceordde flac aql sE sesBeJJuI stsaqlufs gZJp:)'tsrlC 'llldv eqt qllm uollelcosse sll pue gzcp:) alepBar sassacord o^^l ]seeltV'(VSZ-ZI arn8rg aas) srsolru ul O/ldvaq] satPAIlJE pue o] spulq q3IqM'02)pJuratord aqt sarrnbar uorlelr]f,e J/ldV teIIJee pauolluatu sV 'u^ ou>I dpred dposr ramsue aqJ ill3dv aqt sate^rlf,e 1eq,u 'aseqdeue sraSStrl l/JdV aqt JI

'(ratel

passnJsrp) srsauqoldc pue srsolru yo uoqalduoJ aq] ro; parmbar se 'lleJ eq]ur saleJlsqns 1a8re1 1p3 fueru aq] a1e1,{roqdsoqdap o1 saseleqdsoqd srr,rolp uotl-elrlJeur 1p3

'aseqdeue ur ,,i.lr,Lrlce {pJ lsour Jo ssol aq} ot Surpeal 'uollJnr}sep

ro; surlcu{c-1x puB -s aql slaSret osp :)/3dv eq} 'uunras o} uoulppP ul'ftv-Lr

ernt;91 aleredas .r(1snouorqcu.,{s pue ,(pdnrqe splleluorqJ Je}sIS eql pue "{eme

IIPJ sursaqoJ aqJ 'ursaqoJ Jo slrunqns aql Jo auo aÊelf, o1 aa4 uaql sI tlJlq^\

'aseredas sasealal aseqdelaru Jo pue eq] lB uI-InJas Jo uollJnJlsap aql 'asBJ

-edas palpc asealord e yo flvrqce aq] s]lqqul pue o] spulq uIJnJas 'aseqdeuu

eJoJeg 'uor]lnrlsep roJ ulrnJas uratord drolrqtqur aql Surla8rel [q ssac-ord aqt salBIlIuI :)/JdV eqJ'alpurds aqt;o salod attsoddo o] aloru pue aleredasot sJatsrs aqt smolle qrlq \ 'uorseqof, plleuorqc-Jalsls Jo uopdnrstp uappns Eqlp,t sur8aq aseqdeuy'pede spqeruorqr ratsls aqr gnd teq] saf,roJ prea'tayod aqllsrsar raqlaSol spupruoJqJ Jalsrs eqt Surploq sulsaqoJ 'aseqdelaru Suunq

'N1Z-LI arn8rg aes) uortJnrlsaprraq] 3urra33r4 ̂,(qaraql pue sutalord ,{rolep8ar cpo}Iu IBJeês Surlelrfirnbrqn,{q uoqeredas pueruoJqJ-Ja}srs se}ellrul }eq} qc}rms aql s^\orq} JerlJee passnJ-slp (J/ldv) xaldruoc Sunourord-aseqdeue eq] 'tuaâ srqt roJ a8els aql slas dlt-^rlre {pl-I q8noqlry'(t7-21 arn8gg) uortrsuerl aseqdeue-o1-aseqdelaru aqt tesp[euorqJ ralsrs ar{l Jo uorleredas aq} q}lm xerurlc s}r saqJeer a1c.{c 1ac aq} 'sIS

-o1ru,{pea ur JnJf,o teqt sluaua8uerrear xalduoJ aq} para83rr1 seq IpO-IN Ja}JV

srsolrw Jo uorloldruo)aql pue uoltpledos ptleruorq)-talsls sle66ul )Ddv aql

'alpurds aqt;o rolenbe eql lB ser.uosoruoJql Jo uorlelnruncop aql ul3ur11nsar.{lpnper8 pue alels alod-aq1-urorg-rteme eql ol qJ}IMS e 3uua33tr1 'a1od

('rafeg marpuy ;o,(sa1rno11 'ullnqn] ]sute6e sarpoqrlup palaqel-plob qt;rur paulets aram ]eql s;1a> r.uredsopu a (All) snLltuowaog 1o sqder6o.rrrurrq61; asaql ur uMoqs se-alpurds )rlolrrrt aqlJo selod alrsoddo preMol aôur pue aleredas,{;ueppns sprleuorq) ralsrs

'(B) aspLldeue ol (V) aseLldelaur ulorJ uortrsuert aql ul 'aseqdeue ge uogle,redas plleuorq)-ralsr5 gy-21 atn6;3tud 0Z

(8)

I

(v)

/80 rstsoltw

fssar6 {lrstenrun lallajal)ou aqf uorluorssruled LltlM'966 t,|,6 t [- [B L t: [tI

'lo!8 ila)'f "le la slalPM )'l worl)'(/AoilD

allLllv Aq pelplrpur '1op a1od1a;od elpurdsaqt ol paq)eDP s! leql plteuorq) JolstsaqlJo aroq)oleutl aql qllM polet)ossp

sl ZpeW Jo lunoule lews V '(/Aut0

pa; {q palertput 'Jop pat) ptlpuloJr.l) lelsrspaq)eDeun aqlJo etoq)olaut) aql ol

punoq sr zppw reqt sale)rput satpoqrluezpew-llue LlllM 6ululels'a;purds

eLlt,o alod auo Iluo o1 peqrelle sr tredplleuorq) relsrs auo'an/g ut sptleulolrl)

rolsfs eql pue uaafi ur a;purds )tloltui eqlr.lllm'espqdelouo.rd ur lle) uetleuJuleul

e smoqs qder6otrlr.u a)ue)salonUsrql'saroq)oleull peq)eDeunuo ulelold 7pe4 99-41 ern6;3

'uoltel^roqdsoqdop

st! 6urMolle Iq uorlenrlre aseledasselouiord osle (uot])nllsop ur;tIt ruor;6ur1;nsar) aseqdeue ut uorte^ll)eu! lp) 'snqI'(uMoqs lou) aseJedes sltqtqut osleqpf ^q uollellroqdsoqd 's;;a>

leugueul 'ueoe splleLuorq) ralsrs eqt llnd uaqla;purds rr1o1rlu oqt Jo sa)roJ 6u;lgnd aq1'ft7-4 an6/'7 aes) reqla6ot splleuolr..l)

ratsrs aqt 6u1p1oq xe;duot utsaqo) aql Jolrunqns e '[)fS a^eal) ol asetedas s,l/\olleuun)as Jo uorl)nrlsep eqI'elels e^tDeul

ue ur eseredas sp;oq {;leutou q:rqm'uun)os

ro uo!])nrlsap pue uoqte;I1;nb;qnaqt ot speal gz)pf ^q )/)dV Jo uorte^tl)paql')/)dV aql ̂q uolletpdes plleuorqr

-ralsls ,o uotletltur eql 99-41 etn6l3

'sllec eseql q es"qdPuE;o Surup aqteulruJalep lsnu 'uv\ou{un }ed sE 'rusrueqserrr Jer[o euos 'sofJque ^u pue 8oqdlree go sllac eql puu slsee.{ sE qJns 'sllec aruos ur lulod{Jeqc dlqruasse apurdseql uo puadep lou seop eseqdEue Jo 3uII'4] IEIuJou aqf flSugsrrdJns 'esEqdeue

pue uorleJEdas ppeurorqc-Jelsrs aJnl?ueJd sesnef, rusruBqJeru ]urodlseqceqlJo uonrqlqul ptueulJedxg'ral?l salnuFu 0z ]noqP surSeq esuqdeue puE 'elp

-urds aql uo paluerJo-lq saruo3eq JIBd prleruoJqJ Jelsrs ls?l eql JaUp sluaruorusurBeq slleJ eseql ur urrnces Jo uortf,nrlsep aql 'asuqdEuE

Jo Sullup lelurou eqlseurruralep ]urod{JeqJ dlqruasse elpuds eql 'slleJ Jl}eluos uErleluluelu uI

'llf,dv-Ozrpf, ltqtqut puu pulq uec zpBIN tBqt os 'zpeIN

Jo uonPruJoJuoJ eql uI aSueqJ e sazfplsc leql au^zue uB eII slJe eJoqcoteuplpeqJeueun aq] lBqt sr 'zpew

Jo ses.[lBue lBJnlf,nJ]s pelrelep uo fluEruud peseqzbllqrssod Suluadde auo '(gt-ZI eJntIJ) uorlrurg ol tulodlraqc ,{lqruassealputds eql JoJ parmbar aJp pue seJoqJoleupl paqJEllPun ol pellnroeJ ere 'ZqDW

Surpnlour 'suralord IpJeAes q8noqrp teelJ lou sr IpuBIs slql Jo slseq Julnf,aloru

eqJ'llar eql lnoqSnorqr &I^llce 3/3dv-Ozrpf, srlqHul lsql IeuSls elqlsnJJlp eetereua8 lv\oqeluos seJoqJoleupl paqopDu ,tlalerrdorddBur ]eqt lq8noqt s1 t1

'JnJJo ol uorleJedes plleruoJqo-Jelsrs Surmolle 'pelorueJ {oolq qql $ peqoeue

.{padord sr aJoqooteupl lsel aqt uaqrvr,(yug 'uo11lsue4 eseqdeup-ot-aseqdelaruaq] s{rolq snql puP uor}?Arl3E 3/ldv-Ozrpc qcolq leql IEuSIs arrpeSau u }nospuas alpurds eql ot paqJeDE dlradord lou sI teqt eJoqJoleupl duy'aroqcolaugeql le 'uorsuel ^lqrssod puE 'lualuqcEils elnqnloJ3lIu Jo qlSueJls aql sJolluoulEqt usruBqsaru Josues P uo spuedep luod{3eqJ ,{.lqluessE alputds aql

'elpurds JIlolFr eql uo palualJo-Iq dllcarrocaJE seruoso(uoJqo IIe glun aseqdeuu Jelue lou op sllal leql seJnsue ruslueqJelulurod{JeqJ aqJ'uoqrsupJ} aseqdeue-ol-eseqdeteu eq} q8norqt uolsserSordqcolq pue lueluleaJt Snrp aql [q palEAI]rB sl tEr{} rusrueqsaru lulod{raqr /qq-ruessu alpugds pJo uoupcglluapr aqt ol pel uolle^Jesqo sIqI's.{ep uela ro srnoqJoJ srsolru ur sllac ]sarJe '(gI Jaldeq3 ur passnf,slp) eupsulqutrr Jo auIcIqJIoJ seqsns 'saFqnloJJrrrr ezlrqElssp lPql sSnJC'alEJedas ol spueruoJqc Jelsls sacnpulleq] IBuBIS O/OdV eql SulllE1!\E '1noqe 8u1pso['aleld aseqdE]elu aql uo pau8rlesaruosoruoJqJ aql qll,\,\ 'aseqdulaur ur srsolru Jo Jpq lnoqB puads dlensn s11a3

lu!odl)eq) (lquassv elpulds eql: uoller edes plleuor q)-Jalsls l)ol g seurosoulol q) peqle$eu n

eseqoeueeseqoele[rJ

aseJeoese^!pe

J/JdV o^rlleul

0z)pl

ap{1 1;a1 eqt:/ [ ra]deLl) 980 t

'a)eJrns lla) eql prPMot'Jaqto q)ea

ujorj ̂eMe salod aqt llnd ot salnqntor)rru

lellse uo ])e alod alpurds q)eareau euerqureui eulselo aqt o] peq)eue

suraloJd Jolour pue'uPde salod oMloql qsnd alpurds lerlua) aql u! raqloue

ouo lsed salnqnlor)rur lelodralur aql

,o 6urprls pup uorlp6uola aql:g aspqdeueroJ olqrsuodsor eq or rq6noqr eje sa)JoJaleredas oMI'tJede aôLl-i salod alpurds

oMl oql'B eseqdeue ul 'xnu alnqnlor)rnlprPl aloo pue salnqnlor)il.! aroq)olaur)i

Jo uorlezuaur^lodep aql uo spuadap vaseLldeue ur salod aql preMol ]uauJaôur

aurosourorq)'slle) ue!leuureui uleseqdeue le splleuorq) Jals!s aleJedes

leql sa)ro, roleur aql gt-l I ain6rl

salnqnloJ)il.rJ relod +o pua snld

ueoe uiaql aôu_r ol saloooql uo ̂lparp sl)p e)ro+ 6ur11nd e (7)

:elebuola osle salnqnloJ)r.!re;odralur aql :Uede salod aq1 qsnd og

se;od alrsoddo uroJ+ salnqnlor)rul re;odtaluruaaMlaq pale:aue6 sr a)ro+ 6urprls e (!)

'leluJouqe aJP esEqdPuu Jo JoIAPqeq

eFqnloJsrru puP sluauaôrrl etuosoruoJqJ eql ]nq sJncJo dlerauaS uoIlEJ-edas pupruoJqc-ralsls-eldtuexe JoJ 'll3dV aql ^q pazluSoJer lou sl leql urroJ

tuutnru P Jo uorlsnpord eql fq-peluanard sI uollsnJlsep ullf,l.J-IÎ JI 'sullJIJ

Jo uor]f,nrtsap luepuedap-3/f,dv aql tuo4 sllnser sllac lsolu uI qslqM 'saleJlsqns

Ipl Jo uopellroqdsoqdap eql uo spuedap aseqdeu? le(uJou E Jo uouelduoJ

aqt ^lluulJodrur 1so141 'g aseqdeue uI uolte8uole epulds pue V eseqdeue ul

sluarueôru euosoruoJqs lJeJJoJ aJnSUa OSp StUSIuBqJeIU Jeqlo 'y aseqdeue 3osluerueôru auosoruoJqc eql selElllul uoIlBJPdes ppPlxoJqc-JelsIS qSnoqllv

'uBdE selod eq] ilnd xel-Jo3 IIac aql o] spua snld eFqnloJsllu IeJlse JoqJuB lEq] sJololu uleufp 'uoglppe

uI 'ilpde salod eql qsnd 'sepqnlorJllu rulodJelu eql Jo spue snld Sqdd€lJeô

aqt {uq-ssoJJ qclq a 'sutatord Jo}otu g-uISauDI pelJerlp pue-snld '(Og-Zf ornBIC

ees) srsolru f.Fea ut seluosoJlual oMl eql aleredas lEql esoqt ol IeIIIurs suISIue-qJaru ue^rJp-Joloru uo spuadep g aseqdeue 8u1rnp uollPJEdas alod apulds

'aJollf,olauDl eql le peteJeueS sesJoJ uro4.{.preurrd slpsar slles 3I}EIuos

elEJqaue^ pue lsee.{ uI luelueôrrr seeJeq A 'aldurexa JoJ 'x[lu eFqnloJJllu uo

f.lureru spuadap luerueloru euosoruoJqJ (sllec Jluo^Jqrue uI:sedfl IIeJ iueJeJJIpur serJel aseqdeue Surrnp secJoJ oml eseql Jo eoueuodrr ellleleJ eql 'sJnof,o

uolluzuaurrtlodap pua snul(x aJewv\ 'alod elpulds eql pJBâol sepqnloJ3lru

eq] Jo luellleôlu pruâelod aql sI qJIqM 'xnu eFqnlorctur Lq paprzrord sl puoJas

aql'a1od eql prEMol saôlu aroqJoleupl eql sE pue snld aql le sllunqns ulnqnt

Jo ssol aq] q slFSaJ qJrqm 'eJoqJolaupl eql lP uouezrraur(1odap eFqntoJclru,,i.q paleraua8 eoroJ eqt sl lsrlJ eql'reIIJEe paqlrJsep secroJ pJemalod rofeu o,vr1

eql Jo uopuulqluof, u uo spuadap y asuqdeue uI luelueloru eluosoluoJqJ'@V-Ztarn8gg) tJEd? eouelslp e{uos palolu eAEq seuosoluoJqJ relq8nep

eql pue paleredas eêq sppeurorqc Jelsls eql JaUP sur8aq qolqm'sellaslueql

salod apurds eq] Jo uorleredas aql sl 'g asuqduuu se ol paJJaJeJ 'puoces eqJ'selnqnloJJrru eJoqooleupl aql;o Sulualroqs dq patuedruocce sI qJIqM 'seluos

-oruoJqo eql Jo luaruelotu prea,ralod pplul aql s1 'y aseqduuu sE ol paJreJeJ']sJg eql 'sassacord Sulddepano pue luapuadepul oâl .{q anou seuosoluoJqcar47'uotrySatBas aurcsouorq, pelleJ-lleJ aIDJo salod alrsoddo o] sJalsls aqt 1ndo1 alpurds Jpollu aq] Jo seJJoJ eqt s^ ollu qJlqm 'uotle.redas plleruoJq3-Jalslsol speel aseqdeue Jo tasuo aql tB uolseqoc ppeluoJqf,-Jelsls Jo ssol ueppns eql

g pue v aseqdeuv ut ele6ar6es seuosourolq)

saioq)oleurl 1e I;ureupelerauaE sa)ro] :salod olseuosourorq) ralq6nep ;o

luauaôu :solnq nloJlr l..lreJoq)olaur) ;o 6utueyoqs

680r

8 ]SVHdVNVV ]SVHdVNV

stsoIW

uI uorlBSeJ8as prleuorqc-Jalsrs a)r.I 'seuosoruoJqo snoSolouoq eqt elEBaJ-3as ol llroq :srsoraru;o ualqord IBJlueo aql sallos uorsrnlp f,lloleru lsJrJ aql

