annual reviews …web.nchu.edu.tw/~chinho/cb/chapters from my life.pdfrian minister, as well as a...

www.annualreviews.org/aronlineAnnual Reviews

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http://www.annualreviews.org/aronline

Annu. Rev. Plant Physiol. Plant Mol. Biol. 1994. 45:1 23Copyright © 1994 by Annual Reviews Inc. All rights reserved

CHAtrI?ERS FROM MY LIFE

James Bonner

Division of Biology, California Institute of Technology, Pasadena, California 91125

CONTENTSIN THE BEGINNING ............................................................................................................... 1UNDERGRADUATE SCHOOL ............................................................................................... 3

We Have Physical Chemistry .............................................................................................. 4Theodosius Dobzhansky ...................................................................................................... 5

GRADUATE SCHOOL ............................................................................................................. 7POSTDOCTORAL WORK ....................................................................................................... 11PROFESSIONAL WORK ......................................................................................................... 13

Photoperiodism Work at the University of Chicago ........................................................... 14The War Over Rubber .......................................................................................................... 16Fresh Beginnings--Cell Biology ......................................................................................... 18

A NEW PARADIGM ................................................................................................................ 19FROM KATMANDU TO TIMBUKTU TO KOTA KINABALU ........................................... 22

IN THE BEGINNING

I was born in Ansley, Nebraska on September 1, 1910. When I was six weeksold my father, mother, and 1 returned by train to Kingston, Ontario, where myfather was an Assistant Professor of Chemistry at Queen’s University. Myfather had obtained his PhD in physical chemistry from the University ofToronto in 1910. My paternal grandfather was a very conservative Presbyte-rian minister, as well as a Nebraska homesteader, and my father had run awayfrom home to go to Nebraska Wesleyan University in Lincoln. There he metmy mother, who had come to Nebraska in a covered wagon when she was sixyears old and who had grown up on a homesteaded farm near Ansley. Aftergraduating from high school, she had become a school teacher to earn moneyfor college. She attended Nebraska Wesleyan for two years, taught schoolagain for a year, and then returned to complete her bachelors degree. AtNebraska Wesleyan my parents met a new member of the faculty, Dr. Freder-ick Alway, who had just returned from Germany, where he had received his

0066-4294/94/0601-0001505.00 1


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2 BONNER

PhD in chemistry. He was a good teacher and he persuaded many of theundergraduates to major in chemistry. He later went on to the University ofMinnesota to perform similar marvels. Dr. Alway persuaded my father andothers, like R. A. Gortner, the biochemist, to apply for graduate school fellow-ships. My father received a fellowship to go to Princeton and become anorganic chemist. While at Princeton, he discovered that he really wanted to bea physical chemist. It was easier to leave Princeton, go to another university,and find a physical chemist who would accept a new graduate student, than itwas to simply change fields at Princeton. So my father transferred to theUniversity of Toronto and became W. Lash Miller’s student. Both of myparents were the first in their respective families to go to and graduate fromuniversity. That they were both students of Dr. Alway and that they both had amissionary zeal for producing more chemists explains at least in part theevents I will subsequently reveal.

My only sister, Priscilla, was born in the fall of 1914. World War I alsostarted that year. We left Kingston in 1915--my father had been invited tobecome head of the Chemistry Department of the University of Utah. After wearrived in Utah, my parents bought a house on the edge of the city, overlook-ing the Wasatch Mountains. Behind the house was a beautiful prairie, boundedby a gigantic gully. A cowboy brought his cows to graze on our prairie everyday and at night the coyotes sang to us.

My mother, who deemed her skill as a teacher to be greater than that of anyelementary school teacher in the Salt Lake City school system, taught me athome until I was eight years old. I learned to read, but not to write. I didn’tlearn any mathematics and I didn’t learn to read music. That all came later.When I was eight years old, my mother placed me in the fourth grade despiteconsiderable resistance from the school. There were disadvantages to startingschool two years younger than others in the class: I was a social misfit. I alsodidn’t know how to play baseball, I couldn’t read music, and my handwriting(which I learned in fourth grade) was then, and still is, terrible.

Of all the things I learned in elementary school, I remember geography themost. In ninth grade, I learned about the geography of Africa, Australia, andAsia, and decided I wanted to visit all those places I’d never heard of before.That same year my parents gave me a copy of My Life as an Explorer by SvenHedin, who was a professional explorer. He knew when he was a little boy thathe wanted to be an explorer, so he trained himself to be used to hardships andto be able to find his way without road signs or asking other people. Hedinexplored a lot of Central Asia and after reading of his adventures, I decidedthat I wanted to become an explorer too. My parents coldwatered this idea,however, by saying that most of the world had already been explored and itwas very hard to find places to try to find out something new about. Of course,I later found that my parents were wrong in this regard and that there were a lot


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PREFATORY CHAPTER 3

of places that still needed to be explored, some of them right there in Utah andlots of them still in Central Asia~

I received another kind of education while living in Utah. My parents hadboth grown up on farms and left their farms to attend college. They both feltstrongly that (a) capable people, such as their children must be, should go college and (b) farms were the best place for children to be brought up becausethere was physical work to do. My father wanted us to understarid "the dignityof labor," so when I was eight years old we moved from the home with theprairie, the cowboys, and the coyotes to a three-acre farmlet, which we wouldnow call a family orchard. My responsibility, as the eldest, was to change thewater from ditch to ditch to irrigate the entire farm over a period of severaldays. My other responsibilities included pruning, spraying for insects, sup-pressing fires, and training my younger siblings to take over the irrigation incase I was shunted off into some other activity. The chores changed with theseasons. They never ended.

UNDERGRADUATE SCHOOL

It was taken for granted in our family that everyone wanted to become achemist, and it turned out to be true. All seven of my parent’s childrenreceived bachelors degrees in chemistry from the University of Utah. Four ofthem became biological chemists, two became physical Chemists, and onebecame an applied mathematician, having given up on chemistry, but onlyafter receiving his bachelors degree in chemistry. As the oldest child, it wasmy duty to start the procession off to the university to get an AB degree inchemistry. I graduated from high school in the summer of 1927 and entered theUniversity of Utah in the fall of that year. I spent two years at the University ofUtah as a chemistry major with a minor in mathematics and played flute in theuniversity orchestra, sitting next to my brother Lyman.who played the firstoboe, all under the auspices of the concert master Simon Ramo who laterbecame the "R" of TRW. The calculus and differential equations were allfascinating and’easy. The chemistry was all fascinating and easy. English wasmore difficult. Each week we had to turn in two essays to Professor Crabtree,who would read them and mark them up before handing them back. After ayear of having my essays scribbled on by an exacting person like him, 1learned a lot about good writing. According to Professor Crabtree, the onlyway you learned about writing was by reading other peoples’ writing and byhaving your own work criticized by people like him.

