Time, Love , Memory

by Jonathan Weiner

  In 1967 Benzer published his first paper on the new work, “Behavioral Mutants of Drosophila Isolated by Countercurrent Distribution,” a paper that scientists in several disciplines now think of as a landmark. He was forty-six. He left Purdue, joined the biology department at Caltech, and moved into the laboratory space in Church Hall where he still works today.

  Benzer had written that first paper as a kind of mission statement for his research program. But the project that proved the plan could actually work was done by a graduate student of Benzer’s, Ronald J. Konopka, from Dayton, Ohio. Konopka joined the laboratory because he wanted to use Benzer’s genetic scalpel to find and dissect the sense of time. He thought there must be a master clock hidden in the clockwork of life, and he thought Benzer’s method was the way to find it. Morning glories know when to bloom. Bears in caves know when to wake up. Grunions know when to spawn. (“And doubtless, when swallows come in the spring, they act like clocks,” says Descartes.) During the Enlightenment the French astronomer Jean-Jacques d’Ortous de Mairan performed a famous experiment with heliotrope, a plant whose Latin name means “turning toward the sun.” The heliotrope’s leaves and stems unfold every morning when the sun comes up and fold again every evening when the sun goes down. In the summer of 1729, the astronomer dug up a single heliotrope plant, brought it inside, put it in a pitch-black room, and peeked in every now and then. Even in the dark room the plant was raising its arms and lowering them again, keeping time with the heliotrope out in the garden. A note about this experiment appeared that year in the Histoire de l’Académie Royale des Sciences: “The sensitive plant follows the sun without being exposed to it in any way. This is reminiscent of that delicate perception by which invalids in their beds can tell the difference between day and night.”

  The astronomer’s experiment inspired innumerable imitations. A French botanist carried “sensitive plants” down into a wine cave and watched them by candlelight—not a bad research project. A Swiss botanist tried growing sensitive plants in a room lit only by banks of lamps, and he managed to change the plants’ behavior by lighting or snuffing the lamps. The great Swedish naturalist Carolus Linnaeus dreamed of putting together a flower clock made of evening primrose, marigolds, childing pink, scarlet pimpernel, hawkbit, bindweed, nipple wort, passion flower, spotted cat’s ear, and the Star of Bethlehem, “by which,” Linnaeus wrote, “one could tell time, even in cloudy weather, as accurately as by a watch.” The passion flower would open at noon, the evening primrose at 6 p.m., and so on. One summer, Darwin drew a series of diagrams of a single leaf of Virginia tobacco as it stirred upward and downward from three in the afternoon to 8:10 the next morning.

  Silverfish, crickets, spiders, scorpions, and squirrel monkeys have a sense of time. Biologists proved this by simple experiments, building exercise wheels like the wheel in a mouse cage and monitoring an animal’s cycles of sleeping and waking on the wheel in windowless rooms that were never dark or never light. Twentieth-century biologists built exercise wheels like these for sea hares, lizards, and cockroaches; and they built balances like seesaws so that every time the creature moved, it tilted the seesaw. The more they looked, the more they discovered that the sense of time is everywhere. Even single-celled animals such as Euglena have a sense of time. Euglena swims like an animal but has green chlorophyll like a plant. In a pond, each cell swims more by day than by night; at night it tends to sink sluggishly downward and downward through the water column. And even in a lab in constant light it keeps to this rhythmic cycle, which biologists call “circadian,” meaning “about a day.”

  The literature on circadian rhythms is full of strange factoids about the sense of time in living things. The cells of a banana divide just after dawn. Some animals have clocks longer than twenty-four hours: a normal human clock, for example, runs a bit slow in a cave or a windowless room; we automatically adjust it every day to keep in time with the sun. Our clocks are reset by the dawn. The clocks of mice run a bit fast, and they are reset by the dusk. Confuse the sense of time of a homing pigeon—shift its clock six hours by tricking it with lights—and it will make a ninety-degree error in the path of its flight. Many people can order themselves to wake up at, say, seven in the morning—and they will wake up within a few minutes of seven. “All this showing,” as Spinoza says, “that the body itself can do many things from the laws of its own nature alone at which the mind belonging to that body is amazed.”

