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    Chapter 08

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    Wilco Rave

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    of 36
    pa jie perce ine yb Adapting to Life ona Rotating World at the Gene Expression Level Time isthe substance from which Iam made. Jonge Luis Borges Introduction: An Important Aspect of Circadian Regulation (f Biological Processes Is Conveying Output Signals at the Gene Expression Level® ‘Many biological processes are timed by the clack in almost ll eukaryotes and in some prokaryotes» hy thc outputs may be manifested at several factional levels in organs. An example ofa ince outpat s the regulation o transcriptional efor pate eur gene At the tase level an example is daly lef movement (igure 83) The Compound leaves of many legume are opable of movement, lcing a night ana opening by day, and the cycle i contoled bya icadia cock The level ofthe ‘whole organism exemplified by regulation of lep-wske pattems Whatever the process andl whatever the evel lately al hye behvie pater ave tele Eris in moeclar events at the elu evel. Programming spect evens to cccurn an organism at appropriate mes fd isby far the most commonly recognized function of endogenous pacemnekers Understanding how thi timing works is a ase problem in celular egulation hat ‘has widespread ramifications in many els of biology, By he time ofthe Cold Spring Harbor Symposium on Biological Clocks in 1960 large literature was i place describing ck controled phenomena, “he focus of sty was recognizing ccadian behaviors hythms and docu renting their physical fntures (ae Chapters Land 3) and thr ecogia advan tape fr orgasms (sce Chapters 2and Boone cocks ha not best lcalized at the molecular level te progress had been made in understanding how the ck conto particule rhythm Regardless of the complexity othe organist the 256 Chapter 6 regulatory output ofthe lock the cicadian clock iu ‘Sealy ambled ah eel othe nce weth ‘ontaling dock opel, The advent of gene expression research in chronobiology fay recent”™ Early workoncradian rythm produced a rich ge ey and eataloging of ehythmic Biological processes. “Tnpring recor tard understanding ho ae! wt the cock regulate had to wait for development in he Feld of maleculr genetics (ove Chapter), However, Pittendigh and colleagues noted in a 1959 report on Newosprm css, "We dono! inteepet the presence ot sence of zonation as a eection ofthe presence Absence ofthe clock but athe 5 coupling or uneou pling om th lock ofthe particular processes leading to znation* They right conclael that the process of znaon aeral ope development in Nt, as Alitinct and independent fom an underlying clock mechan By the ime ofthe Cla Spring Hasbor symposium, scielst were leary diferentiting outpt fom the Basic timekeeping mechanism. In the opening talk, Bing emphasized the importance of understanding thot prin changes were being regulate by the clock nd were not sential with te cock, To make the i Sineton care Renamed thee regulted proses he Fins of the clock. At the same meeting, Halberg ie cussed physiological relation af tytn event 35 anspor the ght mount he ight mater tthe "gh destination atthe right ime He noted hat die: “ntehythmicfunctions could acer silent ves of {ay an therfore have iret phases, yet a may D6 Synchronized by a specific imekeeping mechanism Blochemial and molecular analysis of the hands of the clock was ust begining ‘some of the ells work on biochemical hythms involved cycling of carotenoids and ehorophyl ol les in relation torte of protosjaess. Bore 198, Hasings wns messing "CO, incorporation aa mes tre of photosynthesis He and others were discs the ides that nuclei acd metabolism might pay le inclock function Becnsetythns wee Krown no diving single-celled organisms, he postulated that ‘changes in metals of DNA not associated with di Son or RNA metabolism might playa sole "After examining the incorporation of P-labeled inorganic phosphate ints both RNA and DNA over time; Hastings conced tht mace aid metabolism phosphate dehydrogenase) ‘ctv and sbundance ifr when compare ote 0 protein synthesis nvasured by rection of NADI ‘o NADPH, West Bs analysis ad [*S}ethonine ‘Incorporation, espectively a: ibe discusses 5 chapter) Another sample, the rate of tansriptional Synths of burn miNA. shows very song cca ‘dan yt in at hence, cle decved fom mam malian liver Nevertheless, the mRNA's long halite ‘sults in a neal consant level of wanscip ver the ‘course ofthe day ‘Measurement ofthe tansception rates of randomly selected liver mRNA¥ from complementary DNA [GDNA) clones indieates that up to 20% ofthe genes ‘xpresed in hepatocytes may be subject to decadian ‘Cancrption However he percentage of ver NAc tv sigan yas hl abundance appest © the ircdion signal wil undergo even farther modu ‘on asthe leks outputs are conveyed fe efector els ‘isthe production of tokines, the secretion of Bor ‘ones, ransmisionb synapses and even indely the rythm regulation of Behave Differentiating Molecular Outputs from the Clock Machinery Aids in Construction of a ‘Working Clock Medel?" Clock outputs canbe distinguished from ‘sear componen's® (One implication of simple input-rsiltor—output ‘adel the extnce of Biochemical proses that ae ‘tlle by the cada clock ut tent pat of he lock mechanism tel (te Chapter?) Experimental ‘change inthe concentration or activity ofan output ‘molecule regulated by the oc, including elimination tf the output, should not alte the generation of ht tty pers. This will old true ony the Outpt Under consideration does notin any aye bck 0 ‘heciocko onto cknput Aclocecotoied hth, ‘whether elated o motity, development oe umines- ence, hasbeen considered analogous tothe hand of ‘mechanical clock can be removed without the progres ofthe undetying mechanism Being alered {vigenced by the movement he remaining hand Tn the case of Gags, inhibition of photosythe- ‘edocs