The year after my sophomore year in College, my father had another sabbat-ical leave (his first had been spent at the University of Calilbrnia at Berkeleyin 1922-1923). This time he was going to the new, but rapidly becoming well.known, California Institute of Technology (Caltech). His former student, Don


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4 BONNER

Yost, had received his PhD from Caltech and was now an Assistant Professorthere. My father planned to work with Don Yost on an interesting physicalchemical problem that both of them considered potentially fruitful for them.My parents rented out their house and farmlet to my uncle Truesdale, who wasnow a chemical engineer employed by a company headquartered in Salt LakeCity.

With the entire family packed up into our 1921 Essex, we drove to Pasa-dena in about four days. My parents found a real estate agent who assuredthem that "Pasadena is the city of churches and schools." They rented a housein northwest Pasadena, where they were indeed surrounded by schools. Mybrother Lyman and I had taken the required Caltech admission exams and hadbeen admitted and awarded tuition scholarships. Registration at Caltech wasnot as difficult as you might imagine. There are a limited number of coursesand the curriculum and requirements dictate completely which ones you musttake. Thus I registered for Physical Chemistry, the great bug bear of theCaltech undergraduate chemist.

We Have Physical Chemistry

Physical chemistry was taught using Noyes & Sherrill’s textbook, ChemicalPrinciples. The book contained a lot of problems, and each day we wereassigned a few to work out on paper and hand in at the next class. Youcouldn’t solve subsequent problems unless you’d solved previous ones andtotally understood the principles that you were dealing with. In this course Ilearned how to study and how to be absolutely sure I wasn’t fooling myselfinto thinking I understood something that I didn’t.

The fall term had its signs and portents for the future. On October 29, 1929,ihe great stock market crash occurred. Rich people became poor, etc. None ofthis affected Caltech, my family, or me right away, but such effects did come.Even so, the first term was perfectly satisfactory. I got As in everything,including Physical Chemistry. It was a lot of hard work, but I felt then and Istill feel today that this first term at Caltech really turned me on. The lectureswere outstanding. E. C. Watson’s weekly lectures in physics were alwayswonderful. We learned everything by solving problems. This was true inPhysical Chemistry, Physics, and even in Quantitative Analysis. We weretaught a whole new way--no rote. Or as my colleague of later years, HermanKalckar, used to say, "No rosary."

For the winter term, everything was the same, except for Biology. Thebiology class was being taught for the second time. We had three lectures aweek and laboratory class twice a week. The lectures were given by thechairman of the Division of Biology, Professor Thomas Hunt Morgan. Profes-sor Morgan was famous as the father of modern genetics. He was also famousbecause he was a good geneticist, a good embryologist, a good biologist, and


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PREFATORY CHAPTER 5

he was good at choosing his colleagues. Alfred H. Sturtevant and Calvin B.Bridges, two of the three Columbia University undergraduates who had, withhim, developed genetics, became faculty members at Caltech. Dr. Morgangave absolutely fascinating lectures concentrating on genetics with a bit ofembryology tossed in. Slightly more than halfway through the term, Dr. Mor-gan turned the class over to Henry Borsook, from the University of Toronto,who lectured on biochemistry and metabolism. Dr. Borsook was interested inthe mechanism of protein synthesis and tried to prove that proteins could besynthesized by proteolytic enzymes working in the reverse direction, but hedidn’t succeed. Biology was fascinating and I loved the class, but it didn’tseem to require any thinking and I remember musing at the time that thiswasn’t right. There ought to be some problems!

Theodosius Dobzhansky

Once again, I received As in all my classes, including Physical Chemistry, andat the beginning of the third term, the only decision I had to make was whetherto take Introduction to Astronomy or a new biology class. I chose the latter.The new class had a laboratory, which was presided over by a brand newAssistant Professor, Theodosius Dobzhansky. He had been discovered, I be-lieve, at the University of Leningrad, by a roving scout of The RockefellerFoundation. He was awarded a Rockefeller Foundation Fellowship to come tothe United States to study with Dr. Morgan at Columbia, and had arrived in thesummer of 1927. Hc was amazed to find that at the end of the 1927-1928school year, the whole laboratory was moving to Pasadena, California. Hisfellowship was for only one year, so it would run out sometime soon after hegot to Pasadena. Dr. Morgan invited him to come along, saying, with typicalMorgan offhandedness, "When your fellowship ends I’ll make you an Assis-tant Professor." It could be done that way in those days.

Theodosius Dobzhansky and his wife, Natasha, arrived in Pasadena in thefall of 1928 and moved into the new Kerckhoff Laboratories of Biology (thewest half of what we now know as the Kerckhoff Laboratories). Theodosiusnot only ran the laboratory, but also invented interesting things for us to do.We went on field trips. We went to the Marine Station at Corona del Mar,where we trapped Drosophila using half-pint milk boules with fermentingyeast solutions soaked on a piece of paper towel. We brought the bottles backto the station and counted how many of the flies were Drosophila rnelanogas-ter and how many were other species of Drosophila. We learned a little bitabout the phyla of animals (but not of plants). We talked about the naturalhistory of animals in the wild. Dobzhansky frequently told stories about histwo trips to Central Asia, where he had been sent with some colleagues tostudy the fauna of the region, particularly the Altai and the Tien Shan, and to


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6 BONNER

see whether there were any of the wild horses, Prezhevalsky’s horse, that wereknown to inhabit these remote areas.

Toward the end of the term, I learned that because of the financial strin-gency of the Institute after the beginning of the Great Depression, they wouldbe unable to fund my scholarship for the coming year. A few days later andjust in time to really cheer me up a lot, Dobzhansky asked me ifI would accepta position as a research assistant to him for the summer of 1930. Dr. Morganhad authorized him to offer me the position for something like $250 for fourmonths, and I accepted. The academic procession during commencement wasthe most impressive I had ever seen, and it included my father, wearing thecolorful regalia of the PhD from the University of Toronto. At the end of theacademic year, my parents and siblings packed up all their belongings, shippedwhat couldn’t be carried, and drove back to Salt Lake City. I remained inPasadena.

On the first day of the summer term, I went to Dobzhansky’s laboratory tofind out what I was to do for work. He was working on Drosophila, of course(that’s what he had come to the United States to learn about), and he wasdetermining the breakage points of translocations. Dobzhansky was trying tofind out exactly where the break was in each case, and that meant finding outwhere it was with respect to mutant marker genes along each’ of the chromo-somes. I had to be able to distinguish between mutant flies and wild type flies,and between the different kinds of mutant, and I had to keep good records, allof which I could do easily.

For the first week or so I was immersed in this project but interesting as itwas, it was not as amazing as the next development. On Thursday, I think, ofthe first week, Theodosius said out of the clear blue sky, "We have workedvery hard this week. I think tomorrow we go to the beach." We spent the nextday at the Corona del Mar beach with Natasha, taking a final shower at the endof the day in fresh water at the Caltech Marine Station. After another week inthe lab Theodosius suddenly said, "We have been working very hard now fortwo weeks. I think it is time we take a vacation for a couple of days." So hepacked up his camping gear, I brought a blanket and a sweater, and we went upinto the San Jacinto Mountains and camped there. Dobzhansky always carriedan insect-collecting net with him wherever he went out-of-doors, so he col-lected insects and told me about how many different kinds of insects therewere and how he was a specialist on a particular kind of beetle, theCoccinnellidae. He said that he was going to catch all of the wild beetles ofthis group to be found in California and then he would write a taxonomicdescription of each and write a key on how to distinguish them from oneanother (which he did). This was a seductive p|ot. Biologists got to take tripsoutdoors. They had an excuse to go and see nature in the wild. That wassomething to ponder.