'(ZV-Zy a.rnt;g) Soyouroq qcee Jo splleruorqrJalsIS eql sale8ar8as 'srsolr{u

IeuorlualuoJ e{[ 'II srsorel4l '(sprlpruoJqJ Je]srs3o rrcd pelulldpq8u ego pasodruoc qrpo) sSolouroq aq1 sale8ar8as I srsorahtr T1pue I stsorau pallpJ 'uorle8ar8as euosoruoJqt Jo spunoJ omf z(q pamolloJ 'asnt4d

S irrcnru palleo 'uorlecudnp auosoruoJqJ Jo punoJ e qlp su€aq srsorel4'17 raldeq3

uI pelep aJotu ur passnJsrp sr srsoral4 'srsolrur Jo asoq] qlyvr aredruoc daql .uoq

uo srseqdura uE qllm'srsoreurJo uoqepBar pue susrueqJeu Jrspq aql Jeprsuooe^ 'areH 'lenpr rpur ^ eu p uJoJ ol ppualod aql qlp\ aloBfz ploldp B ruroJ o]asq 33a pue urads E uaq ^ spua aycdc anrlcnpordar aql 'sagcads aseq] uI 'sarcads

]soru ur urrads pue s3Ba-satawaB palleo slleJ anqcnpordar pazqercads oluralpuueJaJJrp slac proldeq aq] 'srusrue8ro .{uuu uI 'atuosoruoJr1J qcpe go ddoca18urs e dpo Sulrfuruc s11ac pto1darl sacnpord qJIq { 'srsonru pellpo ssaJord uors-IAIp Jeelcnu pazqercads e uo spuadap uorlonpoJdar pnxag 'luared qJBe ruo4auo 'aruosoruoJqJ qcea 1o

's&o1owot1 ro 'sardoc luaJaJJrp dpq311s o^ ] uruluoodaq] 's1 wrql pto1dtp.{leraua8 are sursruu8ro eseq} Jo sllar aqJ '(IZ raldeq3 urpessncsrp) luared reqtra ruor; }cuuslp dlecrlaua8 eJe leq} Surrds;;o a}uraua8 olxur sluared omlJo saruoua8 aq] :dgenxas acnpordar sursrueBro cqodrecna 1so141

uo!lrnpordaulenxas u! pa^lo^ul uotsr^to teal)nN Jo LuloJ let)eds e sl stsotaw

'aseqdolat pue aseqdeue ur pello^ur selerlsqns {pl Jo tasqns e saleldroqd-soqdap qJIqM 'rtrpJ pelleJ aseteqdsoqd e;o uortelrlce aq] uo spuadap srsotrru3o uoqalduroc aql 'aldruexa ro; 'lseart Suppnq u1 'saseleqdsoqd yo uorlelrlJe uo,{1ar osp silac euos 'sllal lsotu ur alqrsuodsar r(1umru sr-uoncnJlsap uqcdc uor;fyreurrrd Suppsar-uolle^r]Jeur 1p3 q3noqtiv 'q]oq ro 'sasuleqdsoqd;o uorl-eAIllB eql 's{p3

Jo uorlelr}Jeur aqt dq para38rr1 eq plnoo srsollu Jo uoqaldruoceql pup suoqel,{roqdsoqdap asaqt 'aldrcurrd u1 'aseqdola} ur raltnu ralqSnepJo uorlEruJoJ-al aql pue rtlqruassesrp alpurds ro3 parmbar sr suralord eruEs esaql;o uo4e1,{.roqdsoqdap aq} teq} Sursrrdrns tou snq} sl }I 'slsolpu dpua ur u,u.op-Ipalq adolanua Jealcnu pue 'uorlesuepuoo euosotuoJqc .{yquasse apurdssalourord 1pO-IAI ,(q suralo.rd snorJEAJo uor1u1,{roqdsoqd leqr JarlJEa ^ es elA

'olv\l Olur uorsr^rp slralalduroc ol ilac aq] JoJ sr surerueJ luql IIV'alaldruoc sr srsolrru pue 'paleeJc ueeqseq snalf,nu laau V'erunseJ ol uorldrrcsuerl aua8 Surrvrolp 'alels aseqdrelur JIaq]olut panue8roeJ eJp saruosoruoJqJ crlolrru pasuapuoo eql pue 'spuedxa sneltnuaql 'su1alord JEalJnu ur drund saxalduroc arod aq] 'pauro;-ar seq adolanuaJEalJnu aql eJuO 'unlnJrleJ cnuseldopua aql qllllr snonurluoo sauocaq ure8eacuo adolalue aql pue 'sruJoJ-al purruel reelJnu eqt 'edolelua eql olur peluJ-odrocur are saxalduroc arod JEelonN'adolanua JealJnu alalduroc eql uJoJ-eJ01 aJselPof, ueql pu€ saruosoruoJqc Jo sJalsnlc asolJue ,,tpred ol asn; sluaurSer;euPlqureru eseqJ 'sauosoruorqc pnpr^rpur Jo ef,EJJns eqr qlIM elersossB sluaru-3er; auerqrueu JpalJnu ,{1pp1u1 'adolarrua JBelJnu eql Jo uorle{urog-ar aql dqpe^\olloJ 'alpurds JIlolFu aqr3o.{lqruasspsrp eql sr asuqdolal;o tuala roferu lsrgaql 'IelJnu ralqSnep;o rrcd e olur peBelred are seuosotuoJqJ Jo sles oml aql'slsolrurJo a8uls pur; aq] 'asuqdolel uI 'lleo eqlJo spua alrsoddo 1e sdnor8 pnbaoml olur pale8er8as elpq seuosoruoJqc ralq8nep aq] 'aseqdeue

Jo pua eql dg

le ral)nN relqbne6 ur pa6el)ed erv seuosoruorq, O3|Joqir?':*

'ssacord aql u1 qfual aseqdelaru aql sarup 9I ot dn o1 ale8uola salpurds rraqlpue 'aseqdeue lE saruosoruoJr{f, aq} a}eredas o1 g aseqdeue esn Llrerurrd uoz-olord ureueJ pue slsear(go salpurds aql ']seJluoJ ur lqfua1 aseqdelau slr eolmllnoqe sr alpurds eql uaq \ sdols pue y aseqdeuu rage.,ilpoqs sur8aq g aseqdeue's1ac uerpurueu uI 'adr!

lac eql uo Supuadap .{pear8 dren uoue8ar8asaluosoluoJqc o1 g aseqdpue pue y aseqdeue Jo suorlnqrJluoc aluelal aqJ

a1>(1 1;e1 aqI:/ [ raldeqS 060 t

sSolouoq eqt fqeraqm '&uurud Solouoq qtyvr sur8aq ssacord slqJ 7 asnqdotdrc asaqdotd cu.onlil palpc porrad e ut 'aseqd S JIloIeu JaUE sJnJso teql ssacord,tqfual pue xalduoc ,{1qe4.reruer e Lq sSolouroq ueamleq uro; saBelurl

'llar aqtJo spua alrsoddo ol s8olouroq aqr pdo1 alpurds aql Suuvrolp '1 aseqdeue Jo tesuo aqt le peloruar s a8e1u11 Solourog'sa1od alrsoddo o1 paqcel]e rred e ut sSolotuoq aql qlIM'alputds slloleru lsJU 3q]uo paluerro-lq eq o] srrcd Solouroq eqt ̂ olle sa8u4u11 asaq;'s3o1o{uoq ueemleqsa8e1u11;o uolletuJoJ aql uo spuadap I sISoIau ut uone8ar8as Soloruoq 'slsollur

s11e: ralq6nep proldrpsa1eu.re6 pro;deq

'uorsrnrp a;6urs e ur a1e6ar6assplleurorq) ratsrs eql pue'dn rred

lou op s6o;otuoq 'lserluo),(q 'a1>,(r

lla) )rlolil.u e ul (8)'sprleutoJq) lalsrsaql sale6ar6as 'll slsoreLu 'punor

puo)as aql seoreqM's6o;or.uoqeql sele6ar6es'l srsoraur 'punor lsrU

aq1 'uor1e6ar6as aurosor.lJoi q) ,ospunor oMl {q pamol1o1 sr (aseqd 5)rlorau) uolter!ldnp auosouolq)Jo punor a;6urs e q)rqM ur uorsrnrp

reel)nu Jo lrlroJ P sr srsoraw (v)'a;rI>

;;er )llollur aql pue slsolarrr

1o uoslredurol Lt-LL an6lJ

r60t

m

m

m

a)

-fr

3SVHdVNV IVsor-LVrAouH) ulrsts

lo Notrv9tu93s

l'lcNtds lHl_ NoA]tvnotntcNt dn lNl'l

sllAosot/\rouH)c3_LV)t'tdnc

II ]SVHdVNV.LVsor-LVt/\louH) u3tsrs

lo Not-tv9lu9ls

scoroulou I crrv)rldnc lo 9Nturvd I

No[vrtra;u vruc I

6o;ou.roqleuJaleuJ

6olouroqI euraled

ii{iliitii (v) stsofl l(8)

slsoJtJ

'patrJroJ

aq o] slaôssol) aldtllnul lo, lensnsr lr pue '6o;or-uoq laqlo aql Jo plteulolq)reLllla qllM raôssol) p urlo1 ue) 6o;ouoq

p Jo sptteurorr.l) oMl aqt Jo raqtle lnq'e;duexa s!qt ul splleuolq) lolstsuou omluaaMtaq palln))o seq lalossol) a;6urs y

'sqr6ual arlua ltaql 6uo;e pe1>auuot,(;tq6rr are 6o;ouoq q)ea ut sptleuJoJq)

ralsrs aql'stsoltLu ur sy.s6o;ouroquaamlaq talossol) V gt-ll aln6rl

JOAOSSOI)

s6o1ou-roq

'aseqdolal ur srsollu Jo uo4aldruor eq] JaUE ,(proqsspua puu espqdpue ur sur8eq srseuploldJ 'slleJ

Ieurue lsoru uI 'ralJnu eldqlnuoJInbJp,,(qeJeql pue srseuDlol-{J }noq}ll\/\ slso}Iur o8Jepun 'sllal alJsnr.u Ueeq pupsa$coledaq uerlerurupru etuos pue (Jelel pessnosrp) sodrque qtqdosotq [peasP qJns 'sllal eruos q8noqfp 'srsolrur drarra saruedruoJce srseuDlol,{o 'llat

IEJrddtB uI 'tuseldol,b aqryo uorsrlrp eql 'slsauplolb sr a1c,b IIaJ eq] ul dats IpurJ eql

srsr N r) f

'Dpnu proldaqlo uollautot aq| w Suulnsar'uolla&atBas ataosow-ott4c to spunor ory [q pamo1lot s1 uopacl1dnp auosoruoJtlo to punot a1&uts a qur4mur uotsrnlp na1cnu lo tutot pazl1anads a y srcorcW 'adopnua Joalcnu aqL to uoryau-tol-at aqt pua alputds ary to /qqwassastp aqi Sutpnlcur '|slsotlw alalduoc 1ot41 sluanaaqt rct pannbat v s1aBn1 4pJ lo uorln1ttor4dsoqdap 8ut11nsat aWI'qpJ-W lo uo!run!1-Jam aqi sru4i puo uouJnltsap u17c[c satowotd os1a 2p4y nqnSot sp!]DworqJ Ja$$aqi ppq ryq1 suraptd ar4i to uournr$ap arq sawlnuns Llcllm 'J1J1V at47 tq paraS8ujv asaqdauy [Tquassa a1putds a1o71pcat o7 sautosouual asn osID s77ac tunyl 'tatta n7-odtq a oiw salnqntotJtu anuDBJo oi sunrctd rolout lo [.11pqa aq] uo sa Tgam sn '&qtq-rys pua uouuapnu alnqruoJJlw IOJII aruruwlls 01 sauosolaotqc u1o11ut lo ,ttutqa aqluo fiaBn1 spuadap slp) Iawrua w uotluturot a1puldg 'a1putds ar4l to sa1nqntoJllut otalad ptloutotq) ra$$ aqi to tuawt4callo n1odrq pua 'alpurds ctlollw aqi to lqquassD'uolLosuapuoc awosowotqc Surpn1cur 'srsolrw [pna lo sruana aqt staSStu 4p)-h[

fteurun5

'srsolrru ur seop U sB qJnu-uolleJedas ppeuroJqc-Jelsrs pue a8e,realc urseqoo f,rJatuorlueJ sraSSul eJoJaJeql uorlplrlJe :)/ldv'11 aseqdeue Jo lesuo aql lV 'JI srsoreu ur peôrueJ ale I srsoraru ur eJeruoJlue3aql ]e a8elealo ursaqoJ {Jolq leq} srusrueqcaru aqJ 'II srsoreru ur alpurds aqluo uo[e]ualro-rq ]JarJoc rraq] 3uu,ro1e 'I srsoraur lnoq8norql seJetuoJlueJ JraqltB palu1 urpruar eJoJaraqt srmd pqeurorqo-relsrs '(17 raldeq3 ur pessnJsrp)aseredas ruo4 pelJelord sr uor8ar ]uq] ur urseqof, asneJaq I srsoraru ur pe^Bel3unurcrueJ seJaruoJlueJ eql JBeu saxaldruoc urseqoo 'rana.uoq 'srsolnu o] lsEJ]-uoJ ul 'ftV-LI arn8rg aas) sruJe aql Suop a8enealo ursaqoc pue 'uollelllJe aspr€-das 'uotlcnrlsap urrnJes o] speal qlql 'uorlelrlce

O/f,dV uo spuadap I srsoraruur uorseqoc tuJE Jo ssol aql 'sarcads lsoru uI '1 aseqdeue Jo lesuo eql lE uoll-eJedes Solouoq sraSSul aroJaleql sruJe prleuoJqr-Jalsrs ruo4 uorseqoJ Jo ssolaqJ'(8t-ZI arn8rg aas) uorsaqoJ prleruoJqJ-ralsrs Lq palJauuof, eJe spneuoJqcJelsIS eql Jo srure eql esneoeq .,tpo raqlaSol s;rcd Soloruoq ploq sJelossoJ3

'srsolrru ur are ,(aql se alpurds aql uo palueuo-rq eq uPc srrcd pqeuorqcJelsls aq] II srsorau ur ]eq] os 'I srsoraru JeUe pasJaâl aJe srusrupqJaru esaql'WZVrc ern8rg aas) alod auo ]sn[ ot seqJp]te ]eqt l1un Surpurq-alnqnlorf,rrua13urs e olur pesnJ l oqaruos aJp seJoqooleuDl Jelsrs o,\/\l aq] taâl oq 'I srsoreuruI '(6t-ZI arn8rg aes) srsolnu Surrnp paprolp flpurrou sl lueruqJe]le Jo addlsql'a1od aruus eql ol qcpuu Soloruoq e ur saJoqJoleuDl Jelsrs qloq lpql sarrnbaralod apurds e o1 Soloruoq eJo ]uaruqJepe aq] snq] pue 'sppeurorqJ relsrs pa{urTdpq8p o,,t,r] suretuoc Soloruoq qceg 'alpurds f,poletu lsJrJ eqt o] peqceilB erpsrrcd Solouroq eql ueq^\ pe^los aq ]snu uralqord Jrloleu dlanbrun Jeqlouv