not ate the Bokumineacence hythm On te bis of single clock model, this has bee ntrpreted to mean that photosynthesis represents an output hand ofthe clack Thesame result however, would also be expected if the sythns of photoxyess and bok ‘minercence were deen by two diferent locks. The ‘lock hand analogy should be accepted very autusly for organisms in which known genetic lesions for 5 feedback loop do nat exist Knowledge of output regulation may lead back to osellator components” As discussed in Chapter 7, thee main research ap- proaches have been taken withthe alm of lesen Shout the molecular footings of circadian rhythmicity (One of there approsches fm tack the ny tmic rp Jaton of an output ack tothe ack I the biochemty of acradian-eplatd process ove, then the ol ‘Sule iavelved in is contol are expecedtohave some ‘laionah tothe acne mechanism el Tebould ‘be pase to follow the output pathway backward to ‘lekate thi mechanism “Te eal input-output model specie a strategy to ‘experimentally dent the molecules sting ceilitor ‘ten the reeled malecale were la acomponent of ‘hoa tal” In this ease, a molecule repultng a8 ‘uit pres night ban cclstor component al 2 byafeting a rate-iting step. Anltration of his ‘Shera pnp the rythm of lyase Chapter yin which ATP isa product ofthe yey pathray snd ao component ofthe orilstor In both inact yeast and yeast extract, glycol is shythmie tite fo feedback on the slowest and ‘ateliting sep the atvity ofthe enaje posto feuctlinase (PEK). When metabolic flu is high AT? levels ae and PFKisinhbles,shuting down the path tna: The metabolic xf now reduce, cropping ATP Teves and reactivating PPC Ta his way, PKs the focal point af contol anda pat ofthe rhythm generating ‘machinery Ths by amenting he atv of tesla Adapting tion a Rating World athe Gene Expression Level 8 FOURE KS The cas 9-2 gene isan op gene based {hechendan ce Hee vane sbendonesie ogre freinawidtypesanands deh pends op Pe ‘im yi edie chen) Tengen ‘ststep, the lock could control the activity of am entre ‘utp pathway The eas gene in Neurospora helps elucidate ‘readian gene expression A molecular example ofa clock hand known not to tfc the cloak that replats it const ofthe pods ofthe es (aso known a x2) locus (Figuse #8). In Nearaspor, the esc) gene is ehythialy ta sexptoally contlld by the ry Dave feck loop, ‘Mutations in FRO that chaage clock period length sppropristely change the cyte ung oes 2 tan Spon and mRNA abundance, conten tat EAS (CCG.2) i epulted bythe FRO-Danedenllator(ee Figure 8), The EAS (CCG-2}product isa hydrophbin rosin, conferring an extracel, Ayrophobic surface bu fie roets made trom ces-inked FAS (CCG-2}an fungal spores Various mutasons in ais ext ncading some that confer complet oso function Av expect fc the mutant phenotype isa los of hydeophotey resulting in wettability and clumping of Neer Spores. When examined atthe ver of molecule lev ‘sin these mutans including the los oftuncton muta the lacks four operate normally Both he {ry tanscipt and the RQ protein cyce show a ld type periodicity and amplitude in Northern and Westen blot analy, sexpectively Output rhythms, Ineluding thoes of ether clockcontrlied genes and ‘condition, wil be Gea nti chap ore Sant conditions wer alo inctnguishabl fom wid type shythms, even though the morphology of the spores was altered. "An eatly and tit definition of «rhythmic output ‘ene o: proves had the componens 1) The endoge 2 veut aeons BeOee - see we ‘hyn pests nde const conitons The yr Inetivaion ofthe gene has no lect ote ick (Courtesy nous rhythm persis under constant conditions, 2) the Peo length ofthe utp yim elt the gohatype tthe strain, and (9) alteration oe inactivation of the iene or process has no eet the clock, Tete ate vents 0 ti cefiition, however In pricy asthe level sophitzason and analysis inceased nc fadlan systems examples have emerged in which the rovct ofthe utput regulation fede back onto com PPonens ofthe ssllato o input In his way output $c input cating nested fedback lope sueound= Ing the cow oslo Outputs May Have Effects on Oscillator Components=871 [NEUROSPORA ASAGOOD EXAMPLE. In ation oe ingock tof input a deserted in the previous sac ton, outputs ean feed back to affect the clock teh ‘herby generating tly ested st of oops in the ag ‘er cucadian sysem that sound the core orator The ease ofthe VD protein in Newsy was described earlier oe Figure 85) and terested hee ‘WD ene is et by interacting wih the WE poo tein, whichis ove of the PAS-PAS heterodimeric an scriptional activators nthe Nearesor core feedback, loop, inaction to Belg the Blse-igh photorseptor and therefore integral tothe light input patinray Further the sythesie of VWD i clck rule and therefore could conse a part ofthe oslo oop, ‘excep that VVD expression alo depends on ight 2 ‘ot surprising that VWD hos ile effect onthe lock fre-raing inconstant darkens sl # provides 90 example of tpt tha can alles the clock ise, n ths case creating lop that closes within the esi tory ce 264 Chapter 8 STS as ANOTHER GOOD BxAMMLE, Another Wel tlesbed example of output eesing bck on the os Inte comes rom work in mammals Socal iterstions naff broad spectrum oflock properties a= ‘Sets Clock regulation of locomatot activity in man thal was mentioned in earlier chapters, Locomotor vy the mort commonly used say for ication “hythaiey in mammalian 5ST. Some years ago shady wor being done to scren for clock reatng eects of hypnotic drugs that would induce sep infhurans andthe outcome secrned tobe thatsome ofthe drugs had strong phase specie es Insome rodents but not in others. Of eoute hypnotics ‘otonly put an anima ose, but aa esl they aso TEmporutlly block the locomotor att outputs the Fight conto for» ypaati i to give the drug at the ne circadian phase to an ail that is not active then, something that could be done with urna and ‘octuna asl When the conols wet finally sore FIGUREA? Eset sot iterations on he hamster Salis, he sete