ous, Maynard Smith's postmceting om-ment to Science would certainly meetwith broad agreement: "I thought themeeting was very positive. This was thefirst time for more than 25 years thatthere has been serious discussion be-tween paleontologists, geneticists, andthe like. This can't be anything butgood."

Many people suggested thatlhe meet-ing was a turning point in the history ofevolutionary theory. "I know it sounds alittle pompous," Hallam told Science,"but I think this conference will even-tually be acknowledged as an historicevent." Will it prove to be the currentequivalent to the 1946 Princeton meetingat which the capstone of the Modern

Synthesis was laid?i Will a new synthesisemerge, signaling a true paradigm shiftin the Kuhnian sense?

Perhaps. Gould expressed his ex-pectations in more modest terms: "Ihope that this meeting will lead to a rap-prochement. I hope it will set the basisfor a reconstruction of ideas."

- --ROGER LEWIN

The 1980 Nobel Prize in Chemistry

Three molecular biologists win the prize for discoveriesthat can be used to study gene structure and control

The current Nobel Prize in Chemistry many other molecular biologists becamespotlights contributions to the methodo- interested in applying what is knownlogical revolution that is allowing re- about bacterial gene expression to thesearchers to examine the structure and study of gene expression in higher orga-control of genes of higher organisms in a nisms. "We began to think of using SV40detail previously unimagined. Half of the [an animal tumor virus] to carry genes in-prize was awarded to Paul Berg of Stan- to mammalian cells," Berg says. Theford University; the other half was foreign genes could then be studied andawarded jointly to Frederick Sanger of manipulated to see what controls theirCambridge University and Walter Gil- expression.bert of Harvard. This is Sanger's second In 1971, Berg and his colleagues DavidNobel Prize. Jackson and Robert Symons opened the

Berg is cited for "his fundamental circular SV40 molecule with a restrictionstudies of the biochemistry of nucleic enzyme, Eco RI. This enzyme, whichacids, with particular regard to recombi- was discovered in Herbert Boyer's labo-nant DNA." According to a press re- ratory at the University of California atlease from the Swedish Royal Academy, San Francisco, cleaves DNA at specific-"Berg was the first investigator to con- base sequences. In the case of SV40struct a recombinant DNA molecule, DNA, it cleaves it in exactly one spot.i.e., a molecule containing parts of DNA Berg's group then spliced the linearfrom different species. His pioneering SV40 DNA to the DNA of the bacterialexperiment has resulted in the develop virus X. The A DNA also is circular andment of a new often called Berg's group cleaved it too with Eco RI.genetic enginiq.' -Berg does not Although this was the first time thatknow whether the Nobel committee had DNA's from two different species werea particular experiment in mind but, he joined, it was not the first time that anysays, "I would like to think it [the prize] DNA's were joined. H. Gobind Kho-was for a body of work and not for a rana, of the Massachusetts Institute ofsingle experiment." Arthur Kornberg, Technology, discovered in the 1960'salso of Stanford, thinks the only way to that an enzyme produced by the bacte-interpret the Nobel committee's 'car- rial virus T4 can catalyze the linking to-fully worded citation" is as recognition "gber of-DNA molecules. Berg, Jack-for Berg's 20 years of leadership in the son,' and Symons enzymatically con-molecular biology of nucleic acids. structed complementary or "sticky"

In the 1960's, Berg did a great deal of ends on the two DNA segments to beinnovative work on bacterial protein joined and then used the T4 enzyme tosynthesis, particularly the interaction of do the joining. The method they usedamino acids, with transfer RNA's. His was developed and tested in4ependentlywork helped explain how these RNA's by Berg's group and by Peter Lobbanare used as adapters in decoding. His and Dale Kaiser of Stanford. Althoughgroup and several others also discovered no one knew it at the te, it was unnec-one of the enzymes that copies DNA into essary to construct sticky ends, sinceRNA. they are automatically produced whenThen, about 10 years ago, Berg and Eco RI cleaves DNA. This fact was dis-

covered in 1972 by Janet Mertz and Ron-ald Davis and independently by VittorioSgaramella, all of Stanford University.

