tick-tock the biological clocksgpwe.izt.uam.mx/.../the_tick_tock_of_the...subido.pdf · responding...

You have to fight the urge to fall asleep at 7:00 in the evening. You are rav-

enous at 3 P.M. but have no appetite when suppertime rolls around. You

wake up at 4:00 in the morning and cannot get back to sleep. This sce-

nario is familiar to many people who have flown from the East Coast of the U.S. to

California, a trip that entails jumping a three-hour time difference. During a weeklong

business trip or vacation, your body no sooner acclimatizes to the new schedule than it

is time to return home again, where you must get used to the old routine once more.

Nearly every day my colleagues and I put a batch of Drosophila fruit flies through

the jet lag of a simulated trip from New York to San Francisco or back. We have sever-

al refrigerator-size incubators in the laboratory: one labeled “New York” and another

tagged “San Francisco.” Lights inside these incubators go on and off as the sun rises

and sets in those two cities. (For consistency, we schedule sunup at 6 A.M. and sundown

at 6 P.M. for both locations.) The temperature in the two incubators is a constant,

balmy 77 degrees Fahrenheit.

The flies take their simulated journey inside small glass tubes packed into special

trays that monitor their movements with a narrow beam of infrared light. Each time a

fly moves into the beam, it casts a shadow on a phototransistor in the tray, which is

connected to a computer that records the activity. Going from New York to San Fran-

cisco time does not involve a five-hour flight for our flies: we simply disconnect a fly-

64 Scientific American March 2000 The Tick-Tock of the Biological Clock

The Tick-Tock of

the Biological ClockBiological clocks count off 24-hour intervals

in most forms of life. Genetics has revealed

that related molecular timepieces are

at work in fruit flies, mice and humans

by Michael W. Young

SANFRANCISCO

PETE

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Copyright 2000 Scientific American, Inc.

The Tick-Tock of the Biological Clock Scientific American March 2000 65

GIVING FRUIT FLIES JET LAG is helping researcherssuch as the author (above) understand the molecular basisof the biological clocks of a variety of other organisms, in-cluding humans. The tiny flies are kept in small glass tubes(photograph at left) packed into trays equipped with sen-

sors that record the insects’ activity. When a tray from anincubator kept at New York time, where it is dark at 7:30P.M., is moved to another incubator simulating San Francis-co time, where it is three hours earlier and still light, thelevels of key proteins in the flies’ brains plunge.

PETE

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NEWYORK


filled tray in one incubator, move it tothe other one and plug it in.

We have used our transcontinentalexpress to identify and study the func-tions of several genes that appear to bethe very cogs and wheels in the worksof the biological clock that controls theday-night cycles of a wide range of or-

ganisms that includes not only fruit fliesbut mice and humans as well. Identify-ing the genes allows us to determine theproteins they encode—proteins thatmight serve as targets for therapies fora wide range of disorders, from sleepdisturbances to seasonal depression.

The main cog in the human biologi-

cal clock is the suprachiasmatic nucleus(SCN), a group of nerve cells in a regionat the base of the brain called the hypo-thalamus. When light hits the retinas of the eyes every morning, specializednerves send signals to the SCN, whichin turn controls the production cycle ofa multitude of biologically active sub-


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LIGHT HITTING THE EYE causes the pineal gland of thebrain to taper its production of melatonin (insets), a hormonethat appears to play a role in inducing sleep. The signal to reduce

melatonin secretion is relayed from the retina, through the opticnerve, to a structure called the suprachiasmatic nucleus (SCN).The connection from the SCN to the pineal is indirect.

THE BASICS

THE BIOLOGICAL CLOCKTHE AUTHOR ANSWERS SOME KEY QUESTIONS

Where is the biological clock? In mam-mals the master clock that dictates the day-night cycle of activity known as circadianrhythm resides in a part of the brain calledthe suprachiasmatic nucleus (SCN). Butcells elsewhere also show clock activity.

What drives the clock? Within individualSCN cells, specialized clock genes areswitched on and off by the proteins theyencode in a feedback loop that has a 24-hour rhythm.

Is the biological clock dependent onthe normal 24-hour cycle of light anddarkness? No.The molecular rhythms ofclock-gene activity are innate and self-sustaining.They persist in the absence ofenvironmental cycles of day and night.

What role does light play in regulatingand resetting the biological clock? Brightlight absorbed by the retina during theday helps to synchronize the rhythms ofactivity of the clock genes to the prevail-

ing environmental cycle. Exposure to brightlight at night resets circadian rhythms byacutely changing the amount of someclock-gene products.