emg ‘purse svar ltr tt eres hamster ‘vera acy eves, Tite activities ince exploration of Sevan gtr cal oe tng el The apa angle ssh moti nt ‘ike ict thea ab hese bar fe ee ‘Rihows he entre cr roped ta ne ‘Shswotones nce y the elnctrs() Troe SStrelnovelrindd wheel amin atest paseo! ‘hom’ moh pee Sag Cia aes (a fom Mon, 9888 oon gerber e057) x ut the surprising result was that the drugs them Selves were having tiffs, but the change is loco Inotor acy ad aor effect on te ack at the Foren cock sserstveto variety of mul ha ect Tecomotor activity as igure 82 shows. "The mec ese fhe as boen raed 0 ‘he cniltory ellen the SCN, Locomotor actly Seas the salty othe PER prsine there and can thereby eset the pase ofthe osclator. The resetting ‘mechanism, however, stl largely unknown, Selected Case Studies Show the Molecular and Biochemical Correlations of Physiological and Behavioral Rhythms? ‘An overview indicates that clock regulation of Cellar physiology and behavior must relat in Some way to changes in cellular biochemistry ‘Avecessary part f understanding physiologic! oF ‘chav sans toexplin how these yt are hus relate fo anderyingoseltions in the cls Pochemisry such arto changes i the activity ofa key faye othe levels of kay substateImporiel steps Indeveloping schon understanding are to determine the biochemistry srd molecular biology andedying the regulate proves ind then to ont» focal pont or ‘etelmiting sep where the process might regulated Byte clock os densbed in Chapter? for PEK asa p= al egultor a geass. ‘Anvenayane'scetiity might be altered in many way angi ron dramatichanges in abso levels ‘Sune changes strctre The powerful techniques tf meer olecuas biology have mode it possible to {vest the mecha regulating gene expression, find good examples showing lock contol at iferent Teves in many systems have been found. These ex Des include regulation by trarcrptioal,arslationa End postransationl mechanism, all of which affect | ‘Adapting to Life ona Rotating Word at the Gene Expression Level 265 thei au of gene rot proce nation theclock may us dierent mechani oti conta Points Cofactor, second messengers, ona, pH and pro {Sin modistion cama affect tbe rate pete pe in reaction pathways: ‘n Gonyaulas, behavior patterns and rates of protein synthesis are lock controled SCHEMA CORLATS The velo avis ofthe Biche cola yh mut ayn sso cine pe rlatneip nh he yn elf Adc tnd welsuded arp of such schema core ite i the Bolumietence thy of tes ont pir gee) Dg ht pase the dnlgeistn spies proce ight swe Sand 100s ow light canbe peed bye during the sbjcve igh seo hing hessectv day phate when elle paced non stint conto Thee component arenes ar ight pode te susan uci 1S pone (0) a heey Tere UCP) In Acai persed ase ‘pecan rpnelscl swat The Cons beacon ym provides an cxcellen example ofa fyi contd sie pos ‘anerpona tevl~ht where te cain pe Intion Rappens after the gene hos bee expreseed ss SSRNA Grecrelae of te baluminecen yn {hat the numberof sintlons i oughly en times pene inght poe clean in day plas el eric scans ly mRNA (open cies pre angie) eiabeseeee a nd Wa Thase oganelles ae formed de nov atthe beginning and degraded at the end fe night pee, This acti ity can be quantiied by 3 count of the number of organelles rncing with anodes directed agin LP or LCF in call sctions. The disappessance ofthe ‘eganeles an thelr contents has been confirmed by ‘Wt imungblt analyses of protein extacte ken from algae a iferent tine points within the ican cel (ee Figure 88) Thase studies inicate that the light producing pro- tins within tha scion ae degra rater tan er ply moved toa diferent subeeliinrlocaton Ine on, the levels of both LAP and LCF proteins re nd fallaboat tenfold over the ctcadlan cle. Although no anibodies are avalable to. messure the enayie luciferin this component ofthe system can be conve ently measured by vitue of ts endogenovs Mute: ence, Cucferinfaotescence of intact cll or thet vat alo varies roughly tenfold over the ceadian Te amounts fal three biachemcal components of ‘hebioluminescence eation vary phase wth cellule Doluminescenee, ndeting tht they represent te lo chemical corlats tothe behavior shythm. The molecu bass ofthese rhyme was further pusied ‘ith te expetton that the fhython in protein content ‘wotld be the result of underiving eythane in mRNA ‘Coupled with nonrhythmic translation, Surplingy, however, the reverse was the ose (ae Figur 8) The lock was conoling he att eich protein was made from costnt pool of ii NA, se wel athe rate of UcFsyness. Further esauch showed that this rane latina contol arises from spc sequenees of bases in the 5 unransatd region ofthe lop miRNA that are ‘ound by protein capable of locking anlaton. The lock apparently conta the synthesis ofthese proteins Slr proteins and activites have now been deified In Chlamydononas anther algal mode sytem, Diferent rhythms within a organi exhibit di ferent acrophasts (peaks) and durations Clock regal tion of cellular processes orchestrates and organizes physiology and behavior along the tersporsl dora, suring that htm are appropriately negated ad Sequence for concer ston DUFERENT PHASES IN CONTAULAR, Several dllernt ‘hye have been measured in Cowman general, the peak (aerphase) ofeach activity or behavior ppeers tbe approprits the middle the night fr bholuminescence, and daytime for photosynthesis (igure 8) Gonna exhib readily regu fs melita ogregation. The thythm of ggregtion is probably ascocatd! with daily vertical muvetnent. Cells accumulate nes the suiface and phaeyetesie 266 Chapter 8 \ ys inthe daytime, were aight they descend vertaly {o dep at which inorganic nthints are more abun dant The reason fr confining cll divisions toa certain time of day fe not mmecinely apparent. espetally ‘cause i crophare ifs among fret specs, nan extension ofthe ues andy described for st and LCE the rates ofsytess of many indivi proens have been measur 2 Intervals and found {ovary over the ciadian