'uorsr^rp Jllolalu ]SJIJ aq] sJatua IIaJ aq]ueq^ pa]f,auuoc,(1pcrs.,(qd are rrcd rirana ur sSoloruoq eql lur{l Surrnsua tredSoyoruoq qJpe ur srnJco sJalossoJJ eseqlJo euo lseel lV'(97-ZI arn81g) prleru-orqc sno3olouoq B Jo VNCI eqr qllm snonurluoJ aruoJeq ol JeAo sessoJl prleu-oJqJ B Jo VNCI eql ereqm 'stanossoJl palpc sa8ueqcxa VNC pcordrcar ol peelsluane eseqlJo aurog 'sSoloruoq aq] uaamlaq sluala uorleurqruocal VNCJo sJaq-runu a8rey ur Suqpsar 'ppetuoJqJ Jalsrs qJea ur suorlerol IeJaAas lE paruJoJ eJBsTearq pueJts-elqnop VNq :rred Soloruoq qJEe ul splleruoJqc Jalsrsuou ueemlequorleurqruoJar snoSolouroq ,(q aculd ur pelJol ueql eJe sa8e1u11 Soloruog'sSolouroq oml aq] ur saJuenbes VNCI dreluarualdruoJ ueeâleq suorlcp-Jalur Jo llnsal e se ,(Ireturrd 'snalJnu aq] ur Jeqlo qJPe ol esols eôu ,{1unper8

alrI1 11a3 aql:/ L raldeqS Z60L

('rnlnl olqso^Jo(salrno> '1 1rX er-u615 urorJ uorssl.llad qlm'9/6 t 'O6Z-6LZ:tg pS 'ury'lassa) 'D'U pue suree8 'M'H uroJJ 'B) 's;;el ralq6nep lua)seu aql,o xauo)

aql ut suteuar lsal aql:os auop seq utl)e aqllo auos {;uo'6uu alll)er}uo) aq} ol patnqu}srper seq ;1 ursor(tu alqlsln aqlro 11e seataqtrtr'(uaa.r6)

ll ulsolul pue (pal ult)e loJ paulels eqaoulp ploul aurls 6urprnrp e ;o sqderborrrul e)uo)saronll ()) 'lla) leLulue 6ulplntp e Jo Molln, a6eneal> e

;o a6pa 6urrvror6ur aql 1o qder6orlul uoll)ala uV (g) 'llar 6urp;ngp e ur Morrn; abeneelr aql ;o burmerp V (V) '6u!, olll)elluo) aql OS-Z I orn6ll

urn 0tun s'0

anor:n1 abeneagr ur sluaurelt; utsoi{u.tpue urpe lo 6ut: eltlre:luo:

alpuros lerlue) uroJ,selnqnlorlrnr relodralut 6ututeua.t

(s) ())

(v)

(';y eu615 r.uot; uorssrlulad qtl1l' 9 L6 L' 062-6 LZ:Vq' DS' wV' pssa)'9'U pupstleag'M'H lrtor;') pue g) 'uor1err1u6eu

raqbrq te MornJ e Jo a)e;rnsaqf ()) 'll qteaurepun 6utl a;tlrerluol

aql Jo ̂lr^rl)p aql ̂q pasne) sr auerqLuaur

lla) aLll Jo 6urrnorrn; aql'abre; ,(;;ensnunsr lla) aqt sP'pauuap llaM pue snor^qoI;;erradse sr Mornj a6eneep aqr'66a

6or; 6urneelr e;o qder6orrrr.u uort)ele6uruuers uorlerr;ru6eu-nlrol srql ul (g)

'pJeMur auelquau aql sllno uorl)erluo)ltaql leql os'uMoqs se potuauo

ore 6uu alll)erluo) aqt;o segpunq ursor(ru-ulpe aql (v) 'srsaurl0y(1 69-21 an6t7

urrl 9Z

aprôJd tEql suletoJd Jaqto snorelunu sulPluoJ oslE qJIqM'(09-ZI eJntlf) tuIJeIIlJBrluoJ Sutlqruasse l.lprder aql ul elelnlunoJE ol ulSeq II ulsoltlu puE ulllB'aseqdeue ur elpJedas sprluruoJr{f, ralsrs eql sV'paseeleJ eJE s}ueuEIIJ II ulsol(rupue 'sazruu8JoeJ ullf,e aql Jo qonu :elqulassEslp ulsoful puB ullJe Jo SÊJJEaseql (srsolrru ralue slleJ sv '(9I raldE[{l uI pessnsslp) snq{ ssatls pelIBJ selp-unq orruseldo{c a8rel ruJoJ oslE r(aql's11ac auos uI'euBJqlualu Prusuld eq} 8ul-f.papun >lromlau IeJruoJ e uJoJ slueruelg ulsofiu pue ullJe 'q1ac aseqdrelul uI

s!seu!lov()Jo, e)tol eqt aleJauag 6utu elll)etluo) aql u! ll ulso/(w pue ull)v

'uopaldluoJ pue 'uoluasul eueJquelu 'uopoeJluoc 'uopep

-rur-se8uls JnoJ ur JnrJo 01 peJeplsuoc eq ueJ slseuDlolb 'snql'sgac ralq8nepaql uee^\leq de3 aqr pas uorsnJ pue uoluesul auerqueu'palalduroc sI uollssJl-uo3 3uu uaq |uorsr^rp Jrruseldolr(c seluEdIrloJJe leql EeJu aJeJJns uI asEaJ3ulaqt JoJ salusueduoJ eueJqrueru Jo uoulppu sIqI '3uu aql ol luef,Efpe eueJq-(uaru l eu suesur eueJqruaru eurseld aqr qtIM selclsa^ rPlnlleseJtul Jo uolsnJ'aru11 arues eql lE 'pue 'slJeJluos,,tlpnper8 Surr eq1 '(I-ZI

lauPd osle ees :6V-ZteJngIC) eueJqrueu eruseld eql qtuauaq lsnf selquasse 8uu eql 'eseqd?ue 8ur-JnCI 'suralord,'t-rolep8ar pue IeJnlonJls l(ueru pue tslualuelg II ulsodru 'slueur

-plrJ urlse 3o pasodruoc ,{lqtuassE Jllu?udp e--8utt anpDJluoJ aq] s ssacordslq] 8ul{lJapun eJnlcnJls aq} 'sato^JeJne rElnlleJlun ûeur puu silec plulue uI'o^\l ur ilac eql sepFlp dlalalduloc 1I Ipun IIas aql punoJE speards puu suedeep,(prder r oJJnJ eql 'aJBJJns

IIeJ aql uo 'moJJ( aBnnaa1c Jo ta)cnd P Jo ecue-rEeddp ueppns eq] sl [e] plulup up uI slseuploil.sJo eBuEqJ alqlsnlsru eqJ

urn 002())(8)

€60rsrstNr)ol^f

('su!llnw'w'rJo IsaUno) '8 talqang-lq)alqlV raluang Jo{salrnor 'V) 'alputds

lpJtue) aql Jo suteulalaqt 6urureluot tuse;do/r;o puelts

urqt srql ̂q paq)eue utpurol slla) telqbnepaqt lnq 'alaldulo) lsotule sr a6enee;1

'lle) lpultue 6u;prnrp e;o Ipoqprru

aql ;o qder6onru uoJl)ale leuotluenuo)V (g) 'slle) relq6nep oml eqt sutof lltls

,(poqpru aqt j6utptntp jo ssato.rd aqt ul lla)leurue pornllnr e;o qderbontul uoJl)ola

buruuers V (V) 'Ipoqplur aq1 19-4 g arn6r1

lB pele^rlJE sl voqu 'a8u^BelJ

Jo ells aql l3 3uu alllJeJluoJ eq] Jo uopcuru puE dlq-Iuassp aql slorluoJ '(g-gI elqeJ eas) fgure;radns spg aqlJo esEdJC 1euuse

'.Vol4q

6u;g a;;perluo) aqlJo uoll)erluo) pue {;quessy sta66ul vor{u Jo uotle^lpv le)o'l

luanbasqns aqr ur alpurds eql ]uarro or sdreq rpql xelror eql uo tr#Tt;1t:i5:,teur ,(aqt eJeq ̂ 'lleJ qopa Jo eueJqrxeru uuseyd er{} Jo aprsq eqt uo urerueJuago Lpoqpru IpnprseJ eqt Jo slueuodr.uoJ eqt Jo euros dlaralduoc aterpdasslac ralq8nup aq] JaUV'(I9-ZI a.rntlg) Ierrelpu xrrleru esuep e ulqll a raqtaSol,(pq8p palred 'euozpnu alpurds aqt Jo saFqruoJorru reyodralur lalpredrlueaql ruor; pelrJep eJnlonJls uleloJd a3ru1 u 'alpurds

IeJlueJ aqt Jo suletual aq]suleluoo pue slleJ ralq8nep omt aqt uaalqeq Jeqlet e se slsrsred z(poqpnu eq1'dpoqppu eql ruJoJ ot sr oJJBu raoJJnJ aBerrealc eql Jo aueJqruaru euseld aql se'spua a8enealc uaqr!\ JeqlaBolp qlpvr pasuadsrp dluur; sr Surr alllceJluoJ eql

's1saup1o1b Surrnp [1enu4uoc sa8ueqc luaru-a8uurre reql pue 'crureufp [UBq aJe 8uu eql ul slueuel.rJ urloe eql 'elosnru uru1lcp eIIFn'JaAoaJoIN fppeals eseeJJap suletuoc ll slueu€lrJJo Jaqrunu eql puuerunlol Iulo] slr leql 3u4se83ns 'ssaDlJrql erues eq] suplureu ll 'slJlrlsuoc 3uuaqt sV'qled str ur pauasur $ tuql elpaeu sse13 aug e pueq o1 q8noua a8rel acro;e suexe 3up aqt 'sur8aq uoqJeJluoJ eJuO 'oml u1 ruseldoltJ aq] sepwp leqleoJoJ eql aleraua8 ol tJeJluoo slueuplrJ 11 ursodur pue urlJe go sr(erre Surddul-JeAo aqt 'aseqdeue reUV'(gI raldeq3 ur passnJsrp) sruauelg uqce peqouprqun'reautl

3o sr(erre lalpred ;o dlqruasse aql eleelcnu lpql suraloJd uttutol uospuadap qc1q \ 'sluaureyrg urlop llteu Jo uon€ruroJ leJol eqr ruo4 lred ur sllnseJ8uu allcerluoo eql yo dlqurassy .{lqurasse Surr ur }srssu Jo }roddns IeJnpnJls

urrl IaueJqtreur eurselolerJeleur x!ilet! esuep

turi 0l

a;:I1 ;1a3 aql:/ t raldeql V6OL

'bulJ aql Jo uoll)PJluo)6ultoruord {qeraqr Ilrnrlre pue

uorlpuro, luauelU ;1 utso{tu sa}elnuir}slr'l)ou se q)ns'saseurl uralord pale^tl)e-oqg 6urlenrlre Ig '6urr epllertuo) aql u!sluauelu u!l)e Jo (;qLuasse aqt salou.rold

VoqU pale^tire'suttuloJ 6ulputq,(g 'al!s abeneall arnlnJ aq] ]p pasn)oJ

sr voqu lo LuroJ punoq-df9 a^rl)eaql'uralord dVDoqU e {q palenrlreu;

pue uralord llgoqU e,{q pelentlresl VoqU

'sasedlg {1gtue1 oqg raqlo alrl'VoqU asedlD aql {q 6u1r alll)erluo)

aql;o uotle;n6ag gg-4 1 atn6t1

uorlel^roqdsoqdu r eq:-1q6r;

urso{u.r ,fuo1e;n6ar

(l)ou 6urpnl)ur)seseurl pole^rlle-oqu

\\\

s60 t

voqu a^rpeul

{pO Jo uouellJoqdsoqdep esneceq eurl} llarJoJ aq} 1B sJnJf,o oslE slseuDlol-{J'srsolrru etEI ur srsauDlol/tc 3o Sunup lseJJoc eql ol salnqlrluoc oslB Iuslue-qJaru slql 'elpulds asBqdeue aql uI eleuISIJo sleu8ls aseq] asnE3eg 'satuosolu

-oJqJ palEJEdas;o slas o/\ 1 eql uee^ leq sJnJco uoISIAIp lEq] Suunsua dqaraq]'sa1od alpurds eql uea^ laq ^8,1^plu uoplsod E lP uolleruJoJ MoJJnJ elBIlIq ]PqlslEu8rs salereue8 apurds eql 'eseqdBue Suun0 'elpulds JIlolIu eql uo puedep

]Eqt slusrueqf,alu lueSale ,i.q panatqce eJu sllal IeIuIuB uI slseuDloldJ Jo Suluoq-rsod pup 8unult ]JeJJoJ aql']es alalduoc P seAIeJeJ IIaJ Jatq8nep qcea 1eq1 3ut-Jnsue fqaraqf 'seruosourorqc ralq8nep Jo s]as oÂ1 aq] uaaM]aq paceld aq lsnluuorsrlrp Jo alIS eql pue taqlo qcea uror; pale8ar8as d1ry eJe seruosoluoJqc Josles o^^l aqt Jaue.{1uo rncco lsnlu sISeuDIo{3 'ace1d }q8lr eql uI pue au41 }q8IJeql le sJnJco uoISWp leq] ernsua o1 ̂aoq sI slsauDloldl uI uelqoJd IeJluaJ eqJ

uorsrnrc Ila) leuruvJo aupld aql au!uralac alpurds )!lol!w aql Jo salnqnlor)lw eql

']xeu ssnJsrp el\/\ sE 'paAIoAuI eq ol rueas apulds aseqdPue aql Jo saFqn]-oJsrru eql qSnoqlp 'alls UOISIAIp aql tE pelEÎlcE ro pezlpcol sI ca3oqu eq] A\oq

lnoqP epll ^ ou)I el 'voqu ol dJc Jo Sutputq puB doc Jo esEeler aql selPlnunlspue elrs uorsr^rp ernlnJ aql lP xeuol IIes aql le punoJ sl qJIqM '(Jacoqu) Jo]lEJa8ueqcxa appoelJnu auluen8 oq5 e uo puedep o1 lqBnoql sLry\oJJnJ e8enealceqt le voquJo uolleÎlf,P IBcol aqJ,'(gt rardeql uI pessncslp) dlc ol punoq

uaql\/\ e^rl3E pue dclc ol punoq uaq^ eÎlceul sI voqu 'sasEdlc Jeqto e{I'I

'3urr ursol.ru-ullJe aql Jo uollJeJluof, pue dlqruasse eql Suporu-ord dqaraql ,{lplrce rotolu pup uoItBIuJoJ luaurBIIJ II ulsoÎu JEIodIq seteplulls

l'Ihlu eqlJo uollBl^roqdsoqd'II ulsol.urJo sllunqns aqlJo euo sl qrlq ^'(3Tnlu)ureqc lq8q ursodu fuolep8ar eqt elel.{roqdsoqd saseur) aseql'(Zg-21 arn8g)

{JoU aseup{ pele^r}Je-oqg eql SupnlJul 'seseulr uta}ord aldppur Supe,rpce.{qsuolloBrluoc pue flqurasse 1y utsodur salotuord ]l pue 'sultuJo; Eurlenrlce ̂,(q uolr-euroJ ]uauBIIJ ullce salotuord U'uorlcerluoc Surr pue 'z(lqurasse 11 ursodtu'uor1-eruJoJ lueruelg uure saloruord 1r araqm 'ells UoISIAIp aJnlru eq] le xouoc IIaf, aql

stslNt)or^)

'ulaLll uaaMlaq uotlelujoJ MollnJa)nput ol xallo) lle) Jo uot6aj ]ua)p[pp

aql Jelle Moqaulos slalsP aq] leqt6u!lsa66ns'sralse oml uaamlaq {errnplu

uor6at aql ut sulloJ s{em;e s;1a> asaqtur MollnJ ebeneap aql satnpord lpql 6uU

af lt)eJluo) eql'^lruoreddV .(poaq/l u)Dpa;) use;dot{t aues aql aJpqs

lle) leuilouqe slql u! lnq-alpurds )ttolrLue Iq pa1ur1 lou ale ]eqt slalse luatefpeoM] aql uaeMlaq osle lnq ,(spDaLlMu)D

no11al) sapuds )tloltul luenbesqnsoMl oql Jo q)ea jo auozpil.! aLll

1e I;uo 1ou rn>to saberreelt luenbasqn5'lla) aql Jo aprs ellsoddo aq1 uo

rn))o ol 6ur;re;'alalduotur sr 6urnnoln;auerqueur aql ,ppaq ssel6 p qtlM lla)

aql Jo aprs auo ol peqsnd I;lerrueq:ar,ust alputds )rloltu aqt,l .;1e> 66a

a6.re; e u1 a6enea;r 1o aue;d luanbasqnsaql uo slalse alnqnlol)!ut ]o uol11sod

aql Jo a)uanUu! aql 6ullerlsuourapluaulradxa uy y9-11 arn611

'slusrueblo

]ualaJ,tp ur 6urften sulstueq)aullualaJjtp aql Jo o)ueuodult aLll

qlrM 'sr.r,isrueq)au aseql Jo uotleutqu,o)e Iq pauru.ralap sr 6uruorlrsod

morrnl teq] r(;altl st lt pue'suotle^tasqoaqt ;1e sureldxa ;apoLu e;6urs o5

'6u1r e;r1rer1uo) aql to 6uruolgrsod eq1a)uanUu! leql s;eu61s aleleua6 a;purdsaseqdeue aqlro selnqnlor)lu,t oql MotlJo slapour luarin) earql gg-4 1 arn6r3

peurol are s11e: talqbnep tnolpue '1ua:efpe {1durs ate leq} soulosol}ua)

oMl eqt uaamleq pue salpurds t;tol;tu {q pa1ur1saurosoJlua) eql ueeMleq qloq slntlo e6enea;t

srsolil.! leluaral)nu qloq

^.,

aleal)nurq e 6urrnpotd'lla)

