No doubt the process of decipherment was difficult, but only by accomplishing it could one arrive at whatever truth there was to read.
—MARCEL PROUST,
Time Regained
THE MOST DIRECT WAY to clone a fly gene is to take a needle and cut it right out of one of the giant chromosomes in the fly’s salivary glands. These are chromosomes that carry extra copies of each gene: hundreds of copies side by side, so many that through the microscope each salivary chromosome looks swollen and banded, like an obese coral snake. When drosophilists first discovered those banded chromosomes, it was as if they were seeing their maps made flesh. In a fuzzy way they could make out the locations of white, yellow, and other genes that Sturtevant had mapped on that first night, and the bands were close to the positions where Sturtevant had mapped them.
Under the microscope, with a tiny needle and a micromanipulator to guide the needle, a skilled drosophilist can simply slice out the DNA from the region of the giant chromosome where the gene lies. One of the pioneers of this kind of cloning was Vincent Pirrotta, who was then at the European Molecular Biology Laboratory in Heidelberg, Germany. In 1983, at Hall’s request, Pirrotta sent Hall three overlapping DNA fragments that he had cut out of the X chromosome of an ordinary, wild-type fly from positions very close to white.
Somewhere among these three overlapping fragments was the period gene. Konopka’s old maps were not precise enough to say where. The gene might be on any one of the three ribbons, or it might straddle any two of them. To find the gene, Rosbash and Hall once again decided on the simplest and most direct strategy. They instructed students in their laboratories to make copies of the three DNA ribbons and cut the copies into random assortments of smaller fragments, using a whole battery of restriction enzymes as scissors. In this way they made a library of snipped DNA ribbons, so that they could inject each fragment of DNA into a single fly egg and test the fragments one by one.
Cloning in action. A fly has 15,000 genes. Here a microscopic needle cuts out a single one of them: the period gene, from the fly’s first chromosome. (Illustrations credit 12.1)
When the DNA library was complete, they collected eggs from a mutant female fly that had no sense of time. She carried Konopka’s mutant gene, the allele that makes a fly time-blind. She had two copies of that allele, one on each X chromosome. The father was also time-blind. So their children should have been time-blind too. If Hall and Rosbash could inject period into this family of time-blind flies and give them rhythm, they would be the first to cure a broken piece of behavior by injecting a gene. They would have inserted a gene into an embryo and given its living descendents a complex program of behavior.
Rosbash’s team mixed what is known as a “DNA transformation cocktail.” The cocktail included one of the mystery fragments of X chromosome from their DNA library and a P element to make the fragment restless, so that it might jump into the embryo’s DNA. If the fragment of DNA inserted itself in the right place, and if that fragment included the period gene, then the children of the young fly would have a normal sense of time. Each one of those newborn fly children would pop out of its egg at the normal time, the crack of dawn. Each one would go to sleep that first evening of its life at the normal time, sundown. And if the fly was a male, he would sing in standard time when he met his first female fly.
DNA injection is a straightforward laboratory procedure: wash the young embryo with ordinary household bleach (most genetic engineers prefer Clorox) to remove its chorion, the tough outer shell, then insert the microsyringe in the embryo’s rear end. Of course, the embryo is so small that the injection must be performed under a microscope. One by one, the would-be engineers injected their DNA fragments into the rear ends of mutant fly embryos. According to Konopka’s map, some of these DNA fragments should hold the period gene.
Fragment after fragment of DNA went into embryo after embryo, and students in the Drosophila Arms watched bottle after bottle. The flies they had chosen for this experiment were not only time-blind; they also had no tolerance for alcohol, because they carried a damaged form of a gene that helps flies hold their liquor. However, in the DNA transformation cocktail, Hall and Rosbash had included a normal form of that particular gene, so they could tell quickly and easily which young flies might have a clock worth testing. Any fly that had a normal ability to hold its liquor might also have a normal sense of time. When each young fly was four days old, a postdoc of Hall’s, Will Zehring, put it in a little glass tube with a Kleenex soaked in alcohol. If the fly had no tolerance for alcohol, it would sip from the Kleenex and die. But if the fly had inherited the ribbon of genes on the DNA transformation cocktail, it would live. “You come back overnight and say, ‘Oh, my God, we’ve got transformants!’ ” says Hall. They found about one fly that could hold its liquor for every five hundred flies that could not.
Zehring raised each individual hard-drinking fly in its own bottle, and tested it in the same way that Konopka had tested his mutants. He put it in a little glass tube, so that each time the fly walked around in the tube it would break a beam of infrared light and cause a marking pen to draw a squiggle on a scroll of paper. Then he could look at the squiggles and see when the flies were awake and when they were asleep. Hall and Rosbash kept the transformed flies in the subbasement, away from lights, in what Hall called the “pit.”