le, They ter sen to have Alifertntacrophases, whieh appest to fll nto three ‘nejor time las ate ay 0 ety night, dll of the ‘igh a ae ight oea dayInkrestingy durations of inctesed sythese rate may also df: or some proteins, incensed sys erst ony fe Hours Fut for ober inst opt 12 Te tempting to inter pel this variation as an nication ofthe turover time Er the protein in cation but this pint emai fo Be ‘coblhed SINTHIENC EACTERUM SYNFCHOCOCCUS. Spcles ofthe ‘utotrophiecyancbaceriem obtain carbon abd energy "vough photeynhess ee Figures 220 and 51) Some fala able ox Ny ana has are not dependent on an Inorganic nitogen supplysuch sritrates. The boos Cal challenge that ie trogen fixing enzyme nitro rnowe is inactivated in th presence of oxygen 5 Hon clearly incompatibeorith the Oy produced by photosynthese, Some groups of cyanobocterie have Eve tis problem by ella pecalization in which fells of one typ, calles Hteracjt, fxniogen and do ‘ot photsynthesie, obtain mutans om the oer Celle, which photosystesize only Other ies ‘jonobacers have wad fe creado clock to separate ‘trogen and photesythessin time rather than in ‘Space rittogenase le esc tothe igtime when photesyiess and thus, production, sow “Asia behavior canbe served fr rate metab cm in the evry uel Cone se Figure 24) in thiscae, however he man source oF OBEN ‘not N; but nitrate, othe concurtent presence of irate ‘rebolzing enzymes and photosynthesis poses noi ee amine Enon ea panos dit ack ‘stled phenomena in thesngeeie doi fe Conan pola: Stara open rs con pss dy nd gh phen mec om Mitegerst998)| cul n ft, the simultaneous occurence fritate ream an photons sould be advaages tothe lgae teense the sun's energy can ten be fa reed dee into seduced nitogen, as wll a nto odes carbon The problem that photosynthesis ocursatthe su {ace ofthe water column whereas tate slong with oat ofthe oer minerals ana ates hat he algae ‘equi sabundont mach deeper between 10 and 20m Telow the surface. Ty allow the organism to retrieve a5 Iich nitrate as posible during the daytime, the ayn that estalyzes the st step in irate metabo im mate educa induced inthe daytime ocherlal studies show that nitrate reductase sctivity peaks a the same time, midday, os potosy thesis with an approsimately fourfald shyt. ‘unoabeing experimen sages thatthe amount of protein may sneresse rom tives enol during the {ay phase and also compartentalized tothe ite oF Photosynthesis the chlocoplat, Crester levels of Enzyme during the day than during the night psu ‘Ty reflect the movement ofthe alge inte water cl linn, As thy descend 10m orzo st night, oan area ore ite pln lw nitrate educa acy ‘suid and the levee drop. In Neurospora cockcontlled genes play am important role in the regulation of Per dogeaamirss {Lock ECLLATION OF BOLECICAL PROCESSES. Whether metabolism, eptoduction, or bebwiors considered ‘Tack regulation =a diet recon of he biology of he particule system being studied. The 1959 tencrigh End Bruce paper sated that Nevrospon’s cele pro- ‘ution of sews spores of cnida, had a periecty of about 2 hin constant conditions The 22 periodic iy was said tobe maintained aeoss» physologicl teispessuce ange and was eleved to rect an unde lying biological cock ‘Asexual development in Neuaspor lock con trolled The cock rgelting the asexal developmental {jc of the apcomycete Fungus Neurospor cms has Adapting to ife ona Rotting Word at the Gene Expression Level 2 ‘opmental sich sao endogenously controled by the cireadian clack, In constant conditions, lock ee egultionof developments inated inthe te A Rees ras “Subjective night producing conidia for ¢ eae TEES “Atk proporion ofthe day Inthe eae son Ii subjetveccadian day the gals \onaatve/ 9 wlkchad back and a elon of undre wie ff Memes tated ype lat down unt the net ja i ‘bj night esuling inte band Ney Rae ingpatem of Nese seal devel 1 4 D lopment. The clack controls the initia He cee weak Hon and period af development Dbuthas ad elec he development lees sl. nclokies stains, deve Spmentise anger cseadnnly pero icand thesbundance of conidia ay be Stee rst The cok response ae Azwian _ongnning ine sey of hc cpone $Oiglon _invdbedn seca developmen Alppetie ae ope a “ ep cana st rsx or sexual subcyles, both uit ant the mater of sexu spores sho teen fini decgme phe Senge eon ts a In thee cyteof been wl suid (ee Chapter 7s a Plate Photo ©) Ths developmental cyte requies ist three cll ‘ype gue 8 10) Upon geminatnof anaes felepor the macrotis or coin x rach {te yeriiam grows na syncytia with incomplete cel wall. The eytoplanm snd hice! ate able to low recy within hs yneyti Thee hyphae, he se tnd call type. are produced perpendicular othe sub ate ane they elaborate teh ell ype tbe mac. Conidiophore, though the formation of complete Scuswallsebveen hese vegetative spores ‘Numerous soviet gis capable of iting production ofconid, inating carton depia. Son te sbstte, dscns ny tmsion and naa ght and temperate This devel times inthe subjective day this large celular smodifeaton involves many genes and gene prod st, nd many of these genes othe protein pro tes are expected tobe expreste ina me of ay tanner Although the prouction of sexual spores Igthe best characterize ey in Nero se zal persisting Myths atthe physiological, bio ‘Shia and molecule eves have ben dese “The sexual uting bodes ofthe fungus, called perithecia, contain hundreds of sexvalspores (esempores) which when mature, ar expel ina ne of day-specfe manner through a perithecia neck. A ‘lockccontrolled gene, yt, hasbeen Wentifed as "Newaspon mating-type pheromone gene, suggesting thatthe clock isa dtect regulator of sexual mating ‘Other now shythms apply fo the production of CO, lipid metabo various enzymatic atte, nd Bet Fok protein The tytn production of aerial hyphae and spore etals major ella remodeling, with cone: tio istered 1, with each base pat (p) downstream Sequentilly wmbered 2, +3, and soon. The region “stn of start i lbeled with negative