Naturally I chose the rII genes for the experiment, concentrating on the second one, the so-called B cistron. (Cistron was Benzer’s fancy name for a gene, as defined by the so-called cis-trans test.) I selected a mutant from our stock, tried to find a revertant—one more like the wild type—and then looked to see if this reversion was due to a second mutation somewhere else in the same gene. If I couldn’t find it I went on and tried again with another mutant.
At first I couldn’t find any suppressors. Presumably the change that reverted the mutant to wild type was at, or very close, to the original change, too close for me to pick it up. Leslie Orgel came over to coffee one day. As he looked over my shoulder I told him what I was doing and that so far I had no results. He went off to join the others while I quickly scored the remaining plates. To my delight I found I had a candidate suppressor.
Before long I had three mutants with suppressors, fortunately spaced out along the map. I isolated the suppressors and proceeded to map them. My theory was immediately refuted. Instead of each suppressor mapping at the predicted place, some distance away on the map, each had its suppressor quite near it. The suppressor effect must be due to some other reason.
Unbeknown to me, other people had also noted that a mutant in rII could have a suppressor in the same gene. Perhaps the most striking example occurred at Cal Tech. Dick Feynman, the theoretical physicist, had become sufficiently interested in these genetic problems that he had decided to do some experiments himself. He stumbled upon an example of an internal suppressor. Not knowing what it might imply, he asked his mentor, Max Delbrück. Max suggested that the original mutant had produced a changed amino acid and that the second mutant had changed another amino acid, elsewhere in the protein, which somehow compensated for the first change. It was easy to see that this might happen, but one would not expect it to be too common.
I was certainly aware of this possibility but I was not happy with it, partly because I had a very detailed knowledge of what little was then known about protein structure. I decided to try to see how many different suppressors a particular mutant might have. I had to select one of my three to study further and, sensibly, I chose the one whose suppressor was the least close to the parent mutant, hoping this would give me more elbow room. I also noted that two of my three mutants had been produced by proflavin. Even though this was hardly statistically significant, by any test, it seemed suggestive to me.
By now I was a little more experienced, so the experiments went fairly quickly. Phage genetics has the advantage that experiments are rather fast, once everything is set up. It does not take long to carry out a hundred crosses, since the manipulations are easy and an actual cross takes only about twenty minutes, this being the time for the phage to infect the bacterium, to multiply inside it (exchanging genetic material in the process), and to burst open, thus killing the cell. The results of the cross must then be plated out on petri dishes, to which a thin film of bacteria has been added. Then the dishes have to be incubated, to produce a lawn of bacteria. Where a single phage has landed and infected a cell, a colony of phage will grow, killing the local bacteria as it does so, forming a clear little hole (called a plaque) in the lawn of growing bacteria on the surface of the plate. This process takes a few hours, so one has a brief respite while it is going on. Then the petri dishes have to be taken from the 37 ° C incubator and examined to see whether they have plaques or not and, if so, of what type. Interesting plaques are then “picked"—that is, a few phage are picked up with a little piece of paper or a toothpick; grown further; and the process repeated a second time to make sure the phage stock is a pure one. If one works reasonably hard it is possible to complete one extra set of crosses in one day, and prepare for a new set the next day.
As the experiments got more interesting I found that, with careful planning, I could get through two successive sets of crosses in one day. This involved starting promptly in the morning, going home for lunch, more experiments in the afternoon, home for dinner, and a final set after dinner. Fortunately Odile and I then lived within a few minutes of the laboratory, an easy walk through the center of historic Cambridge, so I did not find the work unpleasant. In fact, Odile has told me she had never seen me so cheerful as during the period when I did experiments all the time, but this may have been partly because, for weeks on end, all the experiments seemed to work perfectly.