How does the molecular clock regulatean individual’s day-night activity? Thefluctuating proteins synthesized by clockgenes control additional genetic path-ways that connect the molecular clock totimed changes in an animal’s physiologyand behavior.

LATER INTHE DAY

SHORTLY AFTERLIGHT EXPOSURE

PINEALGLAND

MELATONIN

FIRST THING IN THE MORNING

SUPRACHIASMATICNUCLEUS

OPTIC NERVE

PINEAL GLAND


stances. The SCN stimulates a nearbybrain region called the pineal gland, forinstance. According to instructions fromthe SCN, the pineal rhythmically pro-duces melatonin, the so-called sleep hor-mone that is now available in pill formin many health-food stores. As day pro-gresses into evening, the pineal graduallybegins to make more melatonin. Whenblood levels of the hormone rise, thereis a modest decrease in body tempera-ture and an increased tendency to sleep.

The Human Clock

Although light appears to “reset” the biological clock each day, the day-

night, or circadian, rhythm continues tooperate even in individuals who are de-prived of light, indicating that the activityof the SCN is innate. In the early 1960sJürgen Aschoff, then at the Max PlanckInstitute of Behavioral Physiology inSeewiesen, Germany, and his colleaguesshowed that volunteers who lived in anisolation bunker—with no natural light,clocks or other clues about time—nev-ertheless maintained a roughly normalsleep-wake cycle of 25 hours.

More recently Charles Czeisler, Rich-ard E. Kronauer and their colleagues atHarvard University have determinedthat the human circadian rhythm is ac-tually closer to 24 hours—24.18 hours,to be exact. The scientists studied 24men and women (11 of whom were intheir 20s and 13 of whom were in their60s) who lived for more than threeweeks in an environment with no timecues other than a weak cycle of lightand dark that was artificially set at 28hours and that gave the subjects theirsignals for bedtime.

They measured the participants’ corebody temperature, which normally fallsat night, as well as blood concentra-tions of melatonin and of a stress hor-mone called cortisol that drops in theevening. The researchers observed thateven though the subjects’ days had beenabnormally extended by four hours,their body temperature and melatoninand cortisol levels continued to func-tion according to their own internal 24-hour circadian clock. What is more, ageseemed to have no effect on the tickingof the clock: unlike the results of previ-ous studies, which had suggested thataging disrupts circadian rhythms, thebody-temperature and hormone fluctu-ations of the older subjects in the Har-vard study were as regular as those ofthe younger group.

As informative as the bunker studiesare, to investigate the genes that under-lie the biological clock scientists had toturn to fruit flies. Flies are ideal for ge-netic studies because they have short lifespans and are small, which means thatresearchers can breed and interbreedthousands of them in the laboratory un-til interesting mutations crop up. Tospeed up the mutation process, scientistsusually expose flies to mutation-causingchemicals called mutagens.

The first fly mutants to show alteredcircadian rhythms were identified in theearly 1970s by Ron Konopka and Sey-mour Benzer of the California Instituteof Technology. These researchers fed amutagen to a few fruit flies and thenmonitored the movement of 2,000 ofthe progeny, in part using a form of thesame apparatus that we now use in ourNew York to San Francisco experiments.Most of the flies had a normal 24-hourcircadian rhythm: the insects were ac-

tive for roughly 12 hours a day and rest-ed for the other 12 hours. But three ofthe flies had mutations that caused themto break the pattern. One had a 19-hourcycle, one had a 28-hour cycle, and thethird fly appeared to have no circadianrhythm at all, resting and becoming ac-tive seemingly at random.

Time Flies

In 1986 my research group at theRockefeller University and another

led by Jeffrey Hall of Brandeis Universi-ty and Michael Rosbash of the HowardHughes Medical Institute at Brandeisfound that the three mutant flies hadthree different alterations in a single genenamed period, or per, which each of ourteams had independently isolated twoyears earlier. Because different mutationsin the same gene caused the three behav-iors, we concluded that per is somehowactively involved both in producing cir-

The Tick-Tock of the Biological Clock Scientific American March 2000 67

Most of the research on the biological clocks of animals has focused on thebrain,but that is not the only organ that observes a day-night rhythm.

Jadwiga Giebultowicz of Oregon State University has identified PER and TIMproteins—key components of biological clocks—in the kidneylike malpighiantubules of fruit flies. She has also observed that the proteins are produced accord-ing to a circadian cycle, rising at night and falling during the day.The cycle persistseven in decapitated flies, demonstrating that the malpighian cells are not merelyresponding to signals from the insects’brains.