numbers 50 Db upsteom of stat would be 10, and soon. Ci fnatyees of promoters ate possible i the region tpatea of at i placed ina ning vector such that the promoters conta the transeription ofan easly "Seajed reporter consict The warseripuonal ctv ‘ofthe promote i then followed through the acvity of the reporter molecle. Many potential ways of 5 inga eporter exit include following the transcript tine poten levels or poten activity of he reporter ene eletion and ci-anayses ofthe transcriptonally conf s -2) promoter have separated sequence Adeting to ife ona Rotating World a the Gene Expression Level specific regions that confer clock, development, and light repulsion. Promoter DNA was at i eticton enzymes into discrete locke and cloned into plasmid ‘octors to control the expression of tre bacteral hygromycin phosphotansteace ip) gene Acuanity af out 2 kilobase pars of DNAS tothe taf ‘eription was found to be sufcient to rssptultenor tal epuation. A SShp saquener, the atating cock semen (ACE), Ioeated at 0 fo “118 bp close tothe Start site of taneription, when deleted, disrupted "ythaicy of he reporter gene igure 819), When the acteating tack element wat lace in the content af eterlogos promote diving th ih gene nd containing no other sequences fom Nera it ‘vas found sulin to dive eyeting of the contact In "dition, the region of DNA sequence ying between “225 and =1900 bp was fund to contr represion af ‘xprsion during myceal growth hats reve upon Inkiaton of sxevial development Astrongactvator of ttanseription exists at sbout “1200 within this region Sequences necensary for psive Light regulation were dieterminod oe betwen -€25 and 46D Bp snd aot {er region betwaen 102 and -218 bp wa ound fo be involved inthe kinetics of light indcton inn, the regulatory information fo lack deveepment end light induction is found In separable moti within the promter othe eg) gene, Transcriptional control in plants is degen PLANTS HARVEST SLNLCHT. Iti eayto imagine reson vohy plants have evolved the ability tl tne in onder — Negi dendoprat = Nene devopments cement FIGURE 815 Hepulton lament oft 9. Exes fh ec 5 Coton ay see eves epi aman See transept! reasinecan unde! aprep canciones Pd Wench eel trig ctor. (Bom bth Pedersen Dasa, an Lace 186) ws to espond appropriately o thelr envionment. As se sileoganisis plats depen on dally changes intel Suroundings tat cannot imply bs taken advantag of ‘or avoided by locomation Importantly, photsyatet: Sunlight for nutrition The sty to antepate daly changes light quality and abundance might realy confer adapive advantage for occupying paral ‘cologin niche. Many clek-regulte processes in higher plants are known, inetuding aspects of photorynthesis somal ‘opening: let cotyledon, and Bower movements cll land hypocoty elongation rates; hormone producto; CO, uptake; Calin cycle reactions and photoperiodic timing of flower incton Figure 816) Many clock conitolled ones have foritously ben discovered one document inthe erate, most paying rythm levels of mANA, but els examples of postreslatonal ‘modifications (phospharylatin) of opts have been oTOSMVTESS. Seer ne ivolvedinthe cont 6 biochemist of photosynthesis have ben pain lniywell chracenee, with cradan expreson wu slly peaking shorty aftr dawn. The clorophyi ub binding gees, which encode proteins lnvelved in light harvesting Lic gee omy copay cxk regulted rates of promoter activation Extensive ese ton end mutants he Lc? promoters have Show mulipeinioenes contling pene expression. Inadtion the cock envionment igh ented through the phytocrome re /lared photoreceptors ‘ad blue light photoceepory, incuding eryptocromen hs stong regulatory inuence. Tg regulation final, with an acute and ansent act: ‘ation of gene expresion fer the ‘ret of ight and adioral ong term contol af both ampinde sr prod nthe presence fight ‘hat smelted by bth cee and wavelength: Tae epustors to interact with each ther The Stock modsltes the eponse 1 Tight and light resets the clock Resection analysis ofa promoter fndieates that many of ese responses may occur within 3 sheet eon of the prometer ith cnatapig ing ss Gre ofthese tepals SOCAN relied wanseiption actor essy for promote pon 276 Chapter 8 © on & ai =o @ && Ao \/, oe a FIGURE S16 Pan clas con nme ile eee (A Tepes lever gees ms cc Eatin ron gc cb et rea NAS ieee Strela Sisncl pelosi ‘Shop chowe te totanor gt expresion pres ‘Rape indicat fh ng of pees owing {alan onal (8) Cyrene conan fe asm ‘Greenstein yan ‘nas Constrng te toprtance of sum as bo guvelmenrEyetch bececlasen ary ot ‘ala proceses (Che ac regula he poopy Sanat Soe pris Eanes cst acdc ng acnon ay as Neal opel eps ence "Sal opening hyposoyslrgaton yan ee. ‘ord amen Anca i cea SRG Rec evita sting ae {neil poss suche foweang me) Mine Ear puttve cock ecracated gum caters poe pedicel fowerng om Brita 0) by phyeehrome CCAt is aio strong candidat for an ‘ulster component inthe plant cece Many clockeco trolled genes of plante may be controled directly though binding ses on heir promoters by ranractg, {actots that ate part of he cresdion oscillator, hee CCAt or the related LHY transcription factor (see Chapter we OS Ue (© contend DOMER PLANT CENES, Many genes not involved in pho osynbese are also tytimialiy expressed. The a Tae genes CAT? and CATS, volved in vemoving, Toxic FO, fom the cel cjle [Kou of phase rth each other peaking inthe subjective roming nd etenng respectively The ‘howls nov anderatood aes ine ase ofthe CAT gone tis not own if they are coupled he ste or diferent “Two closely related genes inthe ces Anais talon have bee late by iemolgy to cycling gene arising rm = sublracivehybnantion experiment wang the white mustard, Sia ala. The Araby genes cyl with peak expres sion around ds sh the DNA sequences of the CCR) (Atgp8) and CCR2 (Alp) genes indicate at thy ore gh smart pyinech RNA-binding protein. The proteins are cape of interacting with RNA and ate localized within the rc, indenting