I soon found that my initial mutant had not one, but several, distinct suppressors, all of which mapped fairly close to the original mutant. I decided that I would have to call them all by a distinctive name. I often worked through the weekend, taking Monday off so that our laboratory kitchen (which did all the washing up and also prepared petri dishes for our use) could catch up. It happened that it was a weekend when I needed a new name, and nobody else was around. Mutants were usually called by a letter, followed by a number. Thus P31 meant the thirty-first mutant in the P series, probably produced by proflavin. Unfortunately I could not remember for certain which letters had already been used, so I decided to rename my mutant FCO, since I was quite sure that no one had used my initials to name mutants. The new suppressives were then named FC1, FC2, and so on. This use of my own initials suggested to some people that I must be conceited, but the real explanation was that I have a rather fallible memory.
The new suppressors all seemed like good, nonleaky mutants. So why not, I argued, see if they too had suppressors? And indeed they had. I even went a step further and found suppressors of suppressors of suppressors.
So what was going on? Fortunately we had the right ideas already at hand. Assume that the genetic message was read (to produce a protein) in steps of three bases at a time, starting from one particular point in the message. To make it clearer, let us take an extremely simple message that merely repeats the triplet TAG over and over again:
. . . TAG, TAG, TAG, TAG, TAG, TAG, . . .
the dots indicating that there is message both before and after such a sequence. Commas have been added to show in which “phase” the sequence has to be read. I assumed that this phase was determined by a special “start” signal, somewhere to the left of the stretch shown.
Assume that our original mutant (now called FCO) had added a base to the base sequence. Then, from that point on, the reading would be out of step (out of phase) and thus would produce a nonsense protein, a protein whose amino acid sequence, following the mutant, was completely incorrect, so that the gene product could not function.
Our simple sequence might have become
(The added base has been shown, for clarity, as a C, but it could have been any of the four bases.)
Then, on this interpretation, a suppressor, such as FC1, was the deletion of one base at a point nearby. In between FCO and FC1 the message would still be incorrect, being read in the wrong phase, but elsewhere the reading would be normal.
Our example might thus become:
If the altered bit of the amino acid sequence was not crucial (and in this case there was other evidence to suggest this), then the protein would still function fairly well and the double mutant (FCO + FC1) would behave more like a wild type than like a nonleaky mutant.
I therefore labeled all the first set of suppressors—. The next set, the suppressors of the first set of suppressors, we labeled +, and their suppressors we labeled—.
I had started these experiments early in May and by now summer was advancing. I had previously arranged to take my family on a summer holiday, almost the first proper holiday we had ever had, since by now my financial position was a little easier. We had rented, for a very small sum, a large villa on the old mountain in Tangier, a town in North Africa, just opposite Gibraltar. Here we lived in splendor, with one Arab servant living in and another coming each day. Odile and our German au pair girl, Eleanora, learned how to shop for food in the Arab market, bargaining, walking away, and so on. Our two daughters improved their swimming on the beach while I usually spent the day on the terrace, in the dappled shade of the palm trees.
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br /> On the way to Tangier I attended a scientific meeting. Even in those days scientists were reluctant to go to a meeting unless it was in some interesting place. This meeting was at Col de Voz halfway up Mont Blanc. I reported my preliminary results, which eventually were published as a very brief communication related to the meeting.
After a month in Tangier I went off to the 1961 Biochemical Congress at Moscow, leaving my family to stay at the villa for another week or so. Moscow then was very different from my first visit in 1945, during the war. Now it was summer, rather than the depth of winter, and everything was brighter and more prosperous than in the drab days of wartime. I stayed in a student’s room in the university, where the meeting was held, and got to know some of our Russian hosts. A dominant figure was Igor Tamm, the Russian physicist. The influence of Lysenko, the man who had, for a period, killed genetics in the USSR, was very much on the wane. I sensed that his eclipse was largely the work of physicists like Tamm who had considerable political influence and who could recognize scientific nonsense when they saw it. A number of us were invited to give talks to the biological section of the Russian Atomic Energy Research establishment, something that could not have happened a few years before. We gave our talks in English, but they were brilliantly translated (in chunks, as we went along) by Bressler, a Russian scientist we had already met when he had visited Cambridge. Bressler not only understood what we were saying but in some cases, as I could tell by listening to him, filled out the “references” the speakers were giving, a truly remarkable performance.