In those days Hall and Rosbash were ideal partners. Hall had the genetics, the fly lore, and the flies. He also had a scrupulous sense of fair play that he had inherited from the Drosophila tradition. Rosbash had the drive and impatience of molecular biology and also a certain unscrupulousness that he had inherited from the molecular biology tradition. “Michael Rosbash was always a little bit like the bad boy of biology,” says a molecular biologist who has known Rosbash for years. “Arrogant, always irreverent, extremely ambitious, and he earned a reputation for being a little cutthroaty—but also for being very smart.” At the time he joined forces with Hall, Rosbash was working on mutants in yeast, and he had published some well-regarded papers, his colleague says. “But I always felt, when he started on the per gene, Michael had finally found the interesting biological story that he had been looking for.” For an ambitious young molecular biologist, yeast was too crowded. “Some fields become so deep that it is hard to rise above the surf. Whereas per was unique. It was the only clock gene that anyone had a clue about. It was a golden problem.”
Even before Rosbash and Hall began working on cloning the period gene, another young molecular biologist had also decided that period might be something wonderful. Like Rosbash, Michael Young of Rockefeller University in New York City had begun to suspect that Drosophila was ready to go molecular, and he also thought period might be the perfect place to start. In a Fly Room at Rockefeller, Young began racing Rosbash and Hall to clone period. He, too, collected a library of candidate ribbons of DNA and began injecting them into fly eggs one by one and watching those flies. He, too, had a big box with a roll of paper and a pen that jiggled whenever a fly moved. When he came into the lab each morning, he would roll out fifteen feet of paper and look for rhythms—the periodic bursts of squiggles in the scrolls. Working on behavior was not like doing an experiment in a petri dish, in which one sees the results in an hour or two. After each egg was injected, there would be a wait of several days. Young knew that he was racing the Boston group, and he knew that Rosbash was a speed demon. He remembers vividly how each day he would run into the lab and unroll those scrolls. In the old days, fly people had left problems alone if other people were working on them, but ever since Watson’s The Double Helix, the ethos of molecular biology had been The Race. Young loved it. “It was movielike,” he says.
Late in 1984, Hall and his students looked at their scrolls and decided they had done it: they had injected the first piece of behavior into the genes. The flies had rhythm—because they had received the instinct for it by injection. The flies would not have had rhythm unless Hall and Rosbash had succeeded in cloning the gene they were trying to clone. Hall drove to upstate New York, where the discoverer of period, Ronald J. Konopka himself, was now working in quasi-exile at a small college called Clarkson. Konopka had failed to get tenure in the biology department at Caltech. Hall blamed Benzer for his lack of generalship; it was one of their first serious fights. But Benzer says his colleagues were disappointed by Konopka’s reluctance to publish. Konopka was a perfectionist, and he did not feel he had anything perfect to say about period.
At Clarkson, Konopka had built a computer setup to monitor period mutants in their test tubes. Before, using scrolls of paper, he had been able to monitor the behavior of half a dozen flies a week. But with his new setup, Konopka could monitor hundreds of flies each week. Hall handed his transformed flies to Konopka without telling him what it was he thought he had found, and he asked Konopka for an independent opinion of their sense of time.
“So we went home,” Hall says now, “and in two or three weeks, Ron called us. He was just doing these tests blind. He didn’t know what they were. He said, ‘They’re rhythmic.’ And that nailed it. That nailed it for us. So then we knew that we had the gene.” They were too full of The Race to think very much about the implications. Hall also sent some of their new mutants to Kyriacou, who by this time had established his own Fly Room at the University of Leicester in England. Kyriacou, also working blind, told Hall that the flies’ love songs had rhythm. So the transformants’ period gene was working properly in every respect: Their sense of time was solid, day by day and minute by minute.
Meanwhile—at virtually the same minute—Young and his group were testing the behavior of their own set of flies. Young’s transformed flies, or transformants, had a solid sense of time too. The rival laboratories raced their papers into print. Hall and Rosbash got their paper accepted for the issue of Cell of December 1984. (For their help, Konopka and Kyriacou were listed on the paper as two of the coauthors.) At the same time, Young and his group got their paper accepted for the year-end issue of Nature—the issue that straddled December 1984 and January 1985. Afterward, Kyriacou liked to twit Young: “Well, actually, Michael, yours was ’85, really.”