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PREFATORY CHAPTER 7

Theodosius also gave me a second task. He was a prolific writer. He oftenstressed that "a month without a paper sent to press is a wasted month," and hefulfilled this objective for all of his scientific life. After he passed away and wecounted the number of papers and how many years he’d been working onthem, it was almost exactly one paper a month for his entire 55 years or so ofscientific productivity. But it was hard work even for him, since he was tryingto write one paper a month while he was learning English. He and Natasha hadarrived in New York City not knowing a word of English. They learned by thetotal immersion method. He recruited me to read the things he wrote andchange them into proper English. I was pretty good at this and I’d had a lot ofwriting experience, so Dobzhansky learned to be an excellent writer. He wrotegrammatically proper English, with an enormous vocabulary, but he alwayskept the real honest-to-God Dobzhansky accent, as long as he lived.

At the end of the summer I had to return to the University of Utah tocomplete my bachelors degree, but I planned to return to Caltech afterward. SoI packed up, paid my rent, and asked my landlady, Maria Planas, to save me aroom for next year. I hitchhiked home. This was early in the fall of 1930 and Ipassed through Las Vegas. President Hoover had announced that he was aboutto begin construction of Boulder Dam and people from all over the UnitedStates had congregated in Las Vegas looking for jobs working on the dam. TheDepression had really started to hit. l walked out of town a little way to NorthLas Vegas and was lucky to get a ride that took me all the way to Salt LakeCity.

GRADUATE SCHOOL

In my senior year I had applied for admission to graduate school in biology atCaltech and just to keep things happy in the family, I applied for admission inthe Division of Chemistry at Caltech as well. I was admitted to both depart-ments. I immediately accepted admission in the Division of Biology and wasawarded a teaching fellowship for $750 per year. It was 1931 and the Depres-sion was deepening. The $750 looked pretty good so I accepted. I graduatedfrom the University of Utah with high honors (with a major in chemistry and minor in mathematics).

Immediately after graduation, I did a few assays of ore samples for a manwho was eager to have their precious metals content in a hurry. This gave me alittle money to start me on my way. Toward the end of the summer, I boarded abus, went back to Pasadena, and reported to the Planas’ house, where I got myold room back. I walked to Kerckhoff but there was not a soul in the building.There was no one at work. I then rode my bicycle out to the Caltech farm inArcadia. E. G. Anderson was there, hoeing the weeds out of his corn, andsomeone else, whom I didn’t know, was there with him. He introduced me to


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8 BONNER

the new person, George Beadle, then a postdoc from Cornell. That was the firsttime I saw George Wells Beadle, my friend and confidant for many years.

After talking with George Beadle for a while, I rode back to Caltech andnoticed that there was somebody working in a new building on the comer ofMichigan and San Pasqual. I walked up to the building, knocked on the door,and was let in. The inhabitants introduced themselves as Herman E. Dolk,Assistant Professor of Biology, a plant physiologist; and Kenneth V. Thimann,an Instructor of Biochemistry. They were working on a problem that soundedquite interesting. They told me that Frits Went in Holland had discovered aplant hormone that caused cells to grow longer in plants and that this hormonehad a lot to do with all kinds of plant growth responses. They were studyingthe responses of plants to the hormone, using a fungus, Rhizopus suinus, as asource of the hormone. After the Rhizopus had grown on a culture medium fora few days and the culture medium was extracted with ether, the ether extractcontained active plant growth hormone. They told me that the structure of thehormone was being worked on in Holland where the whole matter had beendiscovered, but the synthetic stuff was not yet available. We talked for quite awhile and the next day I went back and we talked some more. There was stillno one else in the Division of Biology, so finally Professor Dolk said, "Ofcourse I know you’ve come here to become a geneticist, but since there isn’tany geneticist around to consult with right now, why don’t you pitch in andhelp us produce some more of this plant growth hormone? We really needsomebody more to help us in our work and get it going a little faster." So Iagreed and started working on the production of plant growth substance by thefungus Rhizopus.

Dr. Dolk thought I should try to grow Rhizopus on different media and seeif I could find a medium that would cause the Rhizopus to make a lot moregrowth substance. This would be an enormous help in providing enoughgrowth substance for physiological experiments. I started doing what Dolk hadsuggested. One day I discovered something: Instead of feeding the Rhizopusan ordinary synthetic medium like I’d been doing, and on which the Rhizopusgrew very well, I put various sources of nitrogen into the medium, including,for no particular reason, the bactopeptone that one uses to make bacterialmedia for fastidious bacteria that need a complicated source of nitrogen. Onthe bactopeptone medium the Rhizopus not only grew like mad, but it pro-duced over 50 times as much growth substance per unit volume of growthmedium as it did on any of the media that I’d used before. In fact, if it wasgrown on bactopeptone and also aerated so that the fungus had a lot of oxygenavailable to it, it produced 100 or 200 times as much growth substance per unitvolume of medium as I’d ever seen before. This was spectacular! I had found amedium that would produce growth substance until it would run out of Dolk’ sand Thimann’s ears. I wrote a paper on ~ny discovery and after Dr. Thimann


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PREFATORY CHAPTER 9

edited the paper, we sent it off to be published. I was hooked. I’d seen myname in print and decided that I wanted to write a lot of publications just likeTheodosius Dobzhansky did.

In retrospect, I had discovered something really important. My discovery, iffollowed up, would lead to the identification of the chemical in the bac-topeptone that is responsible for helping Rhizopus make growth substance outof it. We now know that the growth substance is indole-3-acetic acid, so mybest bet would have been that it was tryptophan in the bactopeptone that wasoxidized and decarboxylated and made into indole-3-aeetic acid by Rhizopus,especially in the presence of a lot of oxygen. However, nobody suggested thatit would be good to use this as a springboard from which to determine thenature of the plant growth substance. It was hailed as a great achievement inmaking it easier to produce an active substance to use in experiments, butnobody pointed out to me that I had discovered an important clue and I didn’tsee it for myself. I had an opportunity to become the discoverer of the nature ofthe plant growth substance, but I let it slide and went over and worked withDobzhansky for a couple of months on Drosophila genetics. My unfinishedwork on plant growth substance kept gnawing at my conscience, and ulti-mately, I did return to the study of plant growth substance and its chemistry.