+o aprs auo uo{Juo sur.ro; mortnrL

lepou.r uorlelnur!ls alpurds lerluer (g) lapour uorlelnurls lellse (V)

alputos aqlsa)elostp lla) aql olul

peqsnd peaq ssel6 e ;;a: 66e 6urprnrp

peaq sse;6

Jo so,{Jqua dlrea aql uI 'alrs leql lE uorlJeJ}uo3 lecluoJ Sunourotd snqt tolpnbeepurds aq] ]E Iurururur sr uorl€xBleJ IecrUoJ er4t 'Iapow uorl2xalal IDUSD s!r4l olSutproccy 'xeuoJ

IIaJ aql le salpunq ursoû-urloe Jo uorlEXEIeJ lpJol aq] elotu-oJd sepqnloJJrru IpJlsB aql 'sadil

IIac eruos ur 'leql sesodoJd Iapour pJlql V'srsauDIo]43

Jo eJnlreJ ur IFSaJ (alduexa JoJ 'stuelnru Dlrqdosot1 uD sureloJd eseql Jo uoq-cury eqt ur stf,eJeq '(99-ZI aJnBIfl VoqU ale1nrups deru teql suratord Surpnlcur'sutalord Surpu8rs snoJeurnu qlIM aluroosse aypurds leJlueo aqt Jo saFqnloJJrruJElodJalur Surddelra,ro eqJ 'xeuo3

IIec aql ]E uorlBruJoJ ̂ oJJry Jo etrs eq] saUI-oeds leqt pu8rs Surcnpur-MoJJrg B seleJeue8 'alpurds

[BJlueJ Jo (euozp[u elpurdsaq] lPql s 'pporu uouDlnuus alputds lDil.ua7 eql pellBc ,{lilqrssod puoses v

'fug-Zt arn81g) elpurds rrlotrur B ̂q reqlo qcea o] petJeuuoJ touere sJelse aql SurlPalcnu satuosoJlual oâl eqt uaq^^ ueê (sJelse oû uee Aleqde,r,rprur suJoJ âoJJnJ a8eêalc E leql eleJlsuouap qf,rq^ 'slleJ cruofJqura e8JpluI slueurJedxa snorue8ul luoJJ se{uoc Iapou srql roJ eouepr^g 'selod elpurdseql ueaMtaq dea,gpq Surr e olur pasnJoJ ̂\oqaluos aJe deql eJaq ̂ 'xauoc

ilec aqlo1 qeu8rs Surcnpur-,r,roJrnJ ,ftJec salnqnloJcnu IeJlsE eql ]pql sale1nlsod qJrq ^'ppout uoUaInuUS lDr$a aql paura] sr ]srrJ aqJ'(tg-ZI arntgg) asaqtJo uop-eulquo3 e,r{o1dua o} JEadde sllac lsoru pue 'pesodord uaaq eABq susrueqJetuIPraueB earqJ iuorsr^lp Jo elrs eql ,,i;rcads epurds JIlotIu eql seop ̂toH'uorlPruJoJ MorJnJ IEsol asnpur epurds aql dq petEJeueS spuSrs ]Eq] Eepr eql 8ur-uoddns-alls epurds ^ eu eql qlyvt pJoJJp ur sdolelap euo llrau B pup 'sJEeddE-slp ̂aoJJnJ a8enealc ]uardrJur aq] 'aseqdeue,{1rea ur elpeau sselS eurJ e qlrm uol}-ISod ̂\eu e olut pa83nl sr alpurds eql JI

'edoJsoJJrru e qflm atup IpeJ ur palJesqoaq ueJ elpurds aql 'Jealc sr urspldol-{J aql asneJeg 's11ec Jellptus pue Jellerusolur dn pepplp ,{lalrssarSord sr 33a pur8rro eq} t(eâ srql ul 'qtmor8

Jo sporradSuruanralur ]noqlrm ̂lprder anealc softqura eseql (uonpz1lual JaUV '3urr aplcerl-uoo aq] Jo tuarueJeld aql SuuruJelep ur sapqnloJJru epurds Jo eJuetJodrureql peleelar ]sJrJ selpJqaualur aurJeru ;o s33a pazllpJeJ eql Jo serpnls

'sllal leruruE ur srseuDlol,{c uo srseqdrue uB qll^\'llElap

aloru ur sursrueqJeur drolep8ar esaql eqrJosap laou aM 'srseuDlo/.c saleqrul'aseqdeue pue aseqdeleru ur uorlJnrlsap uqc,{c uo spuadap qJqrlr ,seleJlsqns

lepour uorlexelaJ lellse (f)

el)l() lle) eql:/ [ raldeq) 960 t

(rateg Marpuv lo ^sairno))'slle) tueld-raq6rq ur sau:osorlua) ou arearaql asnP)oq 'salnqntor)rLu

lerlse ou aiearaqt teq] oloN'o^p lua)salonu e qllM

paurels sr saurosouroJq) ralqbnep;o stasoMl aql ur vNo aql pue'ullnqnl lsure6e

sarpoqrlup pelaqe;-plob qllM pau!elsaje alpuros aqlJo salnqnlol)lul aqf

'a6ed aql 1o aue;d aqt ol re;nrtpuadredauPld P ur paurloj seq (tpDaq/Au)D

oml aql uaamloq) a1e;d 1;a>,{;teaaql'qdel6or)r.u rq6ll slqt u1 'aseqdolal ug

11ar 1ue;d e ug s;sau11o1I3 99-2 1 arn6r3

(razlolg leeq)rW pue,{u1ar6 esr;y

1o {salrno3) 'lla) aq} }o aueld ;errolenbaeqt tnoq6norql peralle)s salpunq

alnqnlorlrur rlllM pate!)osse s (uail6)y1I1 uralord a;purds ;e.r1ua: aql apqM'a)eJrns

lla) aql qlpouaq 6uu e suro;

@at) Votl[ 'oraq uMoqs se 6urr a;r1>erluor

aqlJo auelo aql ur pauor])as-ssol) sr lla)aql uaq1 (B) '(B€S-/ t pue 7g-1 1 sern6rg

aes) xelJo) lla) oqt te ,(flnlfre VoqUlorluo) dlaq leql s;eu6rs aleraua6 ol

rq6noql are suralord asaql'salnqnlor)rLurelodlalurJo spua snld 6urdde;rano aql ]psexa;duor uro, lpql suretord ,{role;n6ar

lereôs Jo auo sr q)rqM '(uaatb) p4A1pa;;e> uralord e pue (pa4 VoqU asedlg

aqt Jo suolle)ol aql sleaâr stsautlol,{l 1o6uruur6aq aql te lla) ueuinq parntln) p Joqder6orrrr.u a)ue)saronll (V)'lla) upunq

aql lo alpurds lerlua) aql le srole;n6alsgsaul>1oyb 1o uollpz!lp)ol gg-2 1 arnbgg

L6OL

url 0S

srlouru aq] ruo4 pa^lrap selnqnloJsllu suleluoc qclq^\'lsuldoruSurqd eq] pallEc

arnlJnJls e .{q paprn8 sI pue asBqdEu€ e}el uI sulSaq alEld IIeo aql Jo ̂lquesse

eqJ '(g9-ZI arnBIC) IelJnu Jalq8nBp o^\] eql uea^ leq 'elBld IIaJ eql pallE3 'llP^\

IIas ̂\eu e Jo uorlJnJlsuoc eq] r(q tno aplsu aql uro4 pauouluBd sI IIes lueldeqt Jo rusEldo{c aqt 'uI epls}no eql luo4 lusBldo!{.f, aql SuIpIÎp Sutr aplrerl-uoJ B uEq] raqleu 'lpm

IIas plSlrures E dq pasolsue are slles esaql 'slle3 luBld-raq8q uI sISauDIol-(J JoJ lueuodurl ^lrc1nclued sI uolllsodap auerquraql

MoJJnJ eql Ol SnleJ-edde r31o9 aq] ruo4 selnqntoJollu uo pauodsueJl ale leq] selslsel aueJqrueu

lerus rtq peprôrd ̂lpreue8 sI pue MorrnJ e8enealc aqlJo a8pa rauul eql ]e pappP

sr auPrqureru 1v\aN 'auEJqlualu eusEld aql Jo eeJe aJeJJns aql uI eseeJcul uEuo spuadap MoJJnJ a8e,tea1c aql Jo lueluaôru pJBMUI aql 'silec plulue lsolu uI

slueld raq6lH ur slsaulloy() saplng lseldou6elqd aql

'ssnf,slp A\ou e^ se 'o^/ll uI IIaJ aq] apFlp pue alquessP ilI^ [Pâ IIeseq] aJaqr elrs eql s{retu pue slso}Iu eJoJeq 1sn[ salqruasse 'prruq asuqdoJdaJdaql pallPf, 'slueurEllJ ullcP pue salnqnlorsllu Jo puPq pezlueSJo uE 'slleJ lueldq 'alqruasse '11 ursodtu 3urpnlcur '8utr allrerluoJ aq] Jo sluauodtuoc raqloqclq^\ oluo ploJJecs B IuJoJ o1 1q3noq1 eJe sulldas eql'alIS uolslÎp alnlnJ eq]

tp ID elEI ur salquassu suqdas pelleJ sulalord;o Surr e 'aldtuexe ro; 's1sea,{ Sutp-pnq uI 'srsolrru eroJaq uasoqc st.{lquasse Sutr;o e}IS aql'sadIl 1ac aruos uI

'lepolu sII{lqll^\ ]ualsrsuo3 'xeuoc

IIaJ aql lnoqSnorql &1npce alpsBJluoJ paseeJsul 01 pual

seFqnloJJru [BJlsE Jo ssol aql uI ]Insal lBql s]uau]eaJl 'eldluExa n1 'suaga\a '3

sts3Nt}lol^l

'aseqdolet q FIoC eq] ]cnJlsuoJar ol pepaeu slerJaleru eql sllrequr IIaJ Jatq8nppqJEe teql Surrnsua 'alpurds eqt Jo spua alrsoddo ot pelnqulsrp ,{.qaraq1 arepue sayod alpurds eql qtp{ elprcosse sluaur8e4 €yog 'srsolnu Surrnp paluaru8er;pue pazrue8JoeJ sr srueredde r31og aq; 'srsaurloldc Surrnp o,l,r] ur ]nJ sr pue1ce1ur ^,t1a8rel suIEIxeJ Ug aqt 'slleJ lsoru uI 'UA eql sesealeJ adolanua Jeelonu eqlJo u^aop{BeJq pue seFqnloJf,ru aq} Jo uouezrue8roar eql 'aseqd

IN olur drluauodn 'uo1a1a>1so1,b aFqnloJJnu aql .{q pazue8ro sr pue eueJqrueru JpelJnu eqlqlIM snonurluoo sr s11ac aseqd;arq q Ug aql'a1c,{c qJea ecuo elqnop rtp8no.rsJeqrunu JIaq] 'a8erene uo F peluequr dlages aq ol sJaqunu q8noua a8rel ur luas-ard rtlensn are slseldorolqJ puu prrpuoqrolrru sp qons salaue8rg isepwp IIaJE uel{ A alu8ar8as sallauu8ro pasolJua-euEJqrueu snorJe^ aql op 'ueql 'AroH

'luaserd ,,(pearp sg ll go ped auos ssalun(gg) runpc4ar cnuseydopua lvrau B a{eru louuec qlac 'l.lreyurrg 'sallaue8ro 3u4sr-xaard eqtJo uoIsIAIp pue ql toJB aql .{.q d.yuo asrre uec r{aql lsluauoduroJ Ienpn-IpuI Jleql ruo4 olou ap palqruassu aq louueJ slseldorolqc pue prJpuoqJotrusE qJns sallaueBro '71 raldeq3 ul pessnJsrp sy'sallaue8ro pesolcua-eueJqrueuaql Surpnlcur 'sluauodurof,

IIeo lelluessa reqlo aqt Jo IIe tlJaqur oqe tsnu IIaJralq8nep qcee :ela^ oq 'sapplp

1ao rqoftecne B uer{ A'saruosoruoJqo Jo tueru-a1duloc IInJ e salre3eJ IIeJ ralq8nep qcee tpql seJnsue slsolru 3o ssacord aq;

stseutlol^) 6uunc slla)rolq6nec ol palnqrrtslc eg lsnw sellaue6ro pasol)uf-euerqruew

'(Lg-Ll arnS;g) Ilelrlr IIar ^ au eq1Jo uollcnJtsuoc aq] alayduroc o1 aluyd IIeJ aqt Jo xrJleru eq1 ulqtlm rrmop prpl ereslrrqrJorJrru eso1nllar tale.I 'omt ur IIaJ eqt sapplp pup llellr 1ac pufrro aql pueauuJquelu eurseld aql seqJpeJ 1l Ipun uorsnJ elf,rsel Jequry ztq prervrlno spuedxaa1eld aql 'a1zt1d

77ac tpaa aql paflec aJn]JnJ]s pesoloue-eueJqrueu ,a1r1csrp

e uJoJ ol asrg '1parr 11ac ̂\au ar4 Jo srsaqlufs aql JoJ parnbar surelordocflS pue

aplreqcces,{1od qtr.n pe[g 'selorsel esaqJ 'relueJ IIe] eq] ol snteredde r31og aqr

Iuo4 selnqnlorJllu asaql Suop selorsal leurs podsuerl suralord Jolo4 'alpurds

's;;ar ralq6nep oml aql 6urleredas{1e1a1duol 'osnJ

lleM lla) Mau aq} 6urpunorns euerqrlaul aq} pue auelquau eruseld aq1'1;ean ;1at ;eurbuo pue aueJqtueuteulseld aql q)eal ol ptPMlno sanoi6 qrrqan'lleM lla) Mau oql uloJ ol asnJ pue salnqnlol)tul osoql 6uo;e pallodsuellare 'lerlaleu

llem-llel qllm pallU'sal)rsa^ panuap-r6;o9'rolenba lla) aql le pua lnq de;lano la6uo; ou solnqnlol)lulasaqllo spue sn;d aq1'1se;dou6erqd aql r-uro; aseqdo;et 1e 6urureuret a;purds tr1o1rr.u aqllo salnqnlo.r:ru.re;od.relur aq1 'elputds plo aqlJo lolenbe aq] le lla) aql aprsur alquasse ol suels lleM lle) anau e'pa1e6ar6as aneq seuosoulolLll aql laue 'aseqdo;at1o 6uruur6aq eLll lV'xatlo) llal aqt le (pueq aseqdordatd aql) sluaujelU ult)e pue salnqnlol)lm;o pueq e {qaseqd W aroJaq paLlsrlqelsa sr aueld uotst^!p aql.llo) gueld raq6rq e ur srsauglol() ro seJnlpal ;elrads eq1 19-4 1 ern613

srsourloy{raseqclolel

selnqnlojlrujsel)rsaApa^rrep-!6t09

,: 1se;dou6etqd.\

tg '9

llel/\ llo)Mau

salnqnloJ)tu.t oseqdlalul

1o {et.re ;errpor

selnqnlor)il.! alpurdstelodralur ]o sureuraJ

sluaurelr] urpe puesolnqnlor)il.r.r ]o pueq oseqooJoaJo

op^) lle) eqt:/ [ reldeq3 860 t

('auorts uesns Jo (saunol) 'r"urads

pue s66e ol asu anr6 leql slla) aql ug,(1uodn pua salnuer6-d eql leql os'suorsrnrp

;1ar luanbasqns leJaâs u! paleadalsr sse:ord uolleberbas aues eq1 '1q6y.l

aLll uo umoqs sp'sap!^!p 66a aql uaqmse;nuer6-6 eq] sa^ra)ar ;;ar relq6nep

roualsod aql Iluo lpql aJnsuo ot poluauosr aueld abeneep aql'#a/ oqt uo umoqs

se'66e pezrlrlra; eql;o alod .rolralsodaqt ol pate6or6as auo)aq lnq (uMor.lslou) 66a pazrlryarun aqlJo useldotl)

aql tnoLl6norql Ilr.uopuer patnqUlsrp arp{aq1's;;e> ura6 aqt ouo)aq slla) q)lq^

aururalap pue suraloro pue vNU,o epeurere sa;nuerb ;;eus aseql'Ido)sor)r.rt

arua>saronp,(q paMar^ pue sa;nuel6-6tsulebe ̂poqrlup ue qlrm paurels slla)

aures aql de Molaq s;1er aq1'{dorso.nrr.ua)ua)saronu pue lserluo)-a)uaraJralul

-leltua.lajrlp tlloq ̂q perualn ale {aq}l(serpoq re;od pue) snal)nu aql Moqs

ol olp lua)sarong'6u;pu;q-y56 ?nlge qllM paulels ueeq e^Pq a ogp slla) aqf'suobala