Among those who knew what they meant, these papers caused a stir, like Konopka’s announcement of the clock gene a dozen years before. In principle, what could be done with a fly could be done with a mouse or a human being. The technology of the P elements, the needles, and the markers would be much the same. Hall and Rosbash themselves have since performed an experiment that tested one of their field’s futuristic possibilities, an idea that had been discussed for years both inside and outside their field with both interest and dread: Could genetic engineers someday learn to take a piece of behavior from one species and give it to another? Could they take behavior from one breed of cow and give it to another, or take some part of the temperament of one thoroughbred and give it to another, or eventually take a piece of behavior from one human being and inject it into the egg of another? Or inject a human instinct, a piece of human behavior, into a mouse or a chimpanzee?
Hall and Rosbash cloned the period gene of a D. simulans. Then they mixed one of their DNA transformation cocktails and injected it into the egg of a melanogaster. The piece of behavior jumped from one species to another: mel sang the song of sim.
The experiment left Hall elated: “You change the clock, and you make a different organism! We’ve done minievolution! We’ve turned one species into another!” They had transformed one of an animal’s quintessential pieces of behavior. The clock gene allows it to stay in sync with sunrise and sunset—to keep time with its world, which is a piece of behavior that is essential for the animal’s survival. The clock gene also allows the male fly to keep time when he courts and sings, which is essential if he is to pass on his genes. The injection had changed all that: “Not the same species anymore!”
IN THE HIPPOCRATIC SCHEME of the four humors, Hall would classify himself as choleric and melancholic. He keeps a daguerreotype of General William Tecumseh “War Is Hell” Sherman in his sanctum. (In the daguerreotype Sherman is the twin of Jeff Hall.) He also keeps a portrait of John Brown propped on top of his computer and a blue Union cap on the desk between the computer and his microscope. One of his collection of antique rifles hangs on the wall. The legend he has written across the wall in large block letters says, BE AFRAID, BE VERY AFRAID. In this context it looks like a quotation from the War Between the States. It is in fact a line from the Hollywood poster for the Jeff Goldblum remake of The Fly.
Hall also keeps three small dogs, terriers, in the cave under his desk. (In Benzer’s Fly Room he kept dachshunds.) All day while he works, the dogs growl or wrestle with a knot of rope. Whenever a new face appears at the Dutch double doors of his sanctum, everyone in Hall’s Fly Room can hear the uproar, and they can hear Hall’s voice rising over it: “Now, Zoot, down! Down, down, down!”
Not long ago a reporter asked Hall a sensitive question. It is a question that Hall hears often now that the clock gene has become famous as a sort of flagship in the study of genes and behavior. Why does Hall consider clock genes a genes-and-behavior story? “Some of my friends are wondering why you call the sense of time a form of behavior,” the reporter said.
Hall began calmly. “Rest versus activity is a quintessentially definable property of an organism’s behavior,” he said. “I think it is fair to say that sleeping and waking as instincts are not very interesting. But this is very much behavior.” A fruit fly sleeps during the night, wakes up, has its breakfast, and cruises all morning, he explained. Then it takes a siesta in the middle of the day. Then it cruises until sundown and sleeps through the night. A fly follows this routine every day of its life even if it ecloses in a dark test tube all by itself and never sees a ray of light or another living being. Even if the fly’s ancestors have been born and died in darkness for generations, like the citizens of Plato’s cave, the fly still moves through its life in the dark at the same pace as the sun it never sees. Like fruit flies outside the laboratory or fruit flies in Casablanca, in Cairo, or in the Greek islands, it always takes that siesta in the middle of the day. Since it lives in a test tube in a pitch-black room, it does not need a midday break; but a fly in an open-air fruit market in Morocco needs a siesta to escape the heat, just like the human buyers and sellers in the market.
So waking and sleeping organize all of the animal’s behavior, Hall said, and rising up and lying down are bona fide behavior patterns in themselves. “Not just cause I say so. It really is behavior. I’d argue with your friends for forty-five minutes, and at the end of it, I would demolish them.”
Some people seem to think that behavior is behavior only when it is a mystery, Hall continued. But once any piece of behavior is understood at the molecular level, it all comes down to metabolism, whether we are talking about the way a weaver ant folds a leaf, a weaverbird weaves a hanging nest, a human being learns and speaks Swahili, or a fly rises with the dawn and settles down at dusk. “Benzer was once subjected, in my earshot,” Hall said, “to some dumb question like ‘Is that the mind or the brain?’ But every aspect of mind and brain is ultimately metabolism! What do we think? Some kind of electric aura hovers around our heads?” We still seem to want something outside the mechanism, Hall said, some deus ex machina to save us from the clockwork that we have been exploring above and inside our heads for the last several centuries. It is now time for us to accept that behavior is as much a part of the material world as the stars above us and the atoms inside us. All behavior turns on molecular clockwork, Hall said, yet all behavior is fascinating.