By this time Dr. Morgan had assigned me to Room 307 Kerckhoff andsince there weren’t many graduate students, I had the whole room to myself. Iplayed the flute an hour a day, most often in my office. I opened the windows,of course, because it was hot, so my flute music went wafting out over thesouthern part of Pasadena, but nobody complained. I was also invited to playin the Pasadena Civic Symphony and I did do this for three years until I got myPhD and went to Europe. I worked very hard, but didn’t accomplish any majorgoals. I tried to invent ways to find out how the growth substance made plantsgrow. As it turned out, of course, the science of biology was not advancedenough to permit the study of this subject. I did only two things worthy ofremembrance. First, I found that oat coleoptile sections cut out of the oatcoleoptile grow wonderfully when floated on a solution of plant growth sub-stance. If they aren’t given any plant growth substance (the sections justfloated on plain buffer solution or water), they grow just a tiny bit. If they areput on a solution containing a lot of plant growth substance, they elongaterapidly, and this growth can be measured by looking at 5 mm long sectionsunder a microscope with a gradt~ated scale. This is a very quick, easy, andquantitative way to measure amounts of growth substance in a medium. It’snot as inventive as the method invented by Frits Went, but it is simple and hasbeen used a lot by many people. Second, I discovered that if you put oatcoleoptile sections in a solution of, say, pH 4.5 they grow quite rapidly for awhile, and if they’re put in a similar solution, but at, say, pH 7.5, they don’tgrow. By putting them in solutions of different pHs one can plot a sort of


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10 BONNER

titration curve and find the pK of what it is that is being influenced byhydrogen ions that makes oat eoleoptiles grow. This discovery, independent ofeverything else known about growth substances in plants, has started a wholenew chain of thought in the minds of several people in recent years, and it hasengendered a secondary fall-out of papers on the subject.

Professor Dolk was killed in an auto accident in March 1932 and Frits Wentcame to Caltech early in 1933 as an Assistant Professor of Biology. We hadmany interesting seminars at Caltech. As J. B. S. Haldane talked on evolution,his cigar got too short, so he took out his pipe, stuffed the cigar stump in it, andsmoked it all the way. A seminar by Von Wettstein on maternal inheritance ofchloroplasts was bitterly attacked as unlikely, if not impossible.

My $750 per year tumed out to be a lot more than expected. It wasDepression time, remember, and on principle, I would never buy any food thatcost more than ten cents for three pounds, so feeding myself didn’t cost much.My brother Lyman had come to Caltech to become a graduate student inChemistry, starting one year after I did, and we lived together in a two-room-and-bath apartment on the lower floor of the Planas’ house. I bought a usedPasadena Police motorcycle--a Henderson four cylinder. I rode it a lot, ex-ploring every canyon and riding up to Santa Barbara to visit my aunt. I soonfound a friend who had a Henderson exactly like mine and we rode together.One day when I came home from a Sunday trip I had a message from Mrs.Planas that he had called and asked me to visit him at Huntington hospital. Ifound my friend in a very sad condition: he had been hit by a car when hismotorcycle was standing still. He never recovered fully from this accident. Icould see a whole tragic story coming, so I put a for sale sign on the motorcyle.I never rode it again. In a few days a man offered to trade me a 1924 ChevroletSuperior roadster for my Henderson. I took it. The Chevrolet had a four-cylin-der, overhead valve engine and would go forever without boiling. It wasreliable enough to drive to Salt Lake City and back.

Toward the end of 1933 (at the beginning of my third year of graduatestudy), Dr. Morgan suggested that I go to Europe for the next year. I said would like to go to Utrecht where they knew all about plant growth substance,and maybe other places as well. I received a letter from Professor William J.Robbins, the Chairman of the National Research Council Selection Commit-tee, saying that I had been awarded a one-year NRC postdoctoral fellowshipwith a stipend of $1625/year and all travel expenses. I accepted!

I wrote my PhD thesis and my final PhD exam was scheduled. ProfessorSturtevant was the chairman of my thesis committee in the absence of Dr.Morgan, who had acted as the chairman of all previous PhD examinations, butwho was away collecting his Nobel Prize. Dr. Sturtevant, I remember veryclearly, put his feet on the table and said, "James, when you think of a gene,tell us what you envisage the gene looks like and is." Luckily I’ve forgotten


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PREFATORY CHAPTER 11

what I said in reply. We had a commencement, of course, to which my fathercame. I think that one of the joys of his mature years was seeing his childrenget PhDs one by one. Dr. Millikan hung the PhD hood around my neck and itwas all over.

POSTDOCTORAL WORK

We drove back to Salt Lake City in my 1924 Chevrolet roadster, and after afew days I took the Greyhound bus to New York, where I boarded the Penn-land, the steamer that was to take me to Europe. It was a wonderful trip. Youwould hardly know there was a Depression going. The ship was filled withcollege students going to Europe for the summer. Upon arrival at Antwerp, Ifound that the National Research Council had thoughtfully arranged for mytrain ticket to Utrecht. When I got to Utrecht, I rented a room, put my fewbelongings in it, and found, as I had expected, that the universities are empty inthe summer. From June to September, professors and students take their sum-mer vacations----certainly very non-Caltechian behavior but I had expectedthis, so I was prepared. I bought a bicycle and made an outline of a trip to seeEurope by train and bicycle. I spent about two and a half months on thisEuropean tour, having a wonderful time. I took the train to Berlin and found, tomy amazement, that I could understand people and they could understand myGerman. I knew a few people in Berlin and indeed, this was true everywhere Iwent. I’d written ahead to several people, and I went to the famous laboratoriesin Berlin, Jena, Leipzig, Dresden, Prague, Mtinich, Heidelberg, Innsbruck,Ztirich~ Bern, Basel, KOln~ and finally back to Utrecht.

Now it was time to go to work. I had arranged to go to the laboratory ofProfessor Kruyt, the most notable polymer chemist of the time, and I wasunder the impression that I would learn something about colloid chemistry. Atthat time it was fashionable to think that colloid chemistry would give us anunderstanding of how protoplasm was made and what its properties were. Thiswas an inco.rrect view of the matter, as it turned out, and I certainly didn’t learnmuch by working in that lab. I did learn Dutch quite well, however. I also gotto know all of the notable plant physiologists in Utrecht. I worked in thelaboratory that had just been given by Frits Went’ s father to the new ProfessorKonigsberger. A. N. J. Heyn was the chief assistant in the botany department.Professor K6gl was the head of the organic chemistry department. A. J.Haagen-Smit was the first assistant. K6gl, Haagen-Smit, and Hanni Erxlebenjointly had isolated and were determining the structure of the growth substancethat Frits Went had found. They had isolated and were in the midst of deter-mining the structure of auxin a. At the end of 1934, while I was still in Utrecht,they published tlie isolation of indole-3-acetic acid from urine and showed thatit was active as an auxin~ Since its structure was obviously very different from


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12 BONNER

the putative structure of the putative auxin a, the new auxin was calledheteroauxin. Even though this was published in 1934, I heard nothing about itin Holland. There was no seminar, no celebration of this great new finding. Asit turned out, of course, Krgl and Haagen-Smit had found the correct auxin.The substance is really the auxin of plants. Auxin a and auxin b were fantasiesapparently invented by Hanni Erxleben.