) apoleurou aql,o 660 pazllluoJe Jo uorsrnrp tsrU aql 6urrnp ;1ar ralq6nepauo ot stuauodtuor rlr-useldo1{r trlrads

Jo uolle6ar6as >ulauulse palloJluo)aLll alPrlsnllr sqder6o:rrr,u tq6;1 esaql

';;a> ralq6nep auo I;uo ol slueuodr!o)>1urse;dolfu 6u11e6ar6as uorsrArp;;ar >lr1aurur{se uy gg-41 arnbr3

660t

',{.ea,r slqt ur paleelonult1nru eruoJaq 'aldurexe JoJ 'sllac elcsnu ueaq pue se1lb-oledeq etuos pue 'sleleluld poolq ecnpoJd qrlq a'sepboIre1e8a111 's11ac u€Ileru-rueru Jo sadq euros uI sJn33o osp slsauplot{c lnoqlpt UOISIAIp melcnN

<I)JJ>'(69-2I erntlf,)snelcnu qJee asolcue ol JJo saqcurd '3utr ulsol.Iu-ul]f,8 uP Jo dlaq aql q]!v\ 'pue

premul spuaxe euerqrueu eruspld eq1-'uoltozqulryIac palluc slseuDloltJ paleu-lpJoos Jo punoJ auo uI snelJnu qJBe punoJB palPeJc eJe seuPJqrueu 'suoISIAIp

reelJnu prder asaql Jauv 'uorslÎp qJEa roJ slsauPlolb Jo sdels eql ile qSnorqr

oB ol aup eql e{E} ot elpq lou op slloo eql s€ 'luaudolenep ^lJea dn spaads,(pear8 tuerue8uerp srql 'urn;1buds e pelpc sr ruseldo$o eIrES aql aruqsrelJnu aldrlpru qf,rqm ur IIaJ V'erBJJns eq] Jeau Jaf.Blouoru P ut peBueJJE 'IalJnu

puesnoq] IPJeAes Surureluoc ilac aSJPI al8uts e Jo uoneluJoJ al{l q Suppsar'uorsrlrp crurseldoldc lnoqllm JnoJo uoIsIAIp JEelJnu Jo spunoJ €I lsJg eql'aldruexe JoJ 'ofJqua DlnldosoJe fpee aqt uI 'uoIsIAIp cturseldo{c Suruarrra}ut

lnoqllM uorsr rp Jeelcnu Jo spunoJ eldpFur oSJapun silac etxos 'suopdacxe

ere aJeql 'uolstÎp crurseldolb,(q parr,rollo;,{1pnsn sI UOISIAIp JPelJnu q8noqlry

stsou!Ioy() lnoql!/u\ ln))o ue) slsollw

'salurqeua^ ul eloJ Jelrurs E eêq ol uees suleloJd asaql Jo eluos puu '(zz nl-deqf, ur passnJsrp) suorsrlrp clrlaruurri.se qcns JoJ peJlnbeJ sutalord eqlJo eruospegBuapr atteqn\tqdosotq puesunBala 3 ur sas,(puE clleua0 'uofar aleudordduaql ol 'seFqnloJJrru

lErls€ sll eIA 'selod apurds el{l Jo euo [nd aleql penTBsolsuralord rolou teql pue sluatuelou alpurds qcns tf,aJlp xeuoo IIeJ aq];o suor8ar

lecol ur sa8ueqc lpql ^laIII sruaas lI 'ller Su1pptp eql ulq]$ rauupru pellorluooe ur peloru eq o] seq elpurds eqt ffipculatuudse uoIsIAIp;o aueld eqt uoplsodoI'(g9-ZI amt;g) slueuoduor esaql sllrel{ur 1ac ralq8nep alerrdordde eql lBqlos uorsrlrp ;o aueld eql uoplsod uaql pue IIaJ aql Jo epls euo ol (srunulutlappa1al77ac pelIBJ) slueuodruoc uleuac ale8ar8as tsrg lsnru IIaJ Jeqlou aql 'Lean srqlur salpJ tueJeJJrp qllm sllao ralq8nep ateerc oI's,,(ervrqled lueJeJJIp Suop dola,rapol pauusep erp sllec ralq8nep iueJeJJIp oml eql ,{1pnsn 'qloq ut Jo rllJequl

,taql slualuoc ctuseydo/J aqt ul 'ezIS ul raJJIp leql sllec om1 acnpord ol dlluoulaur-uLse aprnrp slles ueqM teâl oq 'luatudolarrap uI secuelsur,{uuru eJE eJeqJ

'ssacord Surraluac eql ol elnqlJluoJseFqnlorJlru eseql uo 1nd ro qsnd raqtla leql suleloJd Joloru pue seFqruoJOIIu

IeJlsV 'rusuldordc aqr ur Jlaslr JelueJ ol spual seseJ lsoru uI qclqm 'a1pu1ds

Jllol$u eql Jo lueueceld aql luoq sllnseJ drleruurrfu slql 'sluauodruoc aures aqlqllj\/\ pue azrs pnba Jo slleJ ralqSnep o,r4 Sutcnpord '11ac luared aql;o rolenbaeql punom srurog 3uu allppJluoo aql :r(lpcrrleunurts ap1lr1p silec IPurIuP lsolN

{;;errrleuur{sy ep;n;g ot elpulds tlaql uotltsodeu slla) auos

ststNt)ot_Af

oseqd Lg e loJ poJtnbar se,stsolil.! laue^ll^ll)e )pf ,o uorssarddns penurluol

e solnsua stqf '(uMoqs lou lsurelo.rdrollqlLlu! lpf Jo uotlelnunt)p aql

ot se llaM se) )/fdv-tqpJJo uotle^tl)eaql ol speal stsoltu alel u! ,(lrnrtte

lp)-t u! dorp aql'aspqd t9 e 6urureluorslle) ul (g) 'ure6e 6ur1e;nutnt>e ur6aq

ol surP,{>-y1 sMolle q)tqM ,stso}tLU laueuorle^rt)eut l/)dv ol speal url)^)-w Jo

ssol aql xtln!t>e )/)dv-oz)p) sotelnulrls,{ll^ll)e lp)-t4 asne)aB'uotllnJ}sop

ur1:It-y11 6urre66rr1,aseqde1ar,u]o pua aqt le sesrr {}rnrt>e )/)dV-OZ)p)

'sa;rb ;1at rruo,{lqu:a Illee ul (V)

'slsolrul Jatle uorltqtqur 1p1 alqegs {qoseqd t9 p,o uollea.t) aq199-21 a.rn613

ulrl 0 I

cll_l'ldtAo)NOtIVZrUV'tn't'll)

(8)

, &4Q9,,)l)iv:€$f]':.ltt.:],,t] :t,:::tt:::,r:,:t:ttt..,:lt:,tt,tl,t,t:,

oseqd rD ou qll/!^ slla) lluofuqure (V)

'(V09-ZI arntgg) elrdr ueu ar{lroJ ullr,{c-IN.r au 3ur-lelnrunJ3p ur8aq,ltpl3rnb o1 1ac aql s^\ollp }r sp 'salJdr

1ac cruodrqrua prdBJ ur

InJesn.,tlercadsa sr srsolrru raue .{.lalErparurur uor}e^rlceur f,/JdV srr{J 'lleo Jruo-.,fuqrua uE ur .{lrlr}Je l/ldv IIe Jo uortelrlJeul aq} ol speel uoos slsol1u atel uruIIr.{r-N Jo uor}rnr}sap eqt 'snqJ .fipr11ce f,/f,dV-OZOpO setelnruns {pl-N }eqt'ralel\oq 'llEJeU 'JellJee passncsrp 'JlJdV-SZJpJ

Jo uorlce aq] o] dlaJrlue lsorupenp sI srsolrru elel uI {pl-hl Jo uor}E^rlJBur eql 'soz{Jqura

lBrurup .,(Fea u1'uorsr^rp ̂tau E ol Suplpfiuoc eJoJeq lueu

-uoJIAue slr sJolruoru pue s^ or8 IIaf, aqt qJtlm SurJnp 'aseqd de8 IC e saluraua8d.tntlceur {pl Jo elels srql 'slleJ }sotu uI 'elo^3

IIec ̂ eu u Jelue o1 saredard 1acaql se .{t1,r,pleur {pl Jo alels B ot ruets.{s Iorluoc elcfr-llec aqt 8up}asar roJ rusru-eqcau B seprnoJd osp lI 'sur8rJo uorlecrldar VNCI ]e saxalduroc e pEOIIdaJeJd

Jo srseqluds eq1 salqeua pup (srseuplolb selouroJd 'slsolrur alpl Jo sluale eqlsreSSul ll :suorlcunJ dueur seq aseqd 61 el€l ur qpl Jo uope pJeul eq] teldeqrslql ul rerlrea paqrrJsep sV 'uorlcnrlsep urTc.{r }uepuedep-f,/Odv ,,(q ,tpreurrrduallrp sl qJlq^a 's{pl

Jo uorlulrtopul aql s1 aseqd ntr alBI ul tuela .{rolpp8ar da4 y

(1rnr1reu; lp) Jo elels alqels e sl eseqd tg aql

'aseqd I Jo pua eqt lE palarqop sr elpls slql ^\oq Surssncsrp,ri.q uoncas slq] pue

el 'a^Ilceul u(psour eJE s)p] qclqM ur (ID Jatua sllec lsoru 'srseuoloi-,b ragy

('ue^rllnS ue!lllM pup nA eurlsu) jo Isauno)'B)'aseqdelar-u ur lle arp,{aql'araq 1I;snouorqruls a1:{r aql qbnolq}

o6 ra;>nu aql lle leql aloN pal sourosoJluaf aql pue uaa;6 paurels aresalnqntor)r.u aqf uorlezuelnllo) aroleq o{rqura olrqdosotg e ur sa;purds

rrlolru a;dr1;nu-r;o qder6o.r:ru.r a)ua)saronll (B) 'uorlpzuelnlla) pallelssa)oro P ur slla) lenpr^rpur Luroj ol snal)nu q)ea punorjns olJJo saq)uro

pup preMur spualxa auerquraur euse;d aqt pue'xelrol aql ot alerbrr-uuaql ral)nu aqi Jo lso6 'unrll(ru,{s abre; e alear> o} uorsrnrp rrluseldoy(r

lnoqlrM pue {;snouorqru,{s rn>ro suorsr^rp rpal)nu € [ ]srU aqf (V)'o{rqura opqdosotq I;rea aql ug slsau;1oy(r lnoqllm slsollw 6s-l I aln6lJ

tunlv{:u,{s eur rellnu Iueu.r66e pezrlrpal

sNotstn tcuvt'tf nN

(v) aue./quaur

el)^) lle) eql :/ [ ra]deqf 00 t t

CONTROL OF CELL DIVISION AND CELL GROWTH

Rapid cyclin accumulation immediately after mitosis is not useful, however,for cells with cell cycles containing a G1 phase. These cells employ several mech-

anisms to prevent Cdk reactivation after mitosis. One mechanism uses anotherAPC/C-activating protein called Cdhl, a close relative of Cdc20. Although both

Cdhl and Cdc20 bind to and activate the APC/C, they differ in one importantrespect. \Arhereas M-Cdk activates the Cdc20-APC/C complex, it inhibits the

Cdhl-APC/C complex by directly phosphorylating Cdhl. As a result of this rela-

tionship, Cdhl-APC/C activity increases in late mitosis after the Cdc20-APC/Ccomplex has initiated the destruction of M-cyclin. M-cyclin destruction there-

fore continues after mitosis: although Cdc20-APC/C activity has declined,

Cdhl-APC/C activity is high (Figure l7-608)A second mechanism that suppresses Cdk activity in G1 depends on the

increased production of CKIs, the Cdk inhibitory proteins discussed earlier.

Budding yeast cells, in which this mechanism is best understood, contain a CKIprotein called Sicl, which binds to and inactivates M-Cdk in late mitosis and G1.

Like Cdhl, Sicl is inhibited by M-Cdk, which phosphorylates Sicl during mito-

sis and thereby promotes its ubiquitylation by SCF. Thus, Sicl and M-Cdk, like

Cdhl and M-Cdk, inhibit each other. As a result, the decline in M-Cdk activity

that occurs in late mitosis causes the Sicl protein to accumulate, and this CKI

helps keep M-Cdk activity low after mitosis. A CKI protein called p27 (see Figure

l7-I9l17-19) may serve similar functions in animal cells.In most cells, decreased transcription of M-cyclin genes also inactivates M-

Cdks in late mitosis. In budding yeast, for example, M-Cdk promotes the expres-

sion of these genes, resulting in a positive feedback loop. This loop is turned off

as cells exit from mitosis: the inactivation of M-Cdk by Cdhl and Sicl leads to

decreased M-cyclin gene transcription and thus decreased M-cyclin synthesis.

Gene regulatoryproteins that promote the expression of G1/S- and S-cyclins are

also inhibited during G1.Thus, Cdhl-APC/C activation, CKI accumulation, and decreased cyclin gene

expression act together to ensure that the early G1 phase is a time when essentially

all Cdk activity is suppressed. As in many other aspects of cell-cycle control, the

use of multiple regulatory mechanisms makes the suppression system robust, so

that it still operates with reasonable efficiency even if one mechanism fails. So how

does the cell escape from this stable Gr state to initiate a new cell cycle? The

answer is that Gl/S-Cdk actMty, which rises in late Gr, releases all the braking

mechanisms that suppress Cdk activity, as we describe in the next section'

Summary

After mitosis completes the formation of a pair of daughter nuclei, cytokinesis finishesthe cell cycle by diuiding the cell itself. Cytokinesis depends on a ring of actin and

myosin that contracts in late mitosis at a site midway between the segregated chromo-

somes. In animal cells, the positioning of the contractile ring is determined by signals

emanatingfrom the microtubules of the anaphase spindle. Dephosphorylation of Cdk

targets, which results from Cdk inactiuation in anaphase, triggers cytokinesis at the

correct time after anaphase. After cytokinesis, the cell enters a stable Gt state of low Cdk

actiuity, where it awaits signals to enter a new cell cycle.

CONTROL OF CELL DIVISION AND CELL GRO TH

A fertilized mouse egg and a fertilized human egg are similar in size, yet they

produce animals of very different sizes. \Alhat factors in the control of cell behav-

ior in humans and mice are responsible for these size differences? The same fun-

damental question can be asked for each organ and tissue in an animal's body.

\Alhat factors in the control of cell behavior explain the length of an elephant's

trunk or the size of its brain or its liver? These questions are largely unanswered,

at least in part because they have received relatively little attention compared

with other questions in cell and developmental biology. It is nevertheless possi-

ble to say what the ingredients of an answer must be.

1 101


The size of an organ or organism depends mainly on its total cell mass,which depends on both the total number of cells and the size of the cells. Cellnumber, in turn, depends on the amounts of cell division and cell death. organand body size are therefore determined by three fundamental processes: cellgrowth, cell division, and cell death. Each is tightly regulated-both by intracel-lular programs and by extracellular signal molecules that control these pro-grams.

The extracellular signal molecules that regulate cell size and cell number aregenerally soluble secreted proteins, proteins bound to the surface of cells, orcomponents of the extracellular matrix. They can be divided operationally intothree major classes:

1. Mitogens, which stimulate cell division, primarily by triggering a wave ofGr/S-Cdk activity that relieves intracellular negative controls that other-wise block progress through the cell cycle.

2. Growth factors, which stimulate cell growth (an increase in cell mass) bypromoting the synthesis of proteins and other macromolecules and byinhibiting their degradation.

3. suruiual facfors, which promote cell survival by suppressing the form ofprogrammed cell death known as apoptosis.

Many extracellular signal molecules promote all of these processes, whileothers promote one or two of them. Indeed, the term growth faAoris often usedinappropriately to describe a factor that has any of these activities. Even worse,the term cell growthis often used to mean an increase in cell numb er, or cell nro-liferation.

In addition to these three classes of stimulating signals, there are extracellu-lar signal molecules that suppress cell proliferation, cell growth, or both; in gen-eral, Iess is known about them. There are also extracellular signal molecules thatactivate apoptosis.

In this section, we focus primarily on how mitogens and other factors, suchas DNA damage, control the rate of cell division. we then turn to the importantbut poorly understood problem of how a proliferating cell coordinates its growthwith cell division so as to maintain its appropriate size. We discuss the control ofcell survival and cell death by apoptosis in Chapter lg.