  By the middle of the century most biologists assumed that this sense of time must be in the genes. But contrarians argued for nurture, not nature. Living things might learn to keep time with the beat of the sun the way goslings learn to follow Mother Goose or Konrad Lorenz. They argued that each seedling, gosling, and newborn baby might be imprinted by the sun, learning the rhythm of day and night and then keeping the beat, keeping time with the sun for the rest of their lives. Some biologists speculated that even sensitive plants sequestered in a wine cave might keep time by picking up subtle tides in atmospheric electricity or tides of cosmic rays, or perhaps secret signals linked to the phases of the moon, cycles of sunspots, or the rotation of the planet.

  To prove that the sense of time is a matter of nurture, not nature, a biologist at Northwestern University, Frank A. Brown Jr., designed elaborate experiments involving carrots, seaweed, crabs, rats, and much solitude and darkness. He grew potato plugs in sealed jars and monitored the rhythms of their metabolism by sampling carbon dioxide and oxygen with gas detectors. He shipped oysters from New Haven, Connecticut, to his laboratory in Evanston, Illinois, and watched them open and close their shells rhythmically day after day in pans of seawater. In another herculean experiment he watched and timed 33,000 individual mud snails as they crawled out of holes. He also lobbied the U.S. National Aeronautics and Space Administration to put one of his potatoes into orbit. He wanted to see what would happen to its metabolic rhythms when the potato was aloft in a satellite and could no longer feel the rotation of the planet. NASA never took up his potatoes.

  In 1960, a botanist tested Brown’s hypotheses in a cheaper experiment by flying Syrian golden hamsters, among other creatures, to the South Pole. There he put the hamsters and their cages on a turntable so that their rotation would counteract the rotation of the Earth. (The Earth’s rotation cannot be counteracted this way except at the pole.) The hamsters went right on waking and sleeping at the same times and with the same rhythms as hamsters that were not revolving on turntables. Apparently the hamsters did not need any cues from the rotation of the Earth to keep track of time. The botanist also put bean plants, fungi, cockroaches, and fruit flies on his turntables. All of them kept time perfectly.

  After that experiment, it seemed clear to almost every biologist on the planet (except Brown) that living things really are born with some kind of inner clock. But no one knew where the clock might be hidden in the body or how it might work. One test of a true clock is its ability to keep time through a wide range of temperatures. A clock that speeds up in hot weather and slows down in cold weather is not a clock, although it may make a good thermometer. So a drosophilist checked the clock in fruit flies by raising them at different temperatures. Heat and cold did nothing to change their rhythms. Whatever they had inside them, wherever it was concealed, and however it worked, it really did deserve to be called a clock.

  In 1969, at a meeting on the sense of time, the botanist who had gone to the South Pole, Karl Hamner of UCLA, declared that the problem was as mysterious as gravity before Newton: “What we need now is another Newton.”

  MEANWHILE, Konopka was experimenting in Benzer’s laboratory. The behavior the flies are named for is waking up in the morning: Drosophila means “lover of dew.” In fact, the flies display their love of dew from the moment they are born. Each young fly develops inside a pupal case. When it is ready to emerge, the fly does not pip its shell with a beak, like a bird; instead it inflates a tiny balloon on its head, like a steering-wheel air bag, and bursts right out of the pupa. Benzer think
s a fly emerging from a pupa is one of the sweetest things in nature. The fly crawls out wet, like a newborn baby. Its wings are not yet inflated; they are crumpled like a new butterfly’s, and because of the air bag its head is still disproportionately large, again like a newborn baby’s. In nature, the flies usually emerge around dawn, when the world is moist and dewy. Even if a jar full of fly pupae is taken out of the light and placed in total darkness for several days, the young flies will still emerge together in the dark, around the time of their virtual dawn.