I also had an opportunity to work for a couple of months at the Universityof Leiden Medical School in the Department of Biochemistry, which waspresided over by Professor Bungenberg de Jong. Bungenberg de Jong was thegreat expert on coacervates. A coacervate is a colloidal system in which onecolloid is dispersed in a second. I learned little from the two colloid chemistrylaboratories. In the beginning of 1935, I moved to ZiJrich to A. Frey-Wyssling’s laboratory at the Swiss Institute of Technology (ETH). ProfessorFrey-Wyssling was an expert on the use of the polarizing microscope for thestudy of cell wall properties, The manuscript that resulted from this work, andwhich I’m happy to point out was written and published in German, showedquantitatively that under the influence of auxin, the microfibrils of cell wailsbecome much more readily separated into independent fibrils. That is, theinteraction of the fibrils is greatly diminished. This is what makes it easier tostretch the cell wall as a result of auxin action. I think this paper was aconsiderable contribution to science.

During the fall term, Dr. Morgan invited me to come back to Caltech as aresearch fellow, which in time would turn into an instructorship. Of course 1accepted. Where else would I want to go? And anyway, that was the only joboffer I received. ! looked no further.

Later in the fall, I attended an international congress of botany in Amster-dam. It was exciting to see the bigwigs of plant biology--whose names Iknew, but whose faces I did not. I think ! met Dennis Hoagland of theUniversity of California at Berkeley for the first time. I also met and made alife-long friend of Hiroshi Tamiya. Hiroshi was professor of botany at theUniversity of Tokyo and a world’s authority on many aspects of photosynthe-sis. Although at this time, before World War ll, he published papers in Ger-man (and we talked in German) as most Japanese did, he transformed himselfinto an English writer by the end of the war, and it was in his English speakingand writing incarnation that he became well known in America.

At the end of the year, I made a reservation for my return trip on the NorthGerman Lloyd Line. We crossed the Atlantic in less than five days on theBremen, in contrast to the almost two weeks it took on the Pennland. I landedin New York, went to Detroit by train, bought a 1934 Ford roadster, and droveacross the country, visiting my relatives in Nebraska, and finally home to SaltLake.


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PREFATORY CHAPTER13

PROFES SIONAL WORK

By the time I returned to Caltech, and by the time Dr. Morgan had given meback Room 307 Kerckhoff and had confirmed that I was an Instructor inBiology, it was 1936. I was 25 years old and I had to decide what to investi-gate. The study of root growth was one of my initial areas of investigation.Phillip White, of The Rockefeller Institute, had successfully grown tomatoroots in culture through repeated transfers. A 1 cm long tip would grow into abig, long root; he’d cut the tip off of tt~at, put it in fresh medium, and it wouldgrow again. The medium consisted of sucrose, inorganic salts, and yeastextract, and I thought the thing to do was to find out what was in the yeastextract.

I decided to start a root organ culture program. I tried growing isolated pearoots, leaving the tomato roots to White temporarily. I germinated pea seedsaseptically, and after the roots had grown for a week and were several cm long,I cut the apical 1 cm off of each root and transferred the tips to fresh petridishes containing liquid medium. The medium contained an inorganic saltconcoction that I had devised with sucrose as a carbon source. I found that theroots would grow very well for a week, but when transferred to fresh medium,they would give out. So l tried yeast extract, like Phillip White had used, andfound that yeast extract would make pea roots grow quite well. There had to besomething in yeast extract that plants needed, l knew that yeast extract wasgood because it contained the B vitamins, including vitamin B 1, which hadjust recently been made available synthetically by Merck. So I wrote to Dr.Randolph Major, Director of Research at Merck, and asked him if I could get abit of crystalline vitamin B 1, which he kindly sent. I put the crystalline vitaminB 1 in the medium together with sucrose as a carbon source and the inorganicsalts, and I found that pea roots grew wonderfully well with synthetic vitaminB 1 instead of yeast extract as a supplement. They would go through maybe sixor eight transfers and then the rate of growth would decrease slowly. The pearoots needed something else besides vitamin B1, but they certainly neededvitamin B 1. So I wrote a letter to Phillip White telling him the joyous newsthat I had discovered that synthetic vitamin B 1 could replace yeast extract to aconsiderable extent in the growing of roots, but he didn’t answer my letter.Instead, he repeated my experiment and published the results in the Proceed-ings of the National Academy of Sciences, which only took a few weeks fromsubmission to publication in those days. His paper on the subject appeared in1936, whereas mine, which was written earlier, but submitted to Science,.didn’t appear until 1937. My long paper on the subject also didn’t appear until1937, in the American Journal of Botany. So I learned something: Be carefulhow you spread the joyous news.


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14 BONNER

I had a good time with roots. My first graduate student, Fred Addicott,came from Stanford to join in the fun. We grew isolated roots of manydifferent dicotyledonous species. Roots of monocotyledons wouldn’t grow inour medium. Most successfully cultured roots of dicotyledons require thia-mine. Many plant roots require both vitamin B 1 and the B vitamin, niacin. Thetomato requires thiamine and pyridoxine (vitamin B6). Thiamine is synthe-sized in leaves and is transported downward to the roots where it is essentialfor their growth. Even though the B vitamins are so clearly root growthhormones for those species that require them for continued growth in culture,it is interesting that thiamine, for example, has not been generally classified asa plant hormone,

My other initial topic of investigation was the wound hormone. For thestudy of wound hormones, after much consideration and advice from FritsWent, I decided to study the wound hormone of the string bean, whichWehnelt had studied previously. The pod of the string bean may be cut in halflongitudinally along the suture, and the seeds removed. In each little cup fromwhich the seed has been removed there is undamaged epidermis, subtended byundamaged parenchymal cells. If a drop of juice of ground bean pods is placedon the surface of that little cup, it will cause cell division and growth of theuninjured bean tissue under it--the ground pea pod juice contains a woundhormone that can induce undamaged cells to grow. My first postdoctoralfellow, James English, Jr., a chemist fresh from Yale University, joined me inthis study. We found that a good source of wound hormone is the ground upbean pods themselves. The ground up bean pods were extracted with water,the water extract concentrated, and after six fractionation steps, a crystallineactive material was obtained. This crystalline active material was characterizedin a variety of ways, and was shown to possess the structure of 1-decene-l,10-dicarboxylic acid. We called this previously unknown compound traumaticacid. Traumatic acid has biological activity as a wound hormone both on beanpods and on potato tuber slices. Today, it appears that traumatic acid isprobably produced as a non-enzymatic oxidative product of 12-oxo-trans-10-dodecenoic acid, the first compound in the jasmonic acid pathway. Thisrevisionist suggestion was made by Zimmerman & Coudron 40 years after ourgroup made the initial observations. In any case, studies of the B vitamins asplant hormones and the isolation and structure determination of traumatic acidconsumed almost but not quite all of my time up to the beginning of WorldWar II.

Photoperiodism Work at the University of Chicago

After Christmas of the 1937-1938 academic year, I received a letter from E. J.Kraus, inviting me to work on photoperiodism with Karl Hamner in theUniversity of Chicago’s wonderfully outfitted laboratories and greenhouses.