Mitogens Stimulate Cell Division

u-nicellular organisms tend to grow and divide as fast as they can, and their rateof proliferation depends largely on the availability of nutrients in the environ-

PDGF is only one of over 50 proteins that are knornm to act as mitogens. Mostof these proteins have a broad specificity. pDGII for example, can stimulatemany types of cells to divide, including fibroblasts, smooth muscle cells, and

1 [ m

Figure 17-61 A platelet. Platelets areminiature cel ls without a nucleus. Theycirculate in the blood and help st imulateblood clott ing at si tes of t issue damage,thereby preventing excessive bleeding.They also release various factors thatst imulate heal ing. The platelet shownhere has been cut in half to show itssecretory vesicles, some of which containplatelet-derived qrowth factor (PDGF).

microtubule


neuroglial cells. Similarly, epidermal growth factor (EGD acts not only on epi-dermal cells but also on many other cell types, including both epithelial andnonepithelial cells. Some mitogens, however, have a narrow specificity; erythro'poietin, for example, only induces the proliferation of red blood cell precursors.Many mitogens, including PDGE also have other actions beside the stimulationof cell division: they can stimulate cell growth, survival, differentiation, or

migration, depending on the circumstances and the cell type.In some tissues, inhibitory extracellular signal proteins oppose the positive

regulators and thereby inhibit organ growth. The best-understood inhibitory

signal proteins are TGFp and its relatives. TGFB inhibits the proliferation of sev-

eral cell t1pes, either by blocking cell-cycle progression in G1 or by stimulatingapoptosis.

Cells Can Delay Division by Entering a Specialized NondividingState

In the absence of a mitogenic signal to proliferate, Cdk inhibition in Gt is main-

tained by the multiple mechanisms discussed earlier, and progression into a new

cell cycle is blocked. In some cases, cells partly disassemble their cell-cycle con-

trol system and exit from the cycle to a specialized, nondividing state called G6.

Most cells in our body are in Gs, but the molecular basis and reversibility of

this state vary in different cell types. Most of our neurons and skeletal muscle cells,

for example, are in a terminally dffirentinted Ge state, inwhich their cell-cycle

control system is completely dismantled: the expression of the genes encoding

various Cdks and cyclins are permanently turned off, and cell dMsion rarely

occurs. Other cell types withdraw from the cell cycle only transiently and retain

the ability to reassemble the cell-cycle control system quickly and reenter the

cycle. Most liver cells, for example, are in Gs, but they can be stimulated to dMde

if the liver is damaged. Still other t!?es of cells, including fibroblasts and some

Iymphocytes, withdraw from and re-enter the cell cycle repeatedly throughout

their lifetime.Almost all the variation in cell-cycle length in the adult body occurs during

the time the cell spends in G1 or Go. By contrast, the time a cell takes to progress

from the beginning of S phase through mitosis is usually brief (typically 12-24

hours in mammals) and relatively constant, regardless of the interval from one

division to the next.

Mitogens Stimulate Gr-Cdk and GrlS-Cdk Activit ies

For the vast majority of animal cells, mitogens control the rate of cell division by

acting in the Gr phase of the cell cycle. As discussed earlier, multiple mechanisms

act during G1 to suppress Cdk activity and therebyblock entry into S phase. Mito-

gens release these brakes on Cdk activity, thereby allowing S phase to begin.

As we discuss in Chapter 15, mitogens interact with cell-surface receptors to

trigger multiple intracellular signaling pathways. One major pathway acts

through the small GTPase Ras, which leads to the activation of a MAP kinase cas-

cade.This leads to an increase in the production of gene regulatory proteins,

including Myc. Myc is thought to promote cell-cycle entry by several mecha-

nisms, one of which is to increase the expression of genes encoding G1 cyclins(D cyclins), thereby increasing Gr-Cdk (cyclin D-Cdk4) activity. As we discuss

later, Myc also has a major role in stimulating the transcription of genes that

increase cell growth.The key function of Gr-Cdk complexes in animal cells is to activate a group

of gene regulatory factors called the E2F proteins, which bind to specific DNA

sequences in the promoters of a wide variety of genes that encode proteins

required for S-phase entry, including G1/S-cyclins, S-cyclins, and proteins

involved in DNA synthesis and chromosome duplication. In the absence of

mitogenic stimulation, E2F-dependent Sene expression is inhibited by an inter-

action between E2F and members of the retinoblastoma protein (Rb) family.

1 103

mr togen

1 104 Chapter 17: The Cell Cycle

cYrosol I

rflNUCLEUS

I

x n"*11,"*on

I' g e n e

gluyc- regulatory.*f protein

Idelayed-response gene expresston

)

I nas

I

+I

act ivat ion of gene regulatory protein

positive feedback

G 1/S-cycl in(cyciln rlS-cyc l in

(cyc l in A)i nactivatedE2F pro te in

inac t iva ted Rbpro te in

F.?ffi"

\A/hen cells are stimulated to divide by mitogens, active Gr-cdk accumulates and

in turn increase Rb protein phosphorylation and promote further E2F release(see Figure 17-62).

The central member of the Rb family, the Rb protein itself, was identifiedoriginally through studies of an inherited form of eye cancer in children, knonmas retinoblastoma (discussed in chapter 20). The loss of both copies of the Rbgene leads to excessive cell proliferation in the developing retina, suggesting thatthe Rb protein is particularly important for restraining ceU aivision in this tissue.The complete loss of Rb does not immediately cause increased proliferation of

act ive DNAS.CdK

+ SYNTHESIS

Figure 1 7 -62 Mechanisms controllingcel l-cycle entry and S-phase init iat ion inanimal cel ls. As discussed in Chapter 15,mitogens bind to cel l-surface receptorsto init iate intracel lular signal ingpathways. One of the major pathwaysinvolves activation of the small GTPaseRas, which activates a MAP kinasecascade, leading to increased expressionof numerous immediate early genes,including the gene encoding the generegulatory protein Myc. Myc increases theexpression of many delayed-responsegenes, including some that lead toincreased G1-Cdk activi ty (cycl in D-Cdk4),which tr iggers the phosphorylat ion ofmembers of the Rb family of proteins.This inactivates the Rb proteins, freeingthe gene regulatory protein E2F toactivate the transcript ion of G115 genes,including the genes for a G1lS-cycl in(cycl in E) and S-cycl in (cycl in A).Theresult ing GrlS-Cdk and S-Cdk activi t iesfurther enhance Rb proteinphosphorylat ion, forming a posit ivefeedback loop. E2F proteins alsostimulate the transcript ion of their owngenes, forming another posit ivefeedback looo.


retinal or other cell types, in part because Cdhl and CKIs also help inhibit pro-gression through Gr and in part because other cell tlpes contain Rb-related pro-

teins that provide backup support in the absence of Rb. It is also likely that otherproteins, unrelated to Rb, help to regulate the activity of E2E

Additional layers of control promote an overwhelming increase in S-Cdkactivity at the beginning of S phase. We mentioned earlier that the APC/C acti-vator Cdhl suppresses cyclin levels after mitosis. In animal cells, however, Gr-and G1/S-cyclins are resistant to Cdhl and can therefore act unopposed by the

APC/C to promote Rb protein phosphorylation and E2F-dependent gene

expression. S-cyclin, by contrast, is not resistant to Cdhl, and its level is initially

restrained by Cdhl-APC/C activity. However, Gr/S-Cdk also phosphorylates andinactivates Cdhl-APC/C, thereby allowing the accumulation of S-cyclin, furtherpromoting S-Cdk activation. Gr/S-Cdk also inactivates CKI proteins that sup-press S-Cdk activity. The overall effect of all these interactions is the rapid and

complete activation of the S-Cdk complexes required for S-phase initiation.

DNA Damage Blocks Cell Division: The DNA Damage Response

Progression through the cell cycle, and thus the rate of cell proliferation, is con-

trolled not only by extracellular mitogens but also by other extracellular and

intracellular mechanisms. One of the most important influences is DNA dam-

age, which can occur as a result of spontaneous chemical reactions in DNA'

errors in DNA replication, or exposure to radiation or certain chemicals. It is

essential that the cell repair damaged chromosomes before attempting to dupli-

cate or segregate them. The cell-cycle control system can readily detect DNA

damage and arrest the cycle at either of two checkpoints-one at Start in late Gt,

which prevents entry into the cell cycle and into S phase, and one at the GzlM

checkpoint, which prevents entry into mitosis (see Figure 17-21).DNA damage initiates a signaling pathway by activating one of a pair of

related protein kinases called ATM and ATR, which associate with the site of

damage and phosphorylate various target proteins, including two other protein

kinases called Chkl and Chk2. Together these various kinases phosphorylate

other target proteins that lead to cell-cycle arrest. A major target is the gene reg-

ulatory protein p53, which stimulates transcription of the gene encoding a CKI

protein called p21;this protein binds to Gr/S-Cdk and S-Cdk complexes and

inhibits their activities, thereby helping to block entry into the cell cycle (Figure

17-63). <TGAA>DNA damage activates p53 by an indirect mechanism. In undamaged cells,

p53 is highly unstable and is present at very low concentrations. This is largely

because it interacts with another protein, Mdm2, which acts as a ubiquitin lig-

ase that targets p53 for destruction by proteasomes. Phosphorylation of p53

after DNA damage reduces its binding to Mdm2. This decreases p53 degrada-

tion, which results in a marked increase in p53 concentration in the cell. In addi-

tion, the decreased binding to Mdm2 enhances the ability of p53 to stimulate

gene transcription (see Figure 17-63).The protein kinases Chkl and Chk2 also block cell cycle progression by

phosphorylating members of the Cdc25 family of protein phosphatases, thereby

inhibiting their function. As described earlier, these kinases are particularly

important in the activation of M-Cdk at the beginning of mitosis (see Figure

17-25). Thus, the inhibition of cdc25 activity by DNA damage helps block entry

into mitosis (see Figure 17-21).The DNA-damage response also detects problems that arise when a replica-

tion fork fails during DNA replication. \{hen nucleotides are depleted, for exam-

ple, replication forks stall during the elongation phase of DNA synthesis. To pre-

vent the cell from attempting to segregate partially replicated chromosomes, the

same mechanisms that respond to DNA damage detect the stalled replication

forks and block entry into mitosis until the problems at the replication fork are

resolved.The DNA damage response is not essential for normal cell dMsion if envi-

ronmental conditions are ideal. Conditions are rarely ideal, however: a low level

1 105

1 1 06 Chapter 1 7: The Cell Cycle

(discussed in chapter 20). This loss of p53 function allows the cancer cell toaccumulate mutations more readily. Similarly, a rare genetic disease known asataxia telangiectasia is caused by a defect in ArM, one of the protein kinases thatis activated in response to x-ray-induced DNA damage; patients with this dis-ease are very sensitive to x-rays and suffer from increased rates of cancer.

\Mhat happens if DNA damage is so severe that repair is not possible? The

individual cell. Cells that divide with severe DNA damage threaten the life of theorganism, since genetic damage can often lead to cancer and other diseases.Thus, animal cells with severe DNA damage do not attempt to continue division,but instead commit suicide by undergoing apoptosis. Thus, unless the DNAdamage is repaired, the DNA damage response can lead to either cell-cycle

DNA damage

Mdm2

D N A

II

ATM/ATR kinase activation

+Chkl /Chk2 k inase act ivat ion

II+

PHOSPHORYLATIONO F p 5 3

p53 UBIQUITYLATIONAND DEGRADATION

IN PROTEASOMES

stable,act ive p53

ACTIVE p53 BINDS TOREGULATORY REGIONOF p27 GENE

p21 gene

TRANscRrproN IV

p2, mRNA

rnarusmloru {

Figure 17-63 How DNA damage arreststhe cel l cycle in G1. When DNA isdamaged, various protein krnases arerecruited to the site of damage andinit iate a signal ing pathway that causescel l-cycle arrest. The f irst kinase at thedamage site is either ATM or ATR,depending on the type of damage.Addit ional protein kinases, cal led Chkland Chk2, are then recruited andactivated, result ing in thephosphorylat ion of the gene regulatoryprotein p53. Mdm2 normally binds top53 and promotes i ts ubiquitylat ion anddestruction in proteasomes.Phosphorylat ion of p53 blocks i ts bindingto Mdm2; as a result, p53 accumulates tohigh levels and st imulates transcript ionof the gene that encodes the CKI proteinp21. The p21 binds and inactivatesG1lS-Cdk and S-Cdk complexes, arrestingthe ce l l in G1. In some cases , DNAdamage also induces either thephosphorylat ion of Mdm2 or a decreasein Mdm2 production, which causes afurther increase in p53 (not shown).

ACTIVE

G1/S-Cdkand 5-Ldk

p21 (cdkinhib i tor protein)

INACTIVE

GrlS-Cdk and S-Cdkcomplexed wi th p21


arrest or cell death. As we discuss in the next chapter, DNA damage-inducedapoptosis often depends on the activation of p53. Indeed, it is this apoptosis-promoting function of p53 that is apparently most important in protecting usagainst cancer.

Many Human Cells Have a Built-ln Limitation on the Number ofTimes They Can Divide

Many human cells divide a limited number of times before they stop andundergo a permanent cell-cycle arrest. Fibroblasts taken from normal human tis-sue, for example, go through only about 25-50 population doublings when cul-tured in a standard mitogenic medium. Toward the end of this time, proliferationslows dolrm and finally halts, and the cells enter a nondividing state from whichthey never recover. This phenomenon is called replicative cell senescence,although it is unlikely to be responsible for the senescence (aging) of the organ-ism. Organism senescence is thought to depend, in part, on progressive oxidativedamage to long-lived macromolecules, as strategies that reduce metabolism(such as reduced food intake), and thereby reduce the production ofreactive oxy-gen species, can extend the lifespan of experimental animals.

Replicative cell senescence in human fibroblasts seems to be caused bychanges in the structure of the telomeres, the repetitive DNA sequences andassociated proteins at the ends of chromosomes. As discussed in Chapter 5,when a cell divides, telomeric DNA sequences are not replicated in the samemanner as the rest of the genome but instead are synthesizedby the enzymetelomerase. Telomerase also promotes the formation of protein cap structuresthat protect the chromosome ends. Because human fibroblasts, and many otherhuman somatic cells, are deficient in telomerase, their telomeres becomeshorter with every cell division, and their protective protein caps progressivelydeteriorate. Eventually, the exposed chromosome ends are sensed as DNA dam-age, which activates a pS3-dependent cell-cycle arrest that resembles the arrestcaused by other types of DNA damage (see Figure l7-63). Rodent cells, by con-trast, maintain telomerase activitywhen they proliferate in culture and thereforedo not have such a telomere-dependent mechanism for limiting proliferation.The forced expression of telomerase in normal human fibroblasts, using geneticengineering techniques, blocks this form of senescence. Unfortunately, mostcancer cells have regained the ability to produce telomerase and therefore main-tain telomere function as they proliferate; as a result, they do not undergoreplicative cell senescence.

Abnormal Proliferation Signals Cause Cell-Cycle Arrest orApoptosis, Except in Cancer Cells

Many of the components of mitogenic signaling pathways are encoded by genes

that were originally identified as cancer-promoting genes, or oncogenes, becausemutations in them contribute to the development of cancer. The mutation of asingle amino acid in the small GTPase Ras, for example, causes the protein tobecome permanently overactive, leading to constant stimulation of Ras-depen-dent signaling pathways, even in the absence of mitogenic stimulation. Simi-larly, mutations that cause an overexpression of Myc stimulate excessive cellgrowth and proliferation and thereby promote the development of cancer.

Surprisingly, however, when a hyperactivated form of Ras or Myc is experi-mentally overproduced in most normal cells, the result is not excessive prolifer-ation but the opposite: the cells undergo either cell-cycle arrest or apoptosis.The normal cell seems able to detect abnormal mitogenic stimulation, and itresponds by preventing further division. Such responses help prevent the sur-vival and proliferation of cells with various cancer-promoting mutations.

Although it is not knolrm how a cell detects excessive mitogenic stimulation,such stimulation often leads to the production of a cell-cycle inhibitor protein

calledArf which binds and inhibits Mdm2. As discussed earlier, Mdm2 normally

1"t07

1 1 08 Chapter 1 7: The Cell Cycle

excessive Myc production

Figure 17-64 Cell-cycle arrest orapoptosis induced by excessivestimulat ion of mitogenic pathways.Abnormally high levels of Myc cause theactivation of Arf, which binds and inhibitsMdm2 and thereby increases p53 levels(see Figure 17-60). Depending on the cel ltype and extracel lular condit ions, p53then causes either cel l-cycle arrest orapoptosis.act ive Mdm2

stable,active p53

promotes p53 degradation. Activation of Arf therefore causes p53 levels toincrease, inducing either cell-cycle arrest or apoptosis (Figure lZ-64).

How do cancer cells ever arise if these mechanisms block the division orsurvival of mutant cells with overactive proliferation signals? The answer is thatthe protective system is often inactivated in cancer cells by mutations in thegenes that encode essential components of the checkpoint responses, such asArf or p53 or the proteins that help activate them.