regulatory roles fo thse proteins “ranerpt cjling of CER? is robut eth a grester ban ‘Ove amp he protein also cycles witha esi {mpitude of abot 10 "he peaks in steady-state protein are about 4 detyed relative tothe arse pes, nd Wanscrpt levels donot begin to Hse unl protein levels have teach or ough The dele elation othe RNA ‘intng protein eave ois wanscrp could be, but fot cay the elf an regulon odbc {oop sini f those sen in orilatr mechanisms i fang and les. Interertingly, when CCE? is overex pressed, he cycling of tow promt strongly sup Pressed aough overl expression no abled When the cycling of ster plant lck-contoled genes was exatined in ether ee-running ondtons {Gostant igh or LD eyes in he CCR? overexpression Sain (Figure 817) no significant itferences were Found between the conto plants and the oveexprese tng line for eb, CATS, and ther cycling genes. However cyling ofthe relaed CCRT (At) Ree was fully repressed witha concontant recon n ver cexpresion level eroticy ofthe CCR arscrps is under conto “TOL ne Chapter) In TOL shreperod mitan the ECR gene expression ehythms are alo shortened The CORE circadian feedback act as both a tg forthe lock and a modulator of oer downstream dk core lelledgones An atractve model postulates Ns ‘Sulorgultor loop tobe 2 “slave” escllator receiving Input toma maser onllsorapble ofoneling ot ‘modulating only ined set of deckepused ous Adapting to ife ona Rotating Word tthe Gene Expresion Level . . cote awe oT SEER FIGURE AA7 Infunee of CCR At oveenpreton totes ccaaroplated vanstps ON oe eles ‘Src Sept epee tape ne cys (. 18) nd on te td dy sr ose contin Cou. CEN ANALYSS. New ecologies for examin lng gene expresion ae revlatiniing lls of ba. ogy Experiments examining clock repultion of in ‘idual gene, their wanscrips, and protein prodcts iments are abor intensive and many year of alysis bby many experimenters hive gone i the character zation of relatively fee genes, Recent advancements Inhigh throughput DNA ssquencing and the resuling allay of genomic ancONA snc nema has allowed ee development ofeenaogies to mes fe the RNA expresion of housands of genes ina si. beset of experiments ‘ie now ave the capability to begin to understand lock ental oar proportion ofan organi tentirearscrptomeby sinulancously examining the temporal regulation lige sts of genes. One tech nique, microaray-based expression ara i mow in farmmon use. tn DNA miraray pos of DNA, ether ‘liganeleoties or PCR fegmant ae placed ina Ugh Iy ordered pattem ona ge microscope sie Sever Runded to thousana of genes can be represent on 8 Single slide, and the pean and sequence dent of ach gene Is known. jst ab when # single DNA quences sed to pre Southern or Northem oot these DNA sequences that rebound othe sob sree represent the robe agin an RN target. a 2 ee ager rin th 8 probe which espinal tb rds pine pee pre devs fm be anaes impor ofthe CCR Epps) gene), rerpectly Sli an penton dag pote spn Tet and reference mRNA sample are harvested, convert tocDNa wives etc, nd ee Ibeled each wth x disint frescetpbe. The ‘ary and cDNA aed ts Te feran ind spotcan be montre by eamiation te amount Qurence em redo other Sking timed err of RNA cra and etn theme duphcte micas allows a teporal pre feof expresion from the ones represent a he say The meds prod eooms aa et inch ene analyses valved nthe aaa Variance satiscl methods act determine what ‘notes sigan change gee expesion eve ‘Gsster analyte ta method ted to group gees decoding othe iat othr expression pater from mioaay experiments Miro expesment in clock regulated gene expe sion in raids ave shown approximately 6 of he genome tobe expressed rhythmical 53 genes Out of ‘total of 120 genes examined: Many previously char acterized clockontoled genes were among the 45, lening validation othe sattal analyse. When the ying gene ere clustered by the timing of peak ‘expression, al pasos wee found tobe well rxpeent : =] CE ete ‘with some unerrepresentationin themida ofthe ight (igre 8), Numerous bcchemical pathways wer identified a having elakeregulated components, including genes involved in cell wal remodeling and water uptake _Slyuences of promotes that pened late nthe subjee= tivedoy were computationally sconned, and highly fencer rine nuts "evening element” was len ‘ied 3 times n 3 evening gene The CCR2 gone pro- tote onlin four ofthe evening elements A130 bp egon of CCR? promoter DNA conan one of thee m FIGURE RIB Repulitin of coc-contoted gen Abia Then Ent nes thoughout he dy.) Cees imp Sie cael maa ce {ld (© Sper mel or font of roe depetn (10) Altos evening FESS gs puss acommon pomesr ‘Dont Sot open br npr asc pepo rere eae a lems ws used toa ura reporter {nd shown eapable of diving #ythmie erase Thi potntl ever ang, Sequence vas hen mutated to change the Sequence, eulting sn substantial oss of the ability of the 130 bp ciselement to drive rhythmic wansription ‘Several micorray stuces have recent ly been published an are ongoing i ‘any of the well characterized model 95° tema fr clock study (Table 81 and Fagure £19), Clary this and elated eclogis in the ear future will have tremendous Jmput on ur Knowledge of what the clock programe In specie organisms, a8 well as our understanding oF the mechanisms sed to couple the clock out Transcriptional regulation contributes ta rhythmie gone expression in ertloraessierancesec ‘vee, Many studies have shown e broad arty of fnchons under Grcadian regulation inthe vertebrate tye (ee Chapter 5) Retinal adaptation to daily lght-rktranetons complex and iol massive “The extent of cicadian control of gene expression in diferent organisms. a ete Rete fg Petes Peres ERGs on suce Speen janice © ase EES Rosny 2 Nowra 203 Danie io a Gurapecrangta 2m, NeSEed ee ose 0. x/maue fonts Manas 2 sda me cele aodtideg Morals ; inet et NouescNandiver” Marna a Pana 2 Nowelversndtest Manas 0 Soh etal 22 sea tne oe ase HSA ome Mac se > Urpin om ii Obamas MC Hangs ‘Adapting to ile on a Rotating Word