Everyone who studies genes and behavior at the level of genes, molecules, or nerves gets asked questions like this: “Is that all there is?” Anatomists hear the same question when first-year medical students get their first look at the organs inside a cadaver. Behavior geneticists hear this all the time now, and the clock gene in particular attracts the question, Hall said, because a clock is a mechanism that we all understand to be a mechanism. It is the very symbol of mechanism in the heavens and in the body. So the deep assumption of the question, Hall said, is this: “Once you know something about it, it’s not behavior. It remains behavior as long as it sits at the level of mystery and miracle.” In Hall’s opinion, that is why people ask him if keeping track of time is really a piece of behavior when it plainly is, and so is the act of seeing the light from a lightbulb, “some of the most fascinating behavior in the whole history of biological investigation!”
The reporter looked as if he were beginning to be glad he had said the question was his friends’. “Vision!” Hall shouted, waking the dogs under his desk. “It’s not just that you can see! It’s how you respond to the visual world of shapes and movements! And this is not to castigate your friend and say, ‘You’re an idiot!’ This is not to say, ‘Your ignorance is so colossal that you’re doing nothing but sit there and dribble saliva into your lap!’ ” And Hall went through the roof. (“Jeff Hall easily goes through the roof,” says Benzer—which is no more than Hall often says of himself.)
Other clock watchers answer the question in a lower key. Jerry Feldman, who discovered a clock gene in the fungus Neurospora, says calmly, “Call it what you want. You could say any gene that modifies behavior is a behavioral gene. What’s important is the gene. What does it do? Ultimately, how does it couple with behavior? That’s what interests me.”
Kyriacou says cheerfully, “It’s a real behavior gene. What it does, it adds behavior when it’s there. It has rhythms. It’s beautiful.”
TODAY MANY of Benzer’s students credit Hall with the success of the atomic theory of behavior after Benzer’s beginning. “It’s because of what Hall pushed first that the field came of age,” says Tim Tully, one of Benzer’s students’ students.
In the late 1970s and for much of the 1980s, Hall felt embattled and alone, as if he were carrying a fallen banner. Benzer, with his fame, could have been a general not only for Konopka but for the whole field he had started. Benzer could have championed it like another Morgan. But unlike Morgan, Benzer had never had much time for committees or speeches, for writing books or playing scientific politics, and he was not working on behavior anymore.
In those years, Benzer was going more and more deeply into the nervous system of the fly. He had decided that before he went any further with behavior he should study the way genes build bodies. He and his latest students were using genes and mutants to study the way nerves work: doing basic neurogenetics. Tracing the way genes build the nerves in the embryo seemed to Benzer the next logical order of business.
Hall was outraged, but Benzer saw nothing wrong with jumping fields. He had jumped before. At Caltech during a night of skits after Delbrück had won his Nobel Prize, Delbrück’s circle sang a song about Benzer to the tune of “Jimmy Crack Corn.” In the first verses he was a physicist playing with charged particles:
Physics was fun, but I don’t care,
I’m on to something else next year,
I must stick with the new frontier
Until I’m old and gray.
In the next verses he quit physics and mapped the gene:
Genetics was fun, but I don’t care,
I’m on to something else next year,
I must stick with the new frontier
Until I’m old and gray.
In the last verses he quit genetics for behavior:
Behavior was fun, but I don’t care,
I’m on to something else next year,
I must stick with the new frontier
Until I’m old and gray.
It was not only Benzer who was restless; by the late 1970s, many of the founders of the atomic theory of behavior had turned away from the theory. Sydney Brenner was phasing down his studies of worm behavior and studying the wiring diagrams of worm nerves instead. In fact, he had turned in this new direction before Benzer had, and his arguments had helped persuade Benzer that the embryo was the next frontier. Now at meetings, Brenner ragged Hall. “All of you neurogeneticists think you are finding interesting new things from your neural mutants,” he told Hall, “whereas in the end they are all just going to be defective in things like aldolase,” a garden-variety enzyme without which a fly cannot digest glucose and gradually starves to death. This was the same argument that Benzer had heard in the Sperry lab and Hall had heard from Michael Rosbash in the Brandeis locker room: period, fruitless, dunce, and the rest were just sick flies; there was something wrong with their housekeeping genes.