Organism and Organ Growth Depend on CellGrowth

For an organism or organ to grow cell division is not enough. If cells proliferatedwithout growing, theywould get progressively smaller and there would be no netincrease in total cell mass. In most proliferating cell populations, therefore, cellgrowth accompanies cell division. In single-celled organisms such as yeasts, bothcell growth and cell division require only nutrients. In animals, by contrast, bothcell growth and cell proliferation depend on extracellular signal molecules, pro-duced by other cells, which we call growth factors and mitogens, respectively.

Like mitogens, the extracellular growth factors that stimulate animal cellgrowth bind to receptors on the cell surface and activate intracellular signalingpathways. These pathways stimulate the accumulation of proteins and othermacromolecules, and they do so by both increasing their rate of synthesis anddecreasing their rate of degradation. They also trigger increased uptake of nutri-ents and production of the ArP required to fuel increased protein slmthesis. oneof the most important intracellular signaling pathways activated by growth factorreceptors involves the en4rme PI 3-kinase, which adds a phosphate from AIp tothe 3 position of inositol phospholipids in the plasma membrane. As discussed inChapter 15, the activation of PI 3-kinase leads to the activation of a kinase calledToR, which lies at the heart of groMh regulatory pathways in all eucaryotes. ToRactivates many targets in the cell that stimulate metabolic processes and increaseprotein slmthesis. one target is a protein kinase called s6 kinase (s6&, whichphosphorylates ribosomal protein 56, increasing the ability of ribosomes to trans-late a subset of mRNAs that mostly encode ribosomal components. ToR also indi-rectly activates a translation initiation factor called eIF4E and directly activatesgene regulatory proteins that promote the increased expression of genes encodingribosomal subunits (Figure f7-6S).

Proliferating cells Usually coordinate Their Growth and Division

For proliferating cells to maintain a constant size, they must coordinate theirgrowth with cell division to ensure that cell size doubles with each division: ifcells grow too slowly, they will get smaller with each division, and if they grow


amino acids { " i ,

I

1 1 0 9

Figure 17-65 Stimulation of cell growthby extracellular growth factors andnutr ients. As discussed in Chapter 1 5, theoccupation of cell-surface receptors bygrowth factors leads to the activation ofPl 3-kinase, which promotes proteinsynthesis through a complex signal ingpathway that leads to the activation ofthe protein kinase TOR; extracel lularnutr ients such as amino acids also helpactivateTOR by an unknown pathway.TOR employs mult iple mechanisms tost imulate protein synthesis, as shown; i talso inhibits protein degradation (not

shown). Growth factors also st imulateincreased production of the generegulatory protein Myc (not shown),which activates the transcript ion ofvarious genes that promote cel lmetabolism and growth.4E-BP is aninhibitor of the translat ion init iat ionfactor elF4E.

activated growthfactor receptor

Isynthesis 55

+l

too fast, they will get larger with each division. It is not clear how cells achievethis coordination, but it is likely to involve multiple mechanisms that vary in dif-ferent organisms and even in different cell types of the same organism (Figure

r7-66).Animal cell groMh and division are not always coordinated, however. In

many cases, they are completely uncoupled to allow growth without division ordivision without growth. Muscle cells and nerve cells, for example, can grow dra-matically after they have permanently withdrawrl from the cell cycle. Similarly,the eggs of many animals grow to an extremely large size without dividing; afterfertilization, however, this relationship is reversed, and many rounds of divisionoccur without growth (see Figure 17-9).

Compared to cell division, there has been surprisingly little study of how cellsize is controlled in animals. As a result, it remains a mystery how cell size isdetermined and why different cell qpes in the same animal grow to be so differ-ent in size (Figure 17-67) . One of the best-understood cases in mammals is theadult sympathetic neuron, which has permanently withdrarnrn from the cellcycle. Its size depends on the amount of nerue growth factor (NGF) secreted bythe target cells it innervates; the greater the amount of NGF the neuron hasaccess to, the larger it becomes. It seems likely that the genes a cell expresses setlimits on the size it can be, while extracellular signal molecules and nutrients

generegulatory

factors

IV

ribosome

(c)

CELL GROWTH

IIt

CELL DIVISION

GROWTH FACTOR

CELL GROWTH

(B)

MITOGEN

CELL DIVISION

Figure | 7-66 Potential mechanisms for coordinating cel l growth and division. ln prol i ferat ing cel ls, cel l size is

maintained by mechanisms that coordinate rates of cel l division and cel l growth. Numerous alternative coupling

mechanisms are thought to exist, and dif ferent cel l types appear to employ dif ferent combinations of these

mechanisms. (A) In many cel l types-part icularly yeast-the rate of cel l division is governed by the rate of cel l

growth, so that division occurs only when growth rate achieves some minimal threshold; in yeasts, i t is mainly the

levels of extracel lular nutr ients that regulate the rate of cel l growth and thereby the rate of cel l division' (B) In

some animal cel l types, growth and division can each be control led by separate extracel lular factors (growth

factors and mitogens, respectively), and cell size depends on the relative levels of the two types of factors.(C) Some extracel lular factors can st imulate both cel l growth and cel l division by simultaneously activating

signal ing pathways that promote growth and other pathways that promote cel l-cycle progression.

growth factor

,/

II

(A)

1 1 10 Chapter 17: The Cel l Cycle

Figure 17-67 The size difference between a neuron (from the retina) anda lymphocyte in a mammal. Both cel ls contain the same amount of DNA.A neuron grows progressively larger after i t has permanently withdrawnfrom the cel l cycle. During this t ime, the rat io of cytoplasm to DNAincreases enormously (by a factor of more than 10s for some neurons).(Neuron from B.B. Boycott, in Essays on the Nervous System [R. Bel lairs andE.G. Gray, edsl. Oxford, UK: Clarendon Press, 1974.)

regulate the size within these limits. The challenge is to identify the relevantgenes and signal molecules for each cell type.

Neighboring Cells Compete for Extracellular Signal Proteins

\Arhen most types of mammalian cells are cultured in a dish in the presence ofserum, they adhere to the bottom of the dish, spread out, and divide until theyform a confluent monolayer. Each cell is attached to the dish and contacts itsneighbors on all sides. At this point, normal cells, unlike cancer cells, stop prolif-erating-a phenomenon known as density-dependent inhibition of cell diuision.This phenomenon was originally described in terms of "contact inhibition" of celldivision, but it is unlikely that cell-cell contact interactions are solely responsible.The cell population density at which cell proliferation ceases in the confluentmonolayer increases as the concentration of serum in the medium increases.Moreover, if a stream of fresh culture medium is passed over a confluent layer offibroblasts to increase the supply of mitogens, the cells under the stream areinduced to divide (Figure r 7-68). Thus, density-dependent inhibition of cell pro-liferation seems to reflect, in part at least, the ability of a cell to deplete themedium around itself of extracellular mitogens, thereby depriving its neighbors.

This type of competition could be important for cells in tissues as well as inculture, because it prevents them from proliferating beyond a certain populationdensity, determined by the available amounts of mitogens, growth factors, andsurvival factors. The amounts of these factors in tissues are usually limiting, inthat increasing their amounts results in an increase in cell number, cell size, orboth. Thus, the amounts of these factors in tissues have important roles in deter-mining cell size and number, and possibly the final size of the organ or tissue.

The overall size of a tissue may also be governed in some cases by extracel-lular inhibitory factors. Myostatin, for example, is a TGFB family member thatnormally inhibits the proliferation of myoblasts that fuse to form skeletal mus-cle cells. \A/hen the gene that encodes myostatin is deleted in mice, muscles growto be several times larger than normal. Remarkably, two breeds of cattle thatwere bred for large muscles have both turned out to have mutations in the geneencoding myostatin (Figure f 7-69).

conf luent mono layer : ce l l sno longer p ro l i fe ra te

stream of f resh mediumpumped across cel ls

neuron

a

lymphocyte

f low of medium st imulatescel l prol i ferat ion under the stream

Figure 17-68 The effect of fresh medium on a confluent cel l monolayer. Cells in a confluent monolayerdo not divide (graf l . fhe cel ls resume dividing (green) when exposed direct ly to fresh culture medium.Apparently, in the undisturbed confluent monolayer, prol i ferat ion has halted because the medium close tothe cel ls is depleted of mitogens, for which the cel ls compete.

ce l l s p ro l i fe ra te


Animals ControlTotal Cell Mass by Unknown Mechanisms

The size of an animal or one of its organs depends largely on the number andsize of the cells it contains-that is, on total cell mass. Remarkably, animals cansomehow assess the total cell mass in a tissue or organ and regulate it: in manycircumstances, for example, if cell size is experimentally increased or decreasedin an organ, cell numbers adjust to maintain a normal organ size. This has beenmost dramatically illustrated by experiments in salamanders, in which cell sizewas manipulated by altering cell ploidy (in all organisms, the size of a cell is pro-portional to its ploidy, or genome content). Salamanders of different ploidies arethe same size but have different numbers of cells. Individual cells in a penta-ploid salamander are about five times the volume of those in a haploid sala-mander, and in each organ the pentaploids have only one-fifth as many cells astheir haploid cousins, so that the organs are about the same size in the two ani-mals (Figure L7-7O andFigure 17-71). Evidently, in this case (and in many oth-ers) the size of organs and organisms depends on mechanisms that can some-how measure total cell mass. How animals measure and adjust total massremains a mystery, however.

The development of limbs and organs of specific size and shape depends oncomplex positional controls, as well as on local concentrations of extracellularsignal proteins that stimulate or inhibit cell growth, division, and survival. As wediscuss in Chapter 22, we now know many of the genes that help pattern theseprocesses in the embryo. A great deal remains to be learned, however, about howthese genes regulate cell growth, division, survival, and differentiation to gener-ate a complex organism.

The controls that govern these processes in an adult body are also poorlyunderstood. \A/hen a skin wound heals in a vertebrate, for example, about adozen cell types, ranging from fibroblasts to Schwann cells, must be regeneratedin appropriate numbers, sizes, and positions to reconstruct the lost tissue. The

1 1 1 1

Figure 1 7-69 The effects of a myostatinmutation on muscle size, The mutationleads to a great increase in the mass ofmuscle t issue, as i l lustrated in this BelgianBlue bul l . The Belgian Blue was producedby catt le breeders and was only recentlyfound to have a mutation in theMyostatin gene. (From H.L. Sweeney,Sci. Am. 291 :62, 2004. With permissionfrom Scienti f ic American.)

Figure 17-70 Sections of kidney tubulesfrom salamander larvae of differentploidies, In al l organisms, from bacteria tohumans, cel l size is proport ional to ploidy.Pentaploid salamanders, for example, havecells that are much larger than those ofhaoloid salamanders. The animals andtheir individual organs, however, are thesame size because each t issue in thepentaploid animal contains fewer cel ls.This indicates that the size of an organismor organ is not control led simply bycounting cel l divisions or cel l numbers;total cel l mass must somehow beregulated. (Adapted from G. Fankhauser, inAnalysis of Development [8.H. Wil l ier,P.A. Weiss, and V. Hamburger, eds.l,pp. 126-150. Phi ladelphia: Saunders, 1955.)

HAPLOID

11 chromosomes

DIPLOID

22 chromosomes

PENTAPLOID

55 chromosomes

1'112 Chapter 17:The Cel l Cycle

Figure 17-7' l The hindbrain in a haploid and in a tetraploid salamander.(A) This l ight micrograph shows a cross section of the hindbrain of ahaploid salamander. (B) A corresponding cross section of the hindbrain of atetraploid salamander, revealing how reduced cel l numbers compensatefor increased cel l size, so that the overal l size of the hindbrain is the samein the two animals. (From G. Fankhauser, lnt. Rev. Cytol. 1:165-193, 1952.With permission from Elsevier.)

mechanisms that control cell growth and proliferation in tissues are likewisecentral to understanding cancer, a disease in which the controls go wrong, asdiscussed in Chapter 20.

Summary

In multicellular animals, cell size, cell diuision, and cell death are carefully controlledto ensure that the organism and its organs achieue and maintain an appropriate size.Mitogens stimulate the rate of cell diuision by remouing intracellular molecularbrakes that restrain cell-cycle progression in Gy Growth factors promote cell growth(an increase in cell mass) by stimulating the synthesis and inhibiting the degradationof macromolecules. For proliferating cells to maintain a constant cell size, theyemploy multiple mechanisms to ensure that cell growth is coordinated with cetl diui-sion. Animals maintain the normal size of their tissues and organs by adjusting cellsize to compensate for changes in cell number, or uice uersa. The mechanisms thatmake this possible are not known.

(B)100 pm

PROBLEMSWhich statements are true? Explain why or why not.'17-'l Since there are about 1013 cells in an adult human,and about 1010 cells die and are replaced each day, webecome new people every three years.

17-2 The regulation of cyclin-Cdk complexes dependsentirely on phosphorylation and dephosphorylation.

17-3 In order for proliferating cells to maintain a relativelyconstant size, the length of the cell cycle must match thetime it takes for the cell to double in size.'17-4 \A4rile other proteins come and go during the cellcycle, the proteins of the origin recognition complex remainbound to the DNA throughout.

17-S Chromosomes are positioned on the metaphaseplate by equal and opposite forces that pull them toward thetwo poles of the spindle.

17-6 If we could turn on telomerase activity in all our cells,we could prevent aging.

Discuss the following problems.

17-7 Many cell-cycle genes from human cells functionperfectly well when expressed in yeast cells. \A/try do yousuppose that is considered remarkable? After all, manyhuman genes encoding enzymes for metabolic reactionsalso function in yeast, and no one thinks that is remarkable.

17-8 You have isolated anew Cdcmutant of buddingyeastthat forms colonies at 25"C but not at 37.C. You would now

like to isolate the wild-type gene that corresponds to thedefective gene in your Cdc mutant. How might you isolatethe wild-type gene using a plasmid-based DNA library pre-pared from wild-t1pe yeast cells?

17-9 You have isolated a temperature-sensitive mutant ofbudding yeast. It proliferates well at25"C, but at 35'C all thecells develop a large bud and then halt their progressionthrough the cell cycle. The characteristic morphology of thecells at the time they stop cycling is knor,tn as the landmarkmorphology.

It is very difficult to obtain s1'nchronous cultures of thisyeast, but you would like to know exactly where in the cellcycle the temperature-sensitive gene product must func-tion-its execution point, in the terminology of the field-inorder for the cell to complete the cycle. A clever friend, whohas a good microscope with a heated stage and a video cam-era, suggests that you take movies of a field of cells as theyexperience the temperature increase, and follow the mor-phology of the cells as they stop cycling. Since the cells donot move much, it is relatively simple to study individualcells. To make sense of what you see, you arrange a circle ofpictures of cells at the start of the experiment in order of thesize of their daughter buds. You then find the correspondingpictures of those same cells 6 hours later, when growth anddivision has completely stopped. The results with yourmutant are shor.trn in Figure Qf 7-f .A. Indicate on the diagram in Figure Q17-l where the exe-cution point for your mutant lies.B" Does the execution point correspond to the time at whichthe cell cycle is arrested in your mutant? How can you tell?

1 7- 1 0 The yeast cohesin subunit Scc 1, which is essential forsister-chromatid pairine, can be artificiallv reeulated for

EN D-OF-CHAPTER PROBLEMS

expression at any point in the cell cycle. If expression isturned on at the beginning of S phase, all the cells divide sat-isfactorily and survive. By contrast, if Sccl expression isturned on only after S phase is completed, the cells fail todivide and they die, even though Sccl accumulates in thenucleus and interacts efficiently with chromosomes. 'Why doyou suppose that cohesin must be present during S phase forcells to divide normally?

17-1'l If cohesins join sister chromatids all along theirlength, how is it possible for condensins to generate mitoticchromosomes such as that shornm in Figure Ql7-2, whichclearly shows the two sister chromatids as separate domains?

1 * .

Figure Q17-2 Ascanning electronmicrograph of aful ly condensedmitoticchromosome fromvertebrate cells(Problem 1 7-1 1).(Courtesy ofTerryD. Al len.)

17-12 High doses of caffeine interfere with the DNA repli-cation checkpoint mechanism in mammalian cells. \A/hythen do you suppose the Surgeon General has not yet issuedan appropriate warning to heavy coffee and cola drinkers? Atypical cup of coffee (150 mL) contains 100 mg of caffeine(196 gi mole). How many cups of coffee would you have todrink to reach the dose (10 mM) required to interfere withthe DNA replication checkpoint mechanism? (A tlpicaladult contains about 40 liters of water.)

17-13 A living cell from the lung epithelium of a newt isshown at different stages in M phase in Figure Ql7-3. Orderthese light micrographs into the correct sequence and iden-tify the stage in M phase that each represents.

17-14 How many kinetochores are there in a human cell atmitosis?

17-15 Aclassic paper clearly distinguished the properties ofastral microtubules from those of kinetochore microtubules.