atthe Gene Expression Level FIGURES: Circa regulated gens in the SCV ant ver. Tantipt ang Fo MOEN pee ans nee fered cc pid Dt [tose inench owe Thewigese ea [nyspeiicepesion Btwn dngrr sho amount of celular semodeling with atendan changes inmany genes and proteins. Much ofthis change stra Srptonally regulon the early to mid 190 sever reports demonstrate hat intact eyes tes and so Tated eel cultured fom vertebrate eyes contained an independent ccadan oscar In many cues inv unl molecule sythme could bellowed, Crean cco in the etl pote cls of chicka the Afsean awed frog, Kemps ave been Sled in vito. The mRNA levels for sed-senstive dpi, the predominant cone pigment in chicken t= a ae eguated by a creadan cock n embryonic ell ature, with a two" to theefold increase i mRNA hundance ut bore subjective dusk This nen cor ‘responds toa phate just before the peskof cone pho toreceptr dic shedding in va Nuclear run sey Show tht todopsin mak symthesisincreasee 3106 brofre expected Hight offch, demonstrating that the ‘yh is generate, leat in prt at the level fra Serptionlsynhese, {XEN00US RETINA. One ofthe best-studied and best lunderstood models for cicadian biology of photor ceptors is the Hemp etna, Reins contig the pho toreceptive layer of cells canbe isolated fom ther ladles or adults, and thew eyecus (Gee Chapter 5) ‘an be maintained in Now trough eltore for seers) days: Numerous moleelar outptrhythms have been ‘characterized in engpu retin, including the neue hormone melatonin, detestable inthe flaw through ‘medium. Melatonin (ae Chapters 4a 6), whch found tobe high a night an low dug the day inl verlbeae clases fa major pea gland hormone but a9 aw hte a cep henge grt reo tote imporsrs th clock nthe SCN ahd peripheral ca yeni we oun to becnadnly expen not BS ssn ero se ec om Pst itisalso found in tina. The best describe function or ‘melatonin ists te in seasonally periodic behavior but Itisalso known funciona ceadin modulator ‘At ess vo and maybe more step inthe protic tion of melatninare cock regulated and several ines of evidence indicate reduncancy in lock contol oF Imeatonin sys The mRNA fo the tenting 2 ‘roll afisk enzyme i meiatonin nthe, yptopan hydroxylase (TPH), satin and peaks ding the erly night, concent with the production of mela Tonin, Nuciear rumen assays demonsteate transcrip ona regulatiog, with TPH mRNA synthesis rake Increasing over te course ofthe subjective day aly studies had show cicada acivity 0 ater enzyme nthe pea of tinal melatonin synthe [Neacety transferase (NAT) Much of the tytn pat tem and ampltue of melatonin synthesise due tothe activity of ts enzyme Tes now known thatthe ot All regulation of NAT ishighly complex. NAT tong Iy sete by eumerous factors, many of which ave ‘organism depencent. To highly conserved forms oF [NAT regulation clade the ciendan cack igure 820) analight Photicstialon has two effets on NAT. The fist isa rapid negative efet on abundance hat regula ‘din part through a cAMP-dependent patoeny and Second by acting Evo the dock tenn NAT at ity shyt in what fe elioved to bea eAMP-inde pendent mechani. An adlittons level el lok eg recepters. Two sublypes of melatonin receptors ae expressed in the ene tina Mel (1h) an Mel (1), ‘The mIENAS for oth ofthese proteins are known to 280 Chapter 8 omy qeoeoe| gS i: o_o Eringcotinet ong uty en etched {ord days alight cyl (LD 121, white ight cm at ET 0) Se sete ised es) Repre TEETRochcr tans of NAT and CAPD oie {Bingo Ua pes nel (Guarini nay of he Sevier mmaizaten tt CAPD agra The eee eaineshows bevel of NAT MRNA cele eit Spat acanmyen D between BT aad SPEEA ay wasnt a ech te ot howe Sree naan ope, marae Phenol ar eis ere sed PAE experts Sa and eer ts epee Soham ee ch Oe ary ina diumel sytem of expression in light-dark ‘eles suggeting rte thi level of eguation ‘Seiteatenn action, The multiple pins of rhythm ‘cpulation within he telatnin owjetic and meta tlic pathuvoys suggest strong selection for bolic thei of thinbomone, ‘cuock pRoTeins REGULATE OUTPUT. Clack-riven Sipthms in enecriptin factors ean regulate gene ‘Cao tar us Albumin DB he ist en ‘thea member ofthe PAR leucine zipper transcription ‘aco amily, sa egustory protein eniched inter bat found in several ther issues, ncuding the SCN. Two ‘tier membes of this amily hep leaker ‘edly and thyroid embryone factor (TEP) share ‘hteeed sequent sinletes vith DBP in the BZIP {hose eucine ripper) DNA Bindi domain ann the {ardomai, a fagon rh in profes and acc amino pes ‘Dar, TEE and HLF a all ciadianly expressed in ssslus tissues, although DBP shows the strongest “hyinms (gure 821), Levelsofdop miRNA and DBP Potes bsh ext oust crcadan syns with heir Fania phased about 3 and 6 respectively after ma ial DBP transerption Ia hepateytes,amplisdes of Dap mxRNA ythms ace high nd may roch 60d tha pe ia ate evening teary might aed then op TD undetectable lve by morning. Ty nocturnal rodent, cholesterol synthesis and catalim in the iver are ges during the dark eed Sngand ectvty phase, when the arimas require more Dc eids to absorb ingested pits. The rateimiting iijme for chsesterolsyhesiand conversion tobe SET mde by the C¥P? gene encoding chlesteo Jachydronylse, a mene of ihe PA5D superar. Levels of tisenayme azo exhbita robust circadian fiythm ia mNAsbundnce, aeaking at abost mi Tigh To. Tage extent, tis thm i replated by Transcriptional mechanism. Ir he coe of cholesterol Techydroxglase radian trasctpions driven by te ethnic binding of DBP {Gating elements hin he promoter ofthe CY?7 fre Insin, TEF and HLS whose BZIP domains ave the ability fo bind tote same cognate DNA equcreesthat ie DBP protein Hrs to, arable wind Gey DNA and may seo playa significant ren the faye prosucion of C¥P7 RRNA. Supporting eve {done comes fom DBP-Enockout mutant mie whic servile with functioning pacemaker ane also show ‘tconobly normal ampliade of civeadian CYP? {ARN although the phe oftbe yon advanced Th Thcestentfo when, nthe presence of DB, these ‘ther PAR amily members parepate in the osc {tanscrptionl regulation of CYP? versus the bit) 0 ‘Sine wher the DBP prot is absent isnot known. ‘ASOPRESIN INTHESCN, Clo component transcrp- ‘on tacos van regulate gene epession nan SCN ot pul Apne vospresin 3 europe found ithe Erin and involved in peripcral eects of salt and Ivater balance. Vasopressin RNA is hythmic nthe [SeNTreglotedby the CLOCK-BMAL! fedbock osc Inv (eve Chapter 7)_ fa homozygous Clok/ Clock ‘stat animal the shythniny’sabelished and ove Illeveirof tansepteemain a ow nighime evel {Gigure829, though overal levels of gene expreson, ‘Adapting Lifeon a Rotating World atthe Gene expression Level 78 a= aeuarccnae” ‘rl of DEE TB and TEP mi tha Dar nytmy nce ng hs pw Caper ingen ar ere ate tele doubiesranded liganuccoide containing the DP- are not reduced in the SCN of these tants ‘Vopressin sao expres in els of he bai ut side of the SCN na nontytuni fasion. Noneycing expression inthe nearby supraoptic nucle SON) was lege affected by maraton inthe clock gene Examination of 1 kilobases of the 5" promter Sequence ofthe vasopressin gene sequence evel i ‘teuneains he op Eos mod, CAST: postions vg apn epee CTTACSTAAD, Ac mae item lage fees ena DrvA-binding abil in both light-dark cycles (lef and con- Eaten gs ec ~154 0-148 Th phase ofthe mRNA shyt fo vaso presin je sila to mPer mRNA cyte, and mer Expression known t be mediated by ihe CLOCK. transcrip factor through foxes within the mPer regulatory sequences. A 200 bp region ofthe promoter inelding he CACGTC motif wa arayee for binding GECLOCK, ina standard reporter assay, snowing that the CLOCK-BMAL1 heterodimer could svat tan 282 Chapter 8 o| Ge © scxpton frm the vasopressin Ebon. Therefore the c= Cada feck mechs sel kad wl ented gone bythe same postive a negative om ponents that dive the cove eco FIGURE Anas die modelo uy Hot niente ae ie Sinatra, Suey eee Seater eae area eee Sree lettre See eee BoA beatin eet SSNS Meena Cait ie sears damien cit Secrets Bob Setess ame wating Secnerena cern se poirearcienrr a Sue aa ere itatCLocK elresion athe SCN crib eter So Sacre iene ceareeraenar See AFRINETY PDN NING. Many genes that bind shyt: ‘mic Wansceiption factors may nat be ehythmicaly expressed, The nucesrconceteation of hepatic DBP bosilates Between 10 WM and 2 UM rom morning fvening. Consider that a target gene whose promoter binds DBP with an equilibrium dissociation constant (a) considerably ea than 10 would not exit ie cadiantranseription because the ei-acting sleet Wwould be occupied by DBP throughout the ccadian jeden contrast, target genes with low aise for DBP (a inthe miromaar ange) would be smelted ‘by the transcription actor only forthe shor iets ‘al when naceer DBP concentration te closet max imal: For example, the promote ofthe ytocwome PAD igre harbor high afinty DBP-binding ste, yt F450 feretranacripion doesnot exhibit sgnifcant cadian, Sella. Transcription ofthe cholesterol 7ashydro ase gene: whose DBP recognison sequence binds DBP fvithelativel ow afi sstongy radon, ‘The entire Synechococcus tanscriptome is clock eematecerocess! -arunrinac GENOME. Ax noted earlier in this chapter, Some species of cyanobacteria have employed their {internal czeadian clocks to temporally seperate the ‘ncompatible metabolic ets of photosynthesis and slogan metabolism. The mechanism of control was ‘thought oe, at eatin pal reputed gene expression bestia known tht rtrogenase mRNA abu= ance was higher at night han rng the yn at {yanobacteris such a the mal molecular system of ‘Sheclocaces elas se ther clocks to regulate 3 Variety of physiological proses ‘Adapting to tile on Rotating World atthe Gene Expresion Level 283, A sophisticated reporting system to manitor pro moter acy i. eongetan war devslopes by acon Sort of collaborating laboratories A gene cst of ‘he acter lucferese pene ei and as from Vb ley ae paced behind a wel charecteried promot expo, from gts. The pb gene encodes the Di protein, a major component ofthe photosystem I seaconceterin photonnthesis When bugis wat ttansformed with the DNA contrac containing the lucerase gncs under conta of the psbA promoter the cera were found to glow thythincally Tis Bole sinecenc could be store in vv, poreral oo 3 Notsurprisingly the mRNA abundance forte pb gene was lo found tobe cyclic. Theough vara of “promote rap" experiment, ‘ho einology made t poset atk how widesprend ‘readlan regulation of frnceipon in. cong as NDNA Iba a 5 clogatus genome fragments fase "upstream f (in front ofa promateras ux pee xt Sse was contac, The ary was the aed into the 8. elgaris genome, where DNA sequences homologous to those in the ary allowed recombine thn at numerous es hrooghout the genome with igh ficlne: Bacteria were kept constant igh. and lucterase expression coll be monitored from mule colonies spread ona single pete dish, When the AB fev casete ener the geome hind a ar Sexiponally active promoter repon of DNA, the bac “alelewil expres cere rd glow, Change in bo luminescence overtime for exch transformed clon reflected gone expresion ati fom ndvigual pro- ‘motes "trapped upstream f the AB genes More than 3000 colores have now been individu ally examine foe temporally regulated expression pat teens, The astonishing result that all he promoters showed cieadinly thythmle activity: Although the Smplitides, waveforms, and phasing ofthe sythnic pattems showed wide variation (Figure 8.23), the Hythe all shared ingle petiod length, Pak expres Sian levels for 3% of te ene in his organism near subjective dusk thythmc expression patiers have been found, leading tothe hypotesi that the clock is mahating sts of tranecrpton in yanobacteta na global fash, pos Sib n conjunction with csi mating factors Because single type of mltsubunt RNA polymeaae Is esponsbe forall ace ranscripion_ one des thatceme part or pact ofthe core tenscipton machi ey sell might be cock repute. 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