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Several people had suggested that flowering, which is induced in many plantsby the appropriate length of day and night, is mediated by a hormone made inleaves that goes to the buds and induces them to transform from vegetative toflower buds. Kraus asked me to spend a summer and then perhaps considerstaying at Chicago after that. I consulted with Dr. R. A. Millikan, the de factopresident of Caltech, about this proposition. Dr. Millikan had been a Professorof Physics at the University of Chicago and had come to Caltech at theinvitation of Dr. Noyes and of the Trustees to become the chairman of itsexecutive committee. Concerning my invitation to go to Chicago, Dr. Millikansaid, "Yes, go ahead and do it. You won’t like the University of Chicago andyou won’t want to stay there so don’t let them give you any money. We’ll payyou all the time and you come back when you’re all done and want to. But, asI said, you won’t like it. I didn’t. I didn’t stay and I don’t think you willeither." So I prepared my thinking about Chicago, along the lines suggested byDr. Millikan, and I also began to think about photoperiodism. I told mygraduate student, Fred, that I was going to be gone for a little while. After theend of school in 1938, I got in my car (a 1937 Ford sedan), put the campinggear and a suitcase full of clothes in it, and drove to Chicago, where I wasgreeted by Hamner and Kraus. They had arranged a room for me in theQuadrangle Club, the faculty club of the University of Chicago. I was alsointroduced to the botany greenhouses, which were not only extensive, but "alsohad good facilities for working on photoperiodism.

Garner & Allard first recognized in 1920 that the relative lengths of day andnight are important for plants. They worked on Maryland Mammoth tobacco, ashort-day plant (i.e. it flowers only when the days are short and the nights arelong). Further studies showed that the effect of day length on plants is per-ceived by the leaves. The leaves then send a message to the bud to turn into aflower bud. Hamner also had worked on this phenomenon with the cocklebur(Xanthium pennsylvanicum,) another short-day plant. If the day is 15.5 hr orless, the cocklebur will flower. In addition, Hamner’s previous work hadshowed that to induce flowering, it is sufficient to expose a cocklebur plant toone short day. If we then transferred the plants to long days, the cockleburwould flower.

Hamner and I spent the entire summer studying the photoperiodic responseof the cocklebur. We showed that it is the length of the night, and not the day,that determines the flowering response. There is a critical night length that hasto be longer than 8.5 hr to induce the plant to flower. Short-day plants arereally long-night plants.

Let us consider a plant that is left in a long night for 16 hr. It will, of course,produce the floral stimulus and will subsequently flower. Suppose, however,that we interrupt the dark period by a short pulse of light given in the middle ofthe night, thus dividing the 16-hour dark period into 2 slightly shorter than


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16 BONNER

8-hour dark periods. Such a light break, as it is often called, even though it isless than 1 rain long, is sufficient to cause the long, dark night to be ineffectivein causing flowering. The light pulse has effectively replaced continuous lightin making the cocklebur think that it has seen a long day. This middle-of-the-night light-break can be of quite low intensity light, just a few foot candles atthe leaf surface for a minute, let’s say. The discovery during the summer of1938 of the effectiveness of the light break in controlling flowering of cockle-bur was shown subsequently by others to be true of other short-day plants aswell. This finding has had considerable subsequent application. Several yearsafter the publication of the 1938 work, the light-break principle was used bySterling Hendricks and his colleague, Harry Borthwick, at the US Departmentof Agriculture in Beltsville to make an action spectrum (i.e. to determine theeffectiveness of different wavelengths of light in causing the suppression offlowering of cocklebur) and with this knowledge, Hendricks & Borthwickdiscovered phytochrome.

By the end of the summer of 1938, Hamner and I wrote up the work we haddone and sent it in immediately for publication in Botanical Gazette, a Univer-sity of Chicago publication noted for its rapid publication. Our contributionappeared in the December issue. I have no hesitation in describing this paperas a minor classic.

Since this summer-long exposure to the study of photoperiodism, I havemaintained my interest in the subject, l did not get seduced into the study ofphotomorphogenesis and the properties of the pigment, phytochrome. I tried todiscover the nature of the hormone that is produced in leaves and sent to budsto make them turn into flower buds. I tried many approaches. All have beenfruitless. To this day, I do not even have a good idea about the chemical natureof the hormone.

The War Over Rubber

Germany invaded Poland on September 1, 1939. This set in motion the train ofevents of World War II. Japan, which was now counted among the AxisPowers, pronounced the desirability of the Southeast Asim~ Co-ProsperitySphere. This included all of the lands that normally supplied the entire worldwith natural rubber for making automobile and truck tires and all of the otherproducts made of rubber that contributed to our culture. All natural rubber thenand now comes from tropical plants that grow principally in Southeast Asia.The question was, what to do? Organic chemists had not yet produced asuitable polymer for making automobile tires. Rubber trees (Hevea brasilien-sis), the only source of natural rubber, can be grown commercially on a largescale only in Southeast Asia, even though the trees are native to Brazil.

Guayule (Parthenium argentatum) is the one plant in the western wordthat has been a serious rubber producer. Guayule grows in Northern Mexico


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and a bit of Texas. After caucusing on the subject of rubber, Frits Went and Idecided to prepare ourselves for a possible war by making ourselves mastersof all guayule knowledge, learning about how it was grown, and what onecould do to improve it. We would become the world’s authority on the subject.In the fall of 1939, Frits Went arranged for us to meet with Mr. Carnahan, thepresident of the Intercontinental Rubber Company (IRC), which was the onlylarge company involved in the production of rubber from guayule.

On the appointed day, Frits and I met with Mr. Carnahan in Salinas,California, where the IRC had large guayule plantations. Carnahan was amining engineer and an enthusiast about guayule. The IRC, which had been inoperation at that time for over 50 years, had hired a professional plant physiol-ogist and plant breeder, Dr. William MacCallum, who had harvested seedsfrom strongly growing plants of high rubber content, grown them in thenursery, and multiplied their seeds. The strain that he commended most highlywas strain 593. It was said to be the best grower and the highest yielder ofrubber under field conditions. The discouraging thing we found out aboutguayule was that the IRC always planted their guayule out in the field on aten-year rotation basis. In this way they achieved a claimed rubber content of 8to 10% of the dry weight of the plant.

We learned about growing guayule too. Irrigation is said to decrease rubbercontent of the plant and so they are dry farmed. That’s why they take so long togrow. We were informed that drought stress is required for rubber production.

As a result of our meeting, we formalized an agreement to do research onthe guayule in cooperation with the IRC. We would, in return for supplies ofthe plants and seeds of guayule, return to them what we found out in the formof reports. When we returned to Pasadena we brought with us as a gift, a smallbale of rubber seedlings, as well as a supply of seeds of strain 593. We set up aspecial laboratory for the rapid analyses of large numbers of samples per dayfor rubber content. We practiced growing guayule plants. We studied thenutrient requirements of the guayule, as well as how to kill the various peststhat attack it.