Another phage veteran who abandoned the atomic theory of behavior in those years was Gunther Stent, who had been working on the leech. Now Stent began writing pessimistic essays about the field, just as ten years before, in the brief doldrums of the 1960s, he had written elegiacal essays about molecular biology. Stent had now reconsidered Butler’s saying “A hen is only an egg’s way of making another egg.” If life is a circle, why focus on the genes? Stent was beginning to look at life more in the philosophical spirit of Emerson: “The method of nature: who could ever analyze it? That rushing stream will not stop to be observed. We can never surprise nature in a corner; never find the end of a thread; never tell where to set the first stone. The bird hastens to lay her egg: the egg hastens to be a bird.” Stent doubted that the genetic dissection of behavior or even the genetic dissection of development was going to turn out to be much more informative than dissection with a scalpel.
Meanwhile, Delbrück felt completely defeated. Twenty-five years after dictating his ebullient letter to Benzer about “starting a new life,” Delbrück still could not understand how a stalk of fungus grows toward light. He confided to his diary that he was “sick at heart at the unsolved state of the problem.” He had failed to understand the genetics and mechanics of even one simple-seeming piece of behavior. “I think Max indeed was disillusioned,” Crick says now. “The phenomenon he tried to study was very intricate, and I don’t think he ever got to the bottom of it. And it was, I think, not a wise choice in the first place, and a bit of bad luck as well.”
In 1978, Delbrück’s doctors, taking routine X rays, discovered that cancer was chewing away his ribs. Benzer and dozens of other phage veterans took to visiting him in his living room in the afternoons while he sat in his rocking chair quoting Samuel Beckett (“The light gleams an instant …”). Although Beckett and Delbrück had both won the Nobel Prize in the same year, Delbrück had not met his hero in Stockholm (“No, he didn’t come, the dog.”).
At the same time, Benzer’s wife, Dotty, was in the hospital with breast cancer. The two of them had been unusually close ever since Seymour was sixteen and she was twenty-one. She was a lark and he was an owl, but he always strolled into the laboratory in the afternoons holding hands with her. That was unusual behavior at Caltech, though it was a Fly Room tradition. Thomas and Lilian Morgan had worked side by side among the milk bottles as soon as their children were out of the house. Morgan’s Raiders had named one of their female mutants bobbed. She had short bristles, and they named her after Morgan’s technician, Phoebe Reed, who had just bobbed her hair. Sturtevant married Phoebe Reed.
Benzer had begun transforming himself into an expert on cancer biology and treatment from the moment that he and Dotty knew she was sick. Now he neglected the laboratory for long periods to attend national and international conferences on breast cancer. Even when he was at the lab, he spent hours on international phone calls to specialists in Switzerland; and even when he was satisfied that he was taking Dotty to see the best possible doctor, he would still arrive in the doctor’s office with stacks of technical papers to explain and discuss. Martin Arrowsmith had lost Leora to a tragic illness; Benzer was determined not to lose Dotty.
That June, Delbrück gave the commencement address at Caltech. He spoke in the school’s “Court of Man,” flanked by buildings dedicated to behavioral biology on one side and to the humanities on the other. In a sense, the atomic theory of behavior is an attempt to unite those disciplines, behavioral biology and the humanities. In the long view, it aims to unite all of the sciences, all of philosophy, and all the arts. But Delbrück, speaking to Caltech’s graduating seniors from a wheelchair, predicted that the union would never take place. “Indeed,” he said, “we can take it for granted that science is intrinsically incapable of coping with the recurrent questions of death, love, moral decision, greed, anger, aggression.” And he told a weary fable. Science is like Tithonus of the Homeric myth, he said. When Tithonus was young, the goddess of the dawn, Aurora, fell in love with him. She asked Zeus to make him immortal. Zeus did so—but he did not grant him eternal youth. “Tithonus aged and shriveled and talked incessantly,” Delbrück said. At last he turned into a grasshopper, and Aurora had to keep him in a little box.
“Science does chatter and chirp incessantly,” Delbrück concluded, “sweet music to those few who are tuned in to it, but does it satisfy Auroras yearnings, Aurora the morning dawn?” Does it answer the questions we all want answered?
Benzer’s current crew of postdocs sometimes talks about the history of the laboratory during their brief late-morning cigarette breaks, before Benzer comes in. They sit on stone benches under palm trees and jacarandas outside Church Hall, and they pass on the legends they have heard. For them all of this happened in hearsay time. They say that when Max and Dotty died, Benzer’s friends doubted that he would ever recover. They say that for a while he published nothing, as if what he saw through a microscope was intrinsically incapable of touching the recurrent questions.