The Moscow meeting was made especially interesting because of the results reported by Marshall Nirenberg, then almost unknown. I had heard rumors of these experiments but no details. Matt Meselson, whom I ran into in a corridor, alerted me to Marshall’s talk in a remote seminar room. I was so impressed that I asked Marshall to take part in a much larger meeting, of which I was the chairman. What he had discovered was that he could add an artificial message to a test-tube system that synthesized proteins and get it to direct some synthesis. In detail, he had added poly U—the RNA message consisting entirely of a sequence of uracils—to the system and it had synthesized polyphenylalanine. This suggested that UUU (assuming a triplet code) was a codon for phenylalanine (one of the “magic twenty” amino acids), as indeed it is. I later claimed that the audience was “startled” (I think I originally wrote “electrified”) to receive this news. Seymour Benzer countered this with a photograph showing everyone looking extremely bored! Nevertheless it was an epoch-making discovery, after which there was no looking back.
There was also a measure of social life during the week in Moscow. I enjoyed visiting an old-style apartment, with heavy furniture and a bed behind a large bookcase. Also a more modern one, with a much lighter tone. The owner collected modern Russian art. I was amused to notice Alex Rich demonstrating a strange new American dance to our host, a dance I later recognized as the twist. As Alex’s waist is not very pronounced, the twist, as demonstrated by him, was somewhat less than free-flowing.
I returned to Cambridge. The next step was to do further experiments to validate the ideas that there was some sense in labeling each of our new rII mutants as either + or −. The theory predicted that any combination of the type (+ +)or(− −) would be a mutant. My colleagues and I constructed quite a number of such pairs and they were all nonleaky mutants, as predicted. The simple theory also predicted that any combination of the type ( + −) would be wild type, or approximately so. Of course we knew this to be true in some cases, since that was how we had picked up the suppressor in the first place, but many other combinations (of a + with −) had never been tested. These we called “Uncles and Aunts,” since creating them often involved putting together a mutant of one generation with a mutant from a previous generation, but one other than the one it was descended from. I had asked Sydney to see that some of these were tried while I was away but he had other ideas, so I had to do it myself when I returned.
At this point a small complication arose. Some of the (+ −) combinations, predicted to be wild type, turned out to be mutant. We explained these away by assuming that in some cases the small local phase shift between the + and the − produced a “nonsense” mutant. We know now that these nonsense sites were due to a triplet that terminated the polypeptide chain, thus producing a nonfunctional protein fragment. I also realized that this depended on the precise phase of the reading. For a nonoverlapping triplet code there is one correct phase but two incorrect ones, so that a combination (+ −), that is, + followed by −, will be locally different from a (− +) combination.
To return to our simple example, a (+ −) combination might be:
and a (− +) combination
The first has GTA between the two alterations; the second has AGT. We showed that our (+ + −) or (+ − + ) failures obeyed this rule, which made us fairly confident our ideas were along the right lines.
Previous to this Sydney had an idea. He reasoned that a (+ +) mutant might backmutate to a wild type. He tried one, but the back mutation must have been too close to an existing one, since he could not separate it. Another, slightly more laborious approach was to construct a triple mutant, of the form (+ + +) or (− − −). According to our ideas, these should be wild type, since the three successive changes in phase should have restored the correct phase, always assuming, of course, that it was a triplet code.
For our simple sequence, an example might be
A direct but laborious way of constructing such a triple mutant is to choose three mutants, not too far apart and all +, then to construct two pairs, each of which has the middle mutant in common. (See figure 12.2.) This is the laborious part, since there is no way to select for such a combination of mutants. One has to do the cross and laboriously test the offspring having a mutant phenotype, by taking each one apart, till one finds one which is indeed the (+ +) one is looking for. The final step is easy. One simply crosses two doubles together. Since each contains the middle mutant of the three, there is no way that the cross can produce true wild type. If apparently wild-type plaques do arise from the cross, they are highly likely to be the sought-after ( + + +). In any case it is then very easy to check that this is so by taking the presumed triplet apart.