In addition,Steve Kay’s research group at the Scripps Research Institute in La Jol-la,Calif.,has uncovered evidence of biological clocks in the wings, legs,oral regionsand antennae of fruit flies.By transferring genes that direct the production of fluo-rescent PER proteins into living flies, Kay and his colleagues have shown that each

tissue carries an independent,photoreceptive clock. The clockseven continue to function andrespond to light when each tis-sue is dissected from the insect.

And the extracranial biologicalclocks are not restricted to fruitflies. Ueli Schibler of the Univer-sity of Geneva showed in 1998that the per genes of rat connec-tive-tissue cells called fibroblastsare active according to a circadi-an cycle.

The diversity of the various celltypes displaying circadian clockactivity suggests that for manytissues correct timing is impor-tant enough to warrant keepingtrack of it locally. The findingsmight give new meaning to theterm “body clock.” — M.W.Y.

CLOCKS EVERYWHERE

JUER

GEN

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Max

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nck

Inst

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/SPL

/Pho

to R

esea

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rs,I

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HEAD OF A FRUIT FLY contains several bio-logical clocks. Cells taken from the oral regionsand antennae (structures between eyes) showthe same response to cycles of light and dark-ness as those isolated from the brain.

THEY ARE NOT JUST IN THE BRAIN


RIBOSOME

TIM PROTEIN

PER PROTEIN

CYCLE/CLOCK COMPLEX

MIDNIGHT

3

4

DUSK

DAWN

NEWLY TRANSLATEDTIM PROTEIN

NEWLY TRANSLATEDPER PROTEIN

Once in the cytoplasm, the perand tim messenger RNAs dockwith structures called ribo-somes. The ribosomes beginto “read out,” or translate, theinformation in the messengerRNAs to make strings of aminoacids.The strings subsequent-ly fold into mature PER andTIM proteins that stick to oneanother to form new PER/TIMcomplexes at dusk.

The Tick-Tock of the Biological Clock68 Scientific American March 2000

A DAY IN THE BIOLOGICAL CLOCK OF A FLYCELLS FROM A FLY BRAIN HAVE MOLECULAR CLOCKWORKS CLOSELY RELATED TO THOSE OF HUMANS

During the night, the newPER/TIM complexes moveinto the nucleus, wherethey block the activity ofCYCLE and CLOCK, essen-tially shutting down theirown production.When thesun comes up the follow-ing day, the PER/TIM com-plexes break down, andthe cycle starts anew.


cadian rhythm in flies and in setting therhythm’s pace.

After isolating per, we began to ques-tion whether the gene acted alone incontrolling the day-night cycle. To findout, two postdoctoral fellows in mylaboratory, Amita Sehgal and JeffreyPrice, screened more than 7,000 flies tosee if they could identify other rhythmmutants. They finally found a fly that,like one of the per mutants, had no ap-parent circadian rhythm. The new mu-tation turned out to be on chromosome2, whereas per had been mapped to theX chromosome. We knew this had tobe a new gene, and we named it time-less, or tim.

But how did the new gene relate toper? Genes are made of DNA, whichcontains the instructions for makingproteins. DNA never leaves the nucleusof the cell; its molecular recipes are readout in the form of messenger RNA,which leaves the nucleus and enters thecytoplasm, where proteins are made. Weused the tim and per genes to make PERand TIM proteins in the laboratory. Incollaboration with Charles Weitz ofHarvard Medical School, we observedthat when we mixed the two proteins,they stuck to each other, suggesting thatthey might interact within cells.

In a series of experiments, we foundthat the production of PER and TIMproteins involves a clocklike feedbackloop [see illustration at left]. The per andtim genes are active until concentrationsof their proteins become high enoughthat the two begin to bind to each oth-er. When they do, they form complexesthat enter the nucleus and shut downthe genes that made them. After a fewhours enzymes degrade the complexes,the genes start up again, and the cyclebegins anew.

Moving the Hands of Time

Once we had found two genes thatfunctioned in concert to make a

molecular clock, we began to wonderhow the clock could be reset. After all,our sleep-wake cycles fully adapt totravel across any number of time zones,even though the adjustment might takea couple of days or weeks.