1 1 1 3

Figure Q17-3 Light micrographs of a single cell at different stages ofM phase (Problem 17-13). (Courtesy of Conly L. Rieder.)

Centrosomes were used to initiate microtubule growth, andthen chromosomes were added. The chromosomes boundto the free ends of the microtubules, as illustrated in Figure

Ql7-4.The complexes were then diluted to verylowtubulinconcentration (well below the critical concentration formicrotubule assembly) and examined again (Figure Ql7-4).As is evident, only the kinetochore microtubules were stableto dilution.A. Why do you think kinetochore microtubules are stable?B. How would you explain the disappearance of the astralmicrotubules after dilution? Do they detach from the cen-trosome, depoll'rnerize from an end, or disintegrate alongtheir length at random?C. How would a time course after dilution help to distin-guish among these possible mechanisms for disappearanceof the astral microtubules?

17-16 'v\hat are the two distinct cytoskeletal machines thatare assembled to carry out the mechanical processes ofmitosis and cytokinesis in animal cells?

17-17 How do mitogens, growth factors, and survival fac-tors differ from one another?

Figure Q17-4 Arrangements of centrosomes, chromosomes, andmicrotubules before and after di lut ion to low tubul in concentrat ion(Prob lem 17-15) .

budsize at t ime oftemperature

shift

tf

+

Figure Q1 7-1 Time-lapsephotography of a temperature-sensitive mutant of yeast(Problem 17-9). Cells on theinner ring are arranged in orderof their bud size, whichcorresponds to their posit ion inthe cel l cycle. After 6 hours at37"C, they have given r ise tothe cells shown on the outerrlng. No further growth ordivision occurs.

/ \

after d i lut ionbefore di lut ion

REFERENCES

General

Morgan DO (2007) The Cell Cyc{e: Principles of Control London: NewScience Press

Murray AW & Hunt T (1993) The Cell Cycle: An Introduction New York:WH Freeman and Co

Overview of the Cell Cycle

Forsburg 5L & Nurse P (1991) Cell cycle regulat ion ln the yeasts

Saccharomyces cerevisrae and Schizosaccharomyces pombe Annu Rev

Cell Biol 7:227-256Hartwell LH, Culott i J, Pringle JR et al (1974) Genetic control of the cel l

division cycle in yeast Sclence 183:46-51Kirschner M, Newport J & Gerhart J (1 985) The t iming of early

developmental events in Xenopus TrendsGenet 1:41-47

1114 Chapter 17: The Cel l Cycle

Nurse P, Thuriaux P & Nasmyth K(1976) Genetic control of the cel ldivisron cycle in the fission yeast Schizosaccharomyces pombe. l\lolGen Genet 146:167-178.

The Cell-Cycle Control SystemEvansT, Rosenthal ET, Youngblom J et al (1983) Cycl in: a protein

specif ied by maternal mRNA in sea urchin eggs that is destroyed ateach cleavage dtvision Cel/ 33.389-396

Lohka MJ, Hayes MK & Maller Jt (t9BB) Purif icat ion of maturarionpromoting factor, an intracel lular regulator of early mitot ic eventsProc Natl Acad Scl U5A 85.3OO9-30'l 3

Masu i Y and Marker t CL (1971) Cy top lasmic cont ro l o f nuc ear behav io rduring meiotic maturation of frog oocytes J Erp Zaol 177:129-146

Morgan DO (1997) Cycl in-dependent kinases: engines, clocks, andmicroprocessors Annu Rev Cell Dev Biol 13)61-291

Murray AW & Kirschner IVW (1989) Cyc in synthesis drives the earlyembryonic cel l cycle Nature 339.275-2BA

Pav e t ich NP ( ,1999) Mechan isms o f cyc l in dependent k inase regu la t ionstructures of Cdks, their cycl in act ivators, and CIP and Ink4 inhibitorsI l\,/ol Biol 287.821-B2B

Peters lM (2006) The anaphase promoting complex/cyc osomer amachine des gned to destroy Nature Rev Mol Cell Biol L644-656

Petroski MD & Deshaies R.J (2005) Function and regulat ion of cul l in-RING ubiquit in l igases A/cture Rev Mol Cell Biol 6:9-2A

Wittenberg C & Reed Sl (2005) Cell cycle-dependent transcript ion inyeast: promoters, transcript ion factors, and transcriptomesOncogene 24:2746-2755

S Phase

Arias EE & Wa ter lC (2007) Strength in numbers: preventing rerepl icat ionvia mult iple mechanisms rn eukaryotic ce ls Genes Dev 21 ,497-518

Bell SP & Dutta A (2002) DNA repl icat ion in eukaryotic cel ls Annu RevBiochem 71.333-374

Bell SP & Sti l lman B (1992) ATP dependent recognit ion of eukaryotrcorigins of DNA repl icat ion by a mult iprotein complex ,^/drure357:128 134

Diff ley JF (2004) Regulat ion of ear y events in chromosome repl icat ionCurr Biol 14.R778-R786

Groth A, Rocha W, Verreault A et a (2002) Chromatin chal lenges duringDNA repl icat ion and repair Cell 128.721-733

Tanaka S, Umemori I Hiral K et al (2007) CDK dependentphosphorylat ion of Sld2 and Sld3 init lates DNA repl icat ion inbuddrng yeast A/cture 445:328-332

Zegerman P & Diff ley )F (2007) Phosphory arion of S d2 and Sld3 bycyc l in -dependent k inases promotes DNA rep l i ca t ion in budd ingyeasl Naturc 445:28- 285

Mitosis

Cheeseman lM, Chappie JS, Wiison Kubalek EM et al (2006) Theconserved KMN network consti tutes the core microtubule bindingsite of the k netochore Cell 127983-99t

Dong Y, Vanden Beldt KJ, Meng X et al (2002) The outer plate invertebrate kinetochores s a f lexible network with mu t ip emicrotubule interactions Nature Cell Biol 9:516-522

Heald R, Tournebize R, Blank T et al (1996) Self-organization ofmic ro tubu es in to b ipo la r sp ind les around ar t r f i c ia l chromosomes inxenopus egg extracts Nature 382.420-425

Hirano T (2005) Condensins: organizing and segregating the genomeCurr Biol 1 5.R265 R215

Kapoor TM, Lampson MA, Hergert P et al (2006) Chromosomes cancongress to the metaphase p ate before biorientat ion Science31 1 :3BB-391

Mitchison T & Kirschner M (1984) Dynamic instabi l i ty of microtubulegrowth, Nature 312 237 242,

Mitchison TJ (1989) Polewards microtubule f lux in the mltot ic spindle:evidence from photoactivation of fluorescence J Cell Biol109:637 652

Mitchison TJ & Salmon ED (2001) Mitosis: a historyof division NatureCell Biol 3:817-E21

Musacchio A & Salmon ED (2007) The spindle-assembly checkpoint inspace and lme Nature Rev llol Cell Biol 8319-393

Nasmyth K (2002) Segregating sister genomes: the molecular biologyof chromosome separation Science297:559 565

Nigg EA (2007) Centrosome duplication: of rules and l icenses IrendsCell Biol 17.215 221

Nurse P (1990) Universal control mechanism regulat ing onset ofM-phase Nature 344.503-508

Page SL & Hawley RS (2003) Chromosome choreography: the meioticballet Sclence 301 .785-789

Petronczki M, Siomos MF & Nasmyth K (2003) Un menage ; quatre: themolecular biology of chromosome segregation in meiosis Cel/112:4T 444

Salmon ED (2005) Microtubules: a r ing for the depolymerization motorCurr Biol 1 5:R299-R302

Tanaka TU, Stark MJ & Tanaka K (2005) Kinetochore capture and bi-orientation on the mitotic spindle ,\arure Rev ltlol Cell Biol 6:929-942

Uhlmann F, Lottspeich F & Nasmyth K (1999) Sister-chromatidseparation at anaphase onset is promoted by cleavage ofthecohesin subunit Sccl, Nature 40a37-42

Wadsworth P & Khodjakov A (2004) E pluribus unum: towards a universalmechanism for spindle assembly Trends Cell Biol 14:413-419

Cytokinesis

Albertson R, Riggs B & Sull ivan W (2005) Membrane traff ic: a drivingforce in cytokinesis Trends Cell Biol 15:92-101

Burgess DR & Chang F (2005) Site selection for the cleavage furrow atcytokinesis Trends Cell Btol 15.156-162

Dechant R & Glotzer M (2003) Centrosome separation and centralspindle assembly act in redundant pathways that regulatemicrotubule density and tr igger cleavage furrow formatioa. Dev Cell4.333 344

Eggert US, Mitchison TJ & Field CM (2006) Animal cytokinesis: fromparts l ist to mechanisms Annu Rev Biochem /5:543-566

Glotzer M (2005) The molecular requirements for cytokinesis Science307:1735-1739

Gri l l SW, Gonczy P, Stelzer EH et al (200'1) Polari ty conrrols forcesgoverning asymmetric spind e posit ioning in the Caenorhabdit ise I eg a n s embry o, N at u re 4a9.630-633

Jurgens G (2005) Plant cytokinesis: f ission by fusion Trends Cell Biol15:277 283

Rappaport R (1986) Establ ishment of the mechanism of cytokinesis inanimal ce ls, lnt Rev Cytol 105:245-281

Control of Cell Division and Cell Growth

Adhikary S & Eilers M (2005) Transcript ional regulat ion andtransformation by Myc proteins Nature Rev l,,4ol Cell Biol 6.635 645

Campisi J (2005) Senescent cel ls, tumor suppression, and organismalaglng: good c;t ize1s, bad neighbo.s Cell 120:513 52)

Conlon 1 & Raff M (1999) Size control in animal development Cel/96.235-244

Frolov MV, Huen DS, Stevaux O et al (2001 ) Functional antagonismbetween E2F family members Genes Dev 152146 2160

Har r ison JC & HaberJE (2006) Surv iv ing the breakup: the DNA damagecheckpoint Annu Rev Genet 40:209 235

Jorgensen P & Tyers M (2004) How cel ls coordinate growth anddivision Curr Btol 14.R1014,R102/

Levine AJ (1997) p53, the cel lular gatekeeper for growth and divisionCell 88:323-331

Raff MC (1992) Social controls on cel l survival and cel ldeath lVoture356.397-4AA

Sh-.rr Cl & DePinho RA (2000) Cellular senescence: mitot ic clock orculture shock? Cell 102:407-410

Sherr CJ & Roberts JM (1999) CDK inhibitors: posit ive and negativeregu la to rs o f G1-phase progress ion Genes Dev 13 :1501-15,12

Trimarchi JM & Lees )A Q)aD Sibl ing r ivalry tn the E2F family, NatureRev Alol Cell Biol 3:,l 1 -20,

Vousden KH & Lu X (2002) Live or let die: the cel l 's response to p53Nature Rev Cancer 2594-604

Zetterberg A & Larsson O (1 985) Kinetic analysis of regulatory events inG1 leading to prol i ferat ion or quiescence of Swiss 3T3 cel ls, Proc NatlAcad Sci USA 82.5365-5369

stltJoJ 'ruels^s sno^Jeu elpJqeue^ Surdoleêp aql uI 'SuHsluolse eq upc senssrl [Bru-rue llnpu pue Sudolaêp ul srncto lpqr qlpep IIec pauruerSord;o lunoue aqJ

slle) pelue/nun seleulutlll qreao lla) peuuerootd

'eseesrp uEtunq olelnqrJluoJ uec srsoldode luelJgJnsu ro alrsseoxe ,vroq pue ,uoqepSer str puusrsoldode Jo rusrueqJaru Jplncalou eql 'slutulup ul qleep IIec peurrueJSord gosuouJuru aql ssnsslp arrn taldeqc sHl uI 'ueql JeprsuoJ tou [eqs e1\^ pue ,sllac

Ipurue ur srsoldode alelpatu leql asoql ruo4 toullsrp are sesec eseql q pellonursrusruuqoaru JplnJeJoru eqJ 'plJeloeq pue slsuad Bulpnlcur ,srusruetro re1n1-leJrun ur sJnJOo ueê qleep ilec peuruuJSor4 'uorlca;ur pue r{:n[ur o1 esuodse.req] ur se ile^ su 'senuel pue sJervrogJo ecueJsaues aql q pue luaurdolanap 8ur-rnp sJn330 11 's]ue1d uI 'sprurue ol peuuuoJ lou sr qteep lec paruuerS0rd

' (f, I-8I arn8rg) esuodsar .{:oleunuegur uu 3uqrc11epue sroqqBreu rraqt JaAo slueluoJ Jreql 8u1pds 'lsJnq pue IIeMs sllac JItoJceN'slsoJJau

IIaJ pelIEJ ssacord e dq os op Llunsn f1ddns poolq Jo {cEI e Jo €runeJlse qcns 'llnsur elnoe ue ol esuodsar u1 dleluaplccp erp leql sllec Ipurue ,(uur8-ord qluap Jelnllaf,EJtur ue Jo uoueJedo aql saqdun qJrqm) qleep IIaJ paruuurB-ord;o sr.uJoJ pezrJeloprpqc llalr,r ssel Jeqlo pue srsoldode ol lserluoc ,tg

'luelxe slr elerxrlserapun fyqeqord 11t1s pue srear( dueur ro; srsol-dode pa>1oo1rano slsr3o1o1q dryr,,(lqeqord s srql 'srsoldode ,i.q perp eÊq sllac JosJeqrunu a8rel uaqirn uele 'uaes eq ol slleJ peep fl\al f1ensn eJB ereql ,rt14cmb ospalsa8rp puu uelee ale slleJ aql esneceg'asuodsar droleuruugur Sufeurep e 8ur-snec ]noql!v\',{.uir,re parealc rtlprder sr pue rtlleau setp IIaJ aq1 dem srq} uI .(SI-8Iarn8rg) stueluoJ laql pds uec,teql eroJaq 'uaql s;p8ua,{prder (gZ Je}deqf,ur passncsrp 'llac cu,tco8eqd pazrprcads u) aSuqdorceru e Jo IIaJ Suuoqq8raue leql os 'peJelle dlecruaqc seuocaq serpoq clloldode Jo IIec eql Jo eouJrnsaql fllueuodrur 1soy,1 'sa?poq cllo\dodo pelleo slueu8er; pasolcue-eueJqruetuo1u1 dn $leerq uago 'a8re1 sr ilec eqt JI ,pu€ sqelq uego ereJrns IIer eqJ .(VI{Iarn8gfl sluau8er; o1u1 dn $leeJq pu€ sesuepuoo ulleruoJqc JEelJnu eq] pue'selqruessesrp adolanua JEalJnu aq1 'sasdelloc uola1a1sol.,io eql ,asuepuoc pue{urJqs daqt .IOOC>'sa8uuqc pcrSoloqdrou cusrJalceJeqc o8rapun srsoldodedq 8u$p sga3 flqua8uuqJrelu srsoldode pue qteep IIeo perxuerSord sruraleql asn uago slsrSolorq ,(lSursrguoc 'pue 'poolsJepun lseq pu€ uoruruof, lsorueqt rBJ [q sl rl 'U]eap

ilec peuruerSord;o uroJ euo dpo s1 srsoldode qtnoqrry'(aeJl e ruo4 selpal se ,,TJo 3u11e;,, Suruearu prom IeeJC eql uro4) slsoldodudq srncco 'dlarrrsnlcxa lou lnq l.lpnsn slerurue ur qluap 1ac paruurerSor4

'loJluoc sll pue qleap IIec paunuerSord ol peleJrpep saua8 lsrg eql pegnueprrcr4t suaBa1a 3 epolerueu eql ur sepnls cpaua8 uo papuadap pue sruar{ 97 reqtoue1oo1 ecueldacce prauaB sll tnq ,s0Z6I eql ur pasodord sem, urerSord qleep ur-lllnq E anpq slleJ lerurue leql eepl aqJ 'qluep

IIaJ paurursJ8o.rd se urvrou4 ssacorde-derrrr pelJorluoJ e ur sallasrueql ilpl pue ruerSord qleep Jelnllef,pJlur ue elBA-llJu sllec aq] qclqv\ uI 'saprcFs ere sqlsep IIaJ ,([pruJou,, eser0 ]eql Motr{ 1v\ou e a'qleep

[Ie] aql seJuelBq rlpexa uorsr^lp [eJ 'srusruEqcau.,i-rolep8er u,rnoulun dq'asneJeq

{urJqs lou op senssp rno rnoq z{rana aunsalur puB MoJJuru auoq eql uletp sllar Jo suoq[q 'uerunq ]InpE fqlleeq E uI 'pooqrppe o]q senu4uoc dgensn 1rpuu 'luarudolalap luuld prrp lerurrre ur ued tuelrodur,tgercruc e s,{e1d qleep [eC

s!sotdodv

Molecular biology of the cell, 5th ed

Science

Transcript of Molecular biology of the cell, 5th ed