  By the time Konopka went to work with Benzer, biologists had already mounted round-the-clock watches on fruit flies. They had discovered that normal flies live on a daily cycle just like the astronomer’s heliotrope. At sundown a normal fly becomes very quiet. It does not close its eyes, but it stops moving and looks as if it is asleep on its feet. At sunup it begins to move around again. Flies will keep to this daily cycle even in total darkness, just like the heliotrope.

  In Benzer’s laboratory, Konopka poisoned flies with EMS to make random “typos” in their DNA. He used these poisoned flies to establish hundreds of separate lines of mutant flies. When he was finished, he had hundreds of fly bottles, and each bottle contained a separate line of mutants. Konopka spent most of the summer of ’68 watching these mutants’ children eclose in bottle after bottle, looking for flies that missed the dawn. It was a lonely way for a young man in California to spend the summer, and most of the professors and students who passed by his door thought he was wasting his time. The smart money said that Konopka had chosen a piece of behavior too central and too complicated to dissect through the genes. A mechanical clock has hundreds of gears, springs, screws, and ratchets. A living clock might require hundreds of working parts too, and hundreds of genes to make each part. But the parts of a clock are complexly interdependent, and if EMS caused a mutation in any one of those hundreds or thousands of genes, the result for the fly was likely to be a broken clock. That is to say, any of hundreds or thousands of mutations would have the identical effect on the fly, destroying its sense of time. So even if Konopka did find a clock mutant, he still might not have a clue what had gone wrong inside it. A fly without a clock might not even live long enough to eclose.

  The ragging that Konopka endured that summer would later become a legend in the Benzer lab and far outside it. Geneticists and molecular biologists would tell the story to the tune of “They Laughed at Columbus.” “They said, ‘It’s too much work!’ ” says Jeff Hall, who was another of Benzer’s first postdoctoral students. “They said, ‘You’ll never find them!’ They said, ‘If you make one, it will die!’ But he told them, ‘Bugger off!’ ”

  Konopka kept all of his flies at a constant temperature in cycles of twelve hours of white fluorescent light and twelve hours of darkness. For the sake of sanity and simplicity, he checked the bottles only twice a day: just after the lights went on in the morning, and just before the lights went off in the evening. If the flies had a normal sense of time, very few of them would eclose before that first check in the morning. When he inspected their bottle in the morning, it should still be full of eggs. The flies would eclose from those eggs in the next few hours of light, and he would find them creeping, crawling, and flitting around in the bottle when he checked on them that evening. So if Konopka checked one of his fly bottles first thing in the morning and found dozens of newborn flies inside it, he would know that they had eclosed sometime in the night, and he would suspect that there was something wrong with their sense of time.

  In bottle after bottle, the flies behaved normally. But in the two hundredth bottle, when Konopka checked in the morning, he saw that it was teeming with flies. And when he inspected them one by one, he saw that most of them were males. Konopka bred those males. When it was almost time for their children to eclose, he put them into absolute darkness and waited to find out what they would do. Not many eclosed at dawn. They eclosed at all hours of the day and night, just like their fathers. He had found his first clock mutant.

  In a second bottle, Konopka discovered a line of mutants that eclosed at the wrong time too. And when he mounted a careful watch over them he saw that they eclosed too early. Apparently their dawn came sooner than it came for the rest of the world. And in a third bottle he found a line of mutants that eclosed too late.

  Konopka talked over the case of these three mutants during a long, meandering lunch with Benzer and Hotta and a few other students. They all wondered what the mutants’ sense of time would be like after they eclosed. Over lunch, Benzer thought of a quick way to help Konopka find out. As Benzer tells it now, this was another small countercurrent moment. “The people in the lab were split. One guy said, ‘That’ll never work. If that works, I’ll buy you an Indonesian dinner.’ ”

  So Benzer tried it. He went back to a standard gadget from physics and chemistry, a spectrophotometer. The centerpiece of a spectrophotometer is a little glass square-sided cylinder called a cuvette. When physicists or chemists have a mystery substance to identify, they pour a few drops of it into the cuvette and switch on the spectrophotometer. The gadget fires a series of light beams through the cuvette: all the colors of the rainbow, plus ultraviolet and infrared. A sensor analyzes each beam as it passes through the cuvette, and a marking pen makes a series of squiggles on a rolling drum of paper. Sometimes the investigators can identify their mystery substance from the pattern of the squiggles on the paper.