FIGURE 12.2
Each line represents one of the two parental strands. Each X represents a mutation. It is impossible to recombine the two parental strands to give a strand having no mutations at all. The middle mutation will always be there. Moreover, some of the progeny may have all three deletions on the same strand.
Of course, the triplet would look like a wild type only if the code was a triplet code. If the bases were read four or five at a time, which for all we knew was not impossible, the (+ + +) would be a mutant, and we would have to construct a (+ + + +) or even a (+ + + + +)• Not everybody in the lab believed the experiment would work. I was almost certain it would. So was Sydney, who was away at the time in Paris. He had listed three possible (+ + +) combinations to try, but after he had left I fortunately realized that two of them would probably not work because they would produce a chain terminator, so we constructed the third one that was likely to be free from this complication.
By this time I had co-opted Leslie Barnett to help me. The final crosses were duly carried out and the pile of petri dishes put in the incubator. We came back after dinner to inspect them. One glance at the crucial plate was sufficient. There were plaques in it! The triple mutant was showing the wild-type behavior (phenotype). Carefully we double-checked the numbers on the petri dishes to make sure we had looked at the correct plate. Everything was in order. I looked across at Leslie. “Do you realize,” I said, “that you and I are the only people in the world who know it’s .a triplet code?”
The result, after all, was remarkable. Here we had three distinct mutants, any one of which knocked out the function of the gene. From them we could construct the three possible double mutants. Each one of these also made the gene nonfunctional. Yet if we put all
three together in the same gene (and we did separate experiments to show that they had to be in the same virus, not some in one and the rest in another separate virus), then the gene started to function again. This was easy to understand if the mutants were indeed additions or deletions and if the code was indeed a triplet one. In short, we had provided the first convincing evidence that the code was a triplet code.
I exaggerate slightly. The evidence would also fit a code with six bases in each codon, but this possibility, as subsidiary experiments showed, was very unlikely and hardly to be taken seriously.
There still remained a lot of work to fill out our results. We constructed not one but six distinct triples—five of the (+ + +) type and a single (− − −) one—and showed they all behaved like the wild type. I was even busier than before, though by now Leslie was giving me a lot of help. Not that there were not distractions. One evening, after dinner, I was working away in the lab when a glamorous friend of mine turned up and stood behind me while I continued to manipulate the tubes and plates. “Come to a party,” she said, running her fingers through my hair. “I’m far too busy,” I said, “but where is it?” “Well,” she said, “we thought we’d hold it in your house.” Eventually a compromise was reached. She and Odile would organize a small party and I would join them when I’d finished.
Looking back, it seems remarkable how little we worked—I was away for about six weeks in the summer, on my trips to Mont Blanc, Tangier, and Moscow—and yet how hard we worked and how fast. I had started the key experiment early in May. Yet the paper was published in Nature in the last issue of the year.
We didn’t stop there. Sydney in particular did many further ingenious experiments with the system. Eventually we decided we had better publish a really full account of it, so Leslie Barnett and I worked hard at tidying up all the loose ends. This had one remarkable result. It was known by then that the two triplets UAA and UAG were chain terminators. I was convinced that UGA was a third one. Sydney had devised a complicated way of testing this genetically, but the experiments always told us that it wasn’t. When we came to write our results up, we noticed that not all the possible experiments of this type had been done. Rather than have a gap in one of our tables, we asked Leslie, as a matter of routine, to do the ones that had been overlooked. To our surprise, the experiment now worked! We then repeated all the earlier ones, and this time they worked too! It transpired that when they were first done, we had included a set of controls to make sure everything was as it should be. Unfortunately, in each experiment one control or another had been skipped. When all the controls worked correctly, the experiment suggested strongly that UGA was a chain terminator.
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