That is when we began to shuttletrays of flies back and forth between the“New York” and “San Francisco” in-cubators. One of the first things we andothers noticed was that whenever a flywas moved from a darkened incubatorto one that was brightly lit to mimic day-

Scientific American March 2000 69

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CELL FROMFLY BRAIN

1

2

NOON

PER PROTEIN

PER MESSENGER RNA

CLOCK PROTEIN

CYCLE PROTEIN

TIM MESSENGER RNA

By midday, all the PER and TIM proteins have beendegraded, and two other proteins named CYCLEand CLOCK stick to each other to form complexesthat bind to the per and tim genes to turn them on.When the per and tim genes become active, theymake genetic intermediates,called messenger RNAs,that move into the cytoplasm.

When a fly is exposed to light, molecularcomplexes made of two proteins namedPER (for “PERIOD”) and TIM (for “TIME-LESS”) begin to break down in the cellsof the fly’s brain.The PER/TIM complexesare part of a feedback loop that controlsthe activity of the per and tim genes,which contain the instructions for mak-ing PER and TIM proteins.

PER GENE

TIM PROTEIN

TIM GENE

NUCLEUS

CYTOPLASM

The Tick-Tock of the Biological ClockCopyright 2000 Scientific American, Inc.

light, the TIM proteins in the fly’s braindisappeared—in a matter of minutes.

Even more interestingly, we noted thatthe direction the flies “traveled” affectedthe levels of their TIM proteins. If we re-moved flies from “New York” at 8 P.M.

local time, when it was dark, and putthem into “San Francisco,” where itwas still light at 5 P.M. local time, theirTIM levels plunged. But an hour later,when the lights went off in “San Fran-cisco,” TIM began to reaccumulate. Ev-idently the flies’ molecular clocks wereinitially stopped by the transfer, but af-ter a delay they resumed ticking in thepattern of the new time zone.

In contrast, flies moved at 4 A.M. from“San Francisco” experienced a prema-ture sunrise when they were placed in“New York,” where it was 7 A.M. Thismove also caused TIM levels to drop,but this time the protein did not beginto build up again because the molecularclock was advanced by the time-zoneswitch.

We learned more about the mecha-nism behind the different molecular re-sponses by examining the timing of theproduction of tim RNA. Levels of timRNA are highest at about 8 P.M. localtime and lowest between 6 A.M. and 8A.M. A fly moving at 8 P.M. from “NewYork” to “San Francisco” is producingmaximum levels of tim RNA, so pro-tein lost by exposure to light in “SanFrancisco” is easily replaced after sun-set in the new location. A fly travelingat 4 A.M. from “San Francisco” to“New York,” however, was makingvery little tim RNA before departure.What the fly experiences as a prema-ture sunrise eliminates TIM and allowsthe next cycle of production to beginwith an earlier schedule.

Not Just Bugs

Giving flies jet lag has turned out tohave direct implications for under-

standing circadian rhythm in mammals,including humans. In 1997 researchersled by Hajime Tei of the University ofTokyo and Hitoshi Okamura of KobeUniversity in Japan—and, independent-ly, Cheng Chi Lee of Baylor College ofMedicine—isolated the mouse and hu-man equivalents of per. Another flurryof work, this time involving many labo-ratories, turned up mouse and humanforms of tim in 1998. And the genes wereactive in the suprachiasmatic nucleus.

Studies involving mice also helped toanswer a key question: What turns on

the activity of the per and tim genes inthe first place? In 1997 Joseph Takaha-shi of the Howard Hughes Medical In-stitute at Northwestern University andhis colleagues isolated a gene they calledClock that when mutated yielded micewith no discernible circadian rhythm.The gene encodes a transcription factor,a protein that in this case binds to DNAand allows it to be read out as messen-ger RNA.

Shortly thereafter a fly version of themouse Clock gene was isolated, and var-ious research teams began to introducecombinations of the per, tim and Clockgenes into mammalian and fruit fly cells.These experiments revealed that theCLOCK protein targets the per gene inmice and both the per and tim genes inflies. The system had come full circle: inflies, whose clocks are the best under-stood, the CLOCK protein—in combi-nation with a protein encoded by a genecalled cycle—binds to and activates theper and tim genes, but only if no PERand TIM proteins are present in the nu-cleus. These four genes and their proteins


1:00 A.M.• Pregnant women are most likely

to go into labor.• Immune cells called helper

T lymphocytes are at their peak.

2:00 A.M.• Levels of growth hormone are

highest.

4:00 A.M.• Asthma attacks are most likely

to occur.

6:00 A.M.• Onset of menstruation is most

likely.• Insulin levels in the bloodstream

are lowest.• Blood pressure and heart rate

begin to rise.• Levels of the stress hormone

cortisol increase.• Melatonin levels begin to fall.

7:00 A.M.• Hay fever symptoms are worst.