When the Japanese started their penetration of Southeast Asia after Decem-ber 7, 1941, the United States government, with all deliberate speed, devel-oped the "Emergency Rubber Program," which consisted of a multi-prongedattack on what to do about getting rubber. One such prong was to growguayule and grow it as quickly as possible on as large a scale as possible. Thisprong was called the Emergency Rubber Project (ERP).

I was immediately commandeered by the ERP. This project was allotted tothe United States Forest Service (USFS) for administration. Congress directedthe Department of Agriculture and the USFS to immediately purchase thebuildings and holdings of the IRC and to get going on guayule. In February1942 the guayule project of the ERP was set up in Salinas. Major Evan Kelly


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18 BONNER

was the director of the ERP, succeeded later by Paul Roberts of the USFS. Dr.William McGinnies of the USFS became the director of research for theguayule project. My laboratories and greenhouse facilities were declared aspecial laboratory of the ERP, and I was appointed a non-civil servant specialagent of the USFS assigned to the ERP. On January 2, 1942 I began anexperiment that was described in a report to Bill McGinnies concerning thenature of the factors that control the rubber content of guayule. This waspublished in the Botanical Gazzette in 1943 and I believe it was the mostimportant finding we made. We showed that the formation of rubber byguayule is controlled almost entirely by night temperature. The temperatureduring the day has to be high enough for the guayule to photosynthesize, it canbe 60° or 80°F--it doesn’t matter. The temperature at night has to be below50°F, preferably at the optimum of 45°F. The plant only grows in weight--itdoes not grow in height and it does not flower. It concentrates on makingrubber. A high proportion of the photosynthate made during the day is con-verted to rubber under these low night temperature conditions. It has sincebeen shown by Chauncy R. Benedict of College Station, Texas, that low nighttemperature causes the production in guayule of a polyprenyl transferase,which catalyzes the polymerization of isopentenyl pyrophosphate into poly-isoprene. The low temperature induction of the gene for the production ofpolyprenyl transferase is the reason that cold makes guayule make rubber.These findings did not, of course, help the ERP. They came too late. My ownfinding that low night temperature increases rubber formation in guayule wassimply disregarded; even though true, it didn’t jibe with the then currentcentral dogma.

In late 1945 it became quite clear the war was going to end in the not-too-distant future, so it was time to think of other things to do. Early in 1946, theERP was officially declared ended.

Fresh Beginnings--Cell Biology

The end of the war, gasoline rationing, and guayule husbandry all encouragedme to start a new program of research. With the encouragement of my newassociate, Samuel G. Wildman, this new start included what we today call cellbiology. We would isolate chloroplasts, mitochondria, cytoplasm, and lots ofenzymes!

We first wished to isolate chloroplasts. These consisted of membranes,chlorophyll contained in grana, and the soluble stroma that bathes the grana.Spinach leaves were ground in a colloid mill. This device grinds up planttissue wonderfully well and leaves intact grana but no intact chloroplasts. Thegrindate is then centrifuged at 20,000 xg. This completely pellets the grana, aswell as mitochondria, etc. The supernatant from this centrifugation comprisesthe soluble leaf proteins that in turn make up about 16% of the dry weight of


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PREFATORY CHAPTER19

the leaf. An interesting discovery was that upon analysis, whether by centrifu-gation, ultracentrifugation, or electrophoresis in the Tiselius electrophoresisapparatus, over half of the total soluble protein consists of Fraction I, a singlecomponent of molecular weight approximately 500,000. Sam found similarresults with leaf proteins of several other kinds of plants and in 1947, hepublished the first of a series of papers on the proteins of green leaves.

Fraction I was subsequently shown by our former postdoctoral fellow, JohnLittleton of Palmerston North, New Zealand, to be the main constituent of thestroma protein of chloroplasts. Littleton and Paul Ts’o, as well as others, thenwent on to show that Fraction I is, in fact, ribulose-l,5-bisphosphate car-boxylase, the enzyme of the first step in the photosynthetic path of carbon.There is so much of it in leaves because it is not a very good enzyme (i.e. itsturnover number is low). However, Fraction I is the most abundant protein inthe world and it is central not only to the life of plants, but to all life: "All fleshis grass." Sam Wildman has continued to work on Fraction I protein from theday he discovered it to the present time--it’s that important a protein!

I moved next to the mitochondria. Understanding of plant respirationlagged until great forward strides were made in a rush in 1948-1951. AdeleMillerd and I had first met at the University of Sydney in 1949. She then knewthat succinate was oxidized in vitro by cytoplasmic particles of a suspiciouslymitochondria-like nature. While at Caltech from 1950 to 1953, she establishedthat these particles were indeed mitochondria, very much like those of animaltissues. Millerd further showed that these plant mitochondria were capable notonly of using the Krebs cycle to oxidize pyruvate, but they also carried outcoupled oxidative phosphorylation with production of ATP. The paper de-scribing these developments in 1951 brought our previously fractional knowl-edge of plant respiration up to the level of that known for other organisms.

A NEW PARADIGM

Even plant biologists can sometimes take a hint from what is going on in theworld around them. Early in 1960, I said to myself, "It’s rather late now to getto the basic problems of biology. Many other people have started already. Butthere is one basic problem remaining, namely, how does RNA get made? I willwork on that." A new postdoctoral fellow, Ru-chih C. Huang, joined me in themiddle of 1960. She had been a graduate student of Joe Varner’s and camehighly recommended by Joe. I suggested to Ru-chih that she try isolatingnative chromatin (i.e. DNA strands with proteins attached) of the nucleus pea epicotyls, and that she find out if preparations of this kind were capable ofmaking RNA from the four riboside triphosphates. She found very rapidly thatisolated crude nuclear extract was capable of catalyzing the incorporation ofC14-1abeled nucleoside triphosphate into TCA-insoluble material. The next


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20 BONNER

thing was to separate and purify the enzyme responsible for this activity and todetermine whether or not it depended on the presence of DNA. Ru-chihpurified the enzymatic activity, and found that it caused the incorporation ofall four riboside triphosphates into something degradable by RNase and there-fore, RNA. The activity also depended entirely on the presence of DNA in thereaction mixture. We published a short paper on this subject at the end of1960. Demonstrating the importance of this subject, three other groups pub-lished papers at the end of 1960 noting the existence of an enzyme that we nowcall RNA polymerase or DNA-dependent RNA polymerase.

In further study of the enzyme preparatio9, it soon turned out that com-pletely deproteinized DNA is a much better template for DNA-dependentRNA polymerase than is either crude or purified chromatin made from nuclearextracts. This is because DNA in the nucleus is complexed with a class ofproteins called histones. These proteins are complexed with DNA by ionicbonds and can be dissociated from DNA by high concentrations of salt such as2 M sodium chloride. It is therefore possible to remove histones from the DNAof nuclear extracts and to recover the DNA histone-free. Histone-free DNA isa 10- or 20-fold more effective template for RNA synthesis than the originalhistonc-covered DNA.