  Benzer put two strips of black tape on the outside of the cuvette, with a little space between them for the beam to pass through. Then he put one of Konopka’s flies inside the cuvette, and corked it with a cotton plug. He set the spectrophotometer to infrared, which flies can’t see. Whenever the fly moved from one end of the cuvette to the other it would pass between the two strips of tape and block the infrared beam, and that would make the pen jiggle up and down on the drum of paper. When he was finished, Benzer turned on the machine and let it run all night.

  The next morning, Konopka arrived at the second floor of the Church Laboratory and found paper spewed out all over his floor in an almost endless scroll. He fished through loop after loop of paper and looked at the bursts of inky squiggles. The trick worked. He could see exactly how-busy the flies had been every minute of the night.

  (“So that was a very good dinner, actually,” Benzer says. “J.J.’s Little Bali, near the airport, in Inglewood.”)

  Later, Benzer’s postdoc Yoshiki Hotta built a whole set of gadgets based on the same principle, so that Konopka could monitor many flies at once. Now Konopka could find out what his mutants were doing in the dark. A few days of monitoring told him that his first line of mutants was consistent in its inconsistency. Not only did these mutants eclose at all hours of the day and night; for the rest of their lives they woke and slept, wandered and paused at all hours of the day and night. They acted like insomniacs. They seemed to be time-blind.

  The line of mutants that eclosed early was also consistent. The day after they eclosed, they woke up about five hours too early; they did the same thing for the rest of their lives. Apparently these mutants did have clocks, but the clocks ran too fast. Their days had a period of nineteen hours. And Konopka’s third line of mutants, the line that eclosed late, was just as consistent. They woke up late every day of their lives. Their clocks ran slow. Their days had a period of twenty-nine hours.

  Konopka examined the mutants through a microscope. All of them, females and males, throughout all the stages of their life cycles, from egg to larva to pupa to adult, looked absolutely normal. And when they bred, they passed on their sense of time from generation to generation. He could see that in bottle after bottle only the mutant flies had a warped sense of time.

  If Konopka had been working with chicks, chameleons, monkeys, guinea pigs, potatoes, or heliotrope, he might not have been able to push this work any further. But with Drosophila the next step was obvious. He crossed his short-period mutants with a few classic mutants from the Fly Room, including white, singed, yellow, and
miniature. Methodically, using cross after cross, he began trying to map the short-period mutation, using the same method that Sturtevant had invented in Morgan’s first Fly Room. The method was the same, but now he was trying to map a gene that was manifest not in the color of the fly’s eyes or the shape of its wings but in its behavior: a mutation that changed the way it moved through time.

  Konopka found that the short-period mutation mapped to the far-left end of the X chromosome, less than one map unit from white. When he mapped the arrythmic mutation, he found that it too was on the far-left end of the X chromosome, also next to white. He mapped the long-period fly, and again it was on the far-left end of the X chromosome, also next to white.

  By now geneticists called the map unit the centimorgan, in honor of the man who had started their science. If there is a 1 percent chance that two genes will be separated by crossing-over, those two genes are said to be separated by one centimorgan. Konopka’s three mutants were less than one centimorgan away from white, and they were zero centimorgans apart from each other.

  Now Konopka was amazed. These were the first three time mutants he had found, and they all mapped to precisely the same place. Because they mapped to the same place, they had to be alleles, or variants, of the same gene, like tall and short in Mendel’s peas. Konopka had looked at more than two hundred strains of mutants to find these three time mutants, and all three of them pointed to the same spot on the same chromosome. He had barely started his search, and already he seemed to have stumbled straight into the center of the living clock.