8:00 A.M.• Risk for heart attack and stroke

is highest.• Symptoms of rheumatoid arthritis

are worst.• Helper T lymphocytes are at their

lowest daytime level.

Noon• Level of hemoglobin in the blood is

at its peak.

3:00 P.M.• Grip strength, respiratory rate and

reflex sensitivity are highest.

4:00 P.M.• Body temperature, pulse rate and

blood pressure peak.

6:00 P.M.• Urinary flow is highest.

9:00 P.M.• Pain threshold is lowest.

11:00 P.M.• Allergic responses are most likely.

BODY CHANGES OVER 24-HOUR PERIOD

AD

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H,S

PRIN

G 1

998

MONITORING THE CIRCADIANRHYTHMS OF FLIESAN APPARATUS TO TRACK FLY ACTIVITY

To identify genes that play a role inthe day-night activity cycle of flies,

researchers expose fruit flies to mu-tation-causing chemicals that affectthe genes of their offspring. Whenthey become adults, each of the off-spring is placed into a small glasstube. The tubes have fly food at oneend and cotton at the other end tolet in air.

The fly tubes are placed into spe-cial trays equipped with infraredlights and detectors. Fruit flies nor-mally rest at night and are active dur-ing the day. The trays, which are con-nected to a computer, monitor eachfly’s movements by recording howmany times it passes through an in-frared beam. Analyzing thousands ofmutant flies in this manner eventual-ly yields some with abnormal circadi-an rhythms. —M.W.Y.


constitute the heart of the biologicalclock in flies, and with some modifica-tions they appear to form a mechanismgoverning circadian rhythms through-out the animal kingdom, from fish tofrogs, mice to humans.

Recently Steve Reppert’s group atHarvard and Justin Blau in my labora-tory have begun to explore the specificsignals connecting the mouse and fruitfly biological clocks to the timing ofvarious behaviors, hormone fluctua-

tions and other functions. It seems thatsome output genes are turned on by adirect interaction with the CLOCKprotein. PER and TIM block the abilityof CLOCK to turn on these genes at thesame time as they are producing the os-cillations of the central feedback loop—

setting up extended patterns of cyclinggene activity.

An exciting prospect for the futureinvolves the recovery of an entire sys-tem of clock-regulated genes in organ-

isms such as fruit flies and mice. It islikely that previously uncharacterizedgene products with intriguing effects onbehavior will be discovered within thesenetworks. Perhaps one of these, or acomponent of the molecular clock it-self, will become a favored target fordrugs to relieve jet lag, the side effectsof shift work, or sleep disorders and re-lated depressive illnesses. Adjusting to atrip from New York to San Franciscomight one day be much easier.

The Tick-Tock of the Biological Clock

The Author

MICHAEL W. YOUNG is a professor and head of the Laboratoryof Genetics at the Rockefeller University. He also directs the Rocke-feller unit of the National Science Foundation’s Science and Technol-ogy Center for Biological Timing, a consortium that connects labora-tories at Brandeis University, Northwestern University, Rockefeller,the Scripps Research Institute in La Jolla, Calif., and the University ofVirginia. After receiving a Ph.D. from the University of Texas in1975, Young took a postdoctoral fellowship at the Stanford Universi-ty School of Medicine to study gene and chromosome structure. In1978 he joined the faculty of Rockefeller, where members of his re-search group have isolated and deciphered the functions of four ofthe seven genes that have been linked to the fruit fly biological clock.

Further Information

The Molecular Control of Circadian Behavioral Rhythmsand Their Entrainment in DROSOPHILA. Michael W. Young inAnnual Review of Biochemistry, Vol. 67, pages 135–152; 1998.

Molecular Bases for Circadian Clocks. Jay C. Dunlap inCell, Vol. 96, No. 2, pages 271–290; January 22, 1999.

Time, Love, Memory: A Great Biologist and His Quest forthe Origins of Behavior. J. Weiner. Alfred Knopf, 1999.

A tutorial on biological clocks—including ideas for home and class-room activities—can be found on the National Science Founda-tion’s Science and Technology Center for Biological Timing’s siteat http://cbt4pc.bio.virginia.edu/tutorial/TUTORIALMAIN.htmlon the World Wide Web.

SA

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INFRARED LIGHTSOURCE

INFRARED BEAM

PHOTOTRANSISTOR

FLY FOOD

1

2

3

4

5

6

COTTON

STOPPER

TRAY FULL OF FLY TUBES

COMPUTERIZEDRECORD OF FLY

ACTIVITY

Scientific American March 2000 71


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