Study of the literature on histones made it clear that not much was knownabout how many kinds of histones there were or whether there were differenthistones in different creatures or in different specialized cells. Nobody knewwhat histones were for and no one had studied histones in plants. My col-league, Paul Ts’o, encouraged me to arrange a conference on histone biologyand chemistry to see if we could make any sense out of the histones. Weobtained money from the Office of Naval Research, the National ScienceFoundation, and private donors, and organized a conference for everyone inthe world who knew something about histones. We planned our conference atthe Rancho Santa Fe, which at that time was a pretty secluded resort hotel, buthas since become a small city. We consumed the whole hotel by our confer-ence of 56 participants. It became clear that there was complete confusionabout the number of molecular species of histones in nature (estimates variedfrom a dozen to thousands), as well as complete confusion about the similarityor non-similarity of the histones of different species and of the histones ofdifferent Cells of a single species. No pure histone had been prepared up to thetime of our conference.

I returned from that meeting, one that included almost every one of theworld’s known experts on histones, certain that the future of histone chemistrywould depend on a new generation of histone chemists. Ru-chih Huang and Iagreed. We picked as a candidate a new graduate student, DouglasFambrough. Doug was first sent to Stanford University for a month to workwith Kenneth Murray, who instructed him on the use of amberlite CG-50


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PREFATORY CHAPTER21

chromatography for the separation of lysine-rich histones from arginine-richhistories. He then came back and got to work. Doug used for the first timepolyacrylamide gel electrophoresis to monitor the purity of individual histonefractions. He ultimately used it as a final purification step for histones destinedfor amino acid sequencing.

In all of this work, we compared the histories of the pea plant (bud) and the calf thymus. Interestingly enough, cysteine, previously thought to be ab-sent from histones, is found in both pea and calf in one fraction, now known asHistone III. Pea Histone III contains a single cysteine per molecule and iscapable of forming dimers in solution. The Histone III of calf thymus containstwo cysteines per molecule and is able to form a great variety of multimersupon oxidation of solutions just by sitting in the refrigerator. The cysteine ofHistone III undoubtedly has been one of the main sources of confusion con-cerning the heterogeneity of the histories.

The histones of pea and of calf thymus are similar with respect to N-termi-nal amino acids, molecular size (as estimated by polyacrylamide gel electro-phoresis in denaturing medium), and amino acid composition. Further, thehistories of different tissues of the same organism appear to be identical with afew exceptions such as the disappearance of Histone I from the erythrocytes ofbirds. As a postdoctoral fellow, Keije Marushige published a much read andstudied paper in the Journal of Molecular Biology, entitled simply "Propertiesof rat liver chromatin." The moral of this article was that what you can do withpea plants you can do with liver. The article found a great many readers.

Now we knew how to prepare pure histones, and now that we saw that thehistones of peas and cows looked so similar in size, terminal amino acids, andso forth, the time had come to do sequence analysis and comparison betweenthe different histones. This was started in collaboration with my long-timefriend Emil Smith, who at that time was Chairman of the Department ofBiological Chemistry of the UCLA Medical School. We decided to start withHistoric IV, which is the smallest of the four species of histone molecules andtherefore the most easily separated from all the others. Emil Smith declaredthat he needed to have two grams, an amount that one would now considerobscenely large, but two grams he got. It’s no trouble preparing two grams ofpure Histone IV from calf thymus glands, but it’s quite a chore to prepare twograms of pure Histone IV from the apical buds of pea seedlings. We mecha-nized the procedures for growing pea seedlings in barrels under a shower ofrain and for subsequently separating the shoots from the two cotyledons,which were discarded. In the year we spent preparing pure Histone IV frompea seedlings, we were the largest user in the western world of the Alaskacultivar of peas--about 25 tons of (dry) pea seeds.

The Histone IV of peas and of cows were eventually sequenced and shownto be essentially identical. There are two conservative amino acid replace-


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22 BONNER

ments between the two species. The conservation of primary structure inhistones and the probability thai similar observations would be made withhistones other than Histone IV caused biologists to reevaluate the role ofhistones. A more specific and important role in some aspect of chromatinstructure or function might be forthcoming.

The sequencing of histones continued in our laboratory for a while and wecontributed substantially to the knowledge concerning Histone II and HistonellI. However, in the world as a whole, historic sequencing became a growthindustry and became one of those things that’s best to turn over to others.

Bill Garrard and Bill Pearson’s program for taking the output from a gelscanner and plotting the acquired information as a series of Gaussians makes iteasy to determine the relative amount of each individual histone species. Bythis method and with our knowledge of the molecular weights of the individualhistones, it is possible to determine their stoichiometry. There are five speciesof histone molecules, with one molecule of Historic I (HI) for each twomolecules of Histone IIA, Histone IIB, Histone III, and Histone IV, andtherefore, one molecule of Historic I for eight molecules of other histones.Douglas Brutlag discovered that if chromatin treated with a low concentration(.02%) of formaldehyde for 24 hr at ice bucket temperature is then banded CsC1, it loses all histones except HI. When the disposition of the remaining HImolecules along the DNA chain is investigated by electron microscopy, itturns out that they are spaced at approximately 200 base pair intervals.

The main hero of histone chemistry is Douglas Fambrough, who madeorder out of chaos. Unluckily, it also made a neuroscientist of our hero.

From this point on, I have followed the now well-traveled trail to geneisolation and study, and to the production of transgenic creatures. Stay tuned.

FROM KATMANDU TO TIMBUKTU TO KOTA KINABALU

Some will no doubt complain that it is more profitable for the serious scientistto stick to his problem and flog it to death. To them I say, for myself, browsingin far-flung pastures is more fun: Dark CO2 fixation by succulents, chemicalplant ecology, the path of carbon from CO2 to rubber, plant taxonomy, andtreatment of plant-chemical interaction by enzyme kinetics are all matters thatI have also touched, and they have all been fun.

Probably, though, my most important contribution to science so far lies inthe 108 graduate students to whom I have acted as advisor and mentor duringtheir work toward the PhD degree and thereafter. The first, Frederick Addicott,received his PhD in 1939. The last, so far, is Carlotta Glackin, 1988. I amproud of my graduate students. A further contribution I have made has beenthrough the some 200 postdoctoral fellows, visiting professors, and others whohave worked in our laboratory and learned new skills and gained new insights


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and who have returned to the great outer world the better for it. I am proud ofthem as well.

Finally, I spoke earlier about the world that awaits exploration. I havestudied it pretty thoroughly. It’s all wonderful. From Katmandu to Timbuktuto Kota Kinabalu and beyond. Do not miss it!!

ACKNOWLEDGMENTS

I thank my wife, lngelore, for her close and ever helpful collaboration and forher wise counsel as well as for her continuing inspiration. The preparation ofthis manuscript could not have been accomplished without the skilled andcheerily helpful work of Stephanie Canada. I thank her both for her help andher enthusiasm.

Any AnnualReview chapter, as well as any article cited in an Annual Review chapter,may be purchased from the Annual Reviews Preprints and Reprints service

1-800-347-8007; 415-259-5017; emaih arpr @class.org


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annual reviews …web.nchu.edu.tw/~chinho/cb/chapters from my life.pdfrian minister, as well as a...

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