It had been Berg's intention to in-troduce the SV40- hybrid molecule intothe bacterium Escherichia coli, which Acan infect. In that way, he could get manycopies of the molecule to be used forfuture experiments in gene expression in

Paul Berg0036 8075/8Q'1121.OS87$00.50/0 Copyright C 1980 AAASSCIENCE, VOL. 210, 21 NOVEMBER 1980

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Frederick Sanger

mammalian cells. In the summer of1971, Mertz, who was Berg's graduatestudent, described the plan at a tumor vi-rus conference held at Cold Spring Har-bor, New York. Robert Pollack of ColdSpring Harbor Laboratories reacted im-mediately with dismay, pointing out thatSV40 transforms human cells in cultureand that E. coli lives in the human gut. Ifany E. coli infected with the SV40-XDNA escaped from the laboratory, theycould be dangerous.

Berg was persuaded by this argumentand decided not to do the experiment.He led molecular biologists in calling fora moratorium on recombinant DNA re-search until the risks could be assessedand the safety of the experiments en-sured. It was a period, Berg recalls, "ofmore controversy than science." In1975, the moratorium was conditionallylifted and the National Institutes ofHealth developed guidelines for the con-duct of recombinant DNA experiments.The guidelines have since been softenedas the experiments turned out to be lessrisky than anticipated.

Ironically, the experiment that Bergoriginally wanted to do would not havesucceeded, and no one at the time wouldhave known why. The SV40-X hybridwould not have replicated in bacteria be-cause Berg inserted the SV40 genes at asite now known to be essential for X'sreplication and thereby interrupted thissite.

In fact, the heart of recombinant DNAtechnology is not just gene splicing butalso gene cloning. It is necessary to findways to get foreign genes into cells, en-sure that the genes are expressed, andthen select for the cells that are ex-pressing those genes. Cloning techniqueswere pioneered by Stanley Cohen andAnnie Chang of Stanford University and

888

Herbert Boyer and Robert Helling of theUniversity of California at San Fran-cisco, who, in the early 1970's, devel-oped a plasmid, which is a small piece ofextra-chromosomal DNA, that couldcarry foreign genes into bacterial cells.The plasmid contained genes that madethe bacteria resistant to the antibiotic tet-racycline, so that the cells which took upthe plasmid and expressed its genescould easily be selected.

In the past decade, recombinant DNAtechniques have become increasingly so-phisticated. Berg has played a major rolein these developments. Most recently,he and others, particularly Daniel Na-thans of Johns Hopkins University Med-ical School, who won a Nobel Prize forhis work in restriction enzymes, exten-sively studied the structure, organiza-tion, and replication of SV40 genes. Af-ter constructing deletion mutants ofSV40 that have proved extremely usefulin studies of SV40 gene functions, Bergwent back to his original idea of usingSV40 to introduce genes into mammaliancells. He spliced to SV40 an E. coli genethat allows cells to use xanthine as a sub-strate in nucleotide synthesis. Then, inseparate experiments, he spliced animalgenes for globin, histone, or the enzymedihydrofolate reductase to this hybridSV40 molecule. When the SV40 carryingthe added bacterial and animal genes wasintroduced into cultured cells, Bergcould pick out the cells that were trans-formed by SV40 by selecting for cellsthat grow on xanthine. In this way, Bergwas able to show that the added animalgenes are expressed in cultured cells.Dean Hamer and Philip Leder of the

National Institute of Child Health andHuman Development have also usedSV40 as a cloning vector in culturedcells. But, says Nathans, "Clearly thenotion that you could construct a vectorwith animal viruses was Berg's idea."An important aspect of Berg's work

has been his extraordinary ability to de-velop methodologies. For example, hewas the first to use nitrocellulose bindingassays to study interactions betweenproteins and nucleic acids. He also de-veloped the nick translation method,which is used to make isotopically la-beled DNA probes and is central to cur-rent studies of gene functions. "His styleof biochemistry helped set the standardsin the nucleic acid field," says Nathans.The second half of the chemistry prize

was also given to developers of method-ologies. Sanger and Gilbert were hon-ored for their discoveries of ways to se-quence DNA. In the past few years,these techniques have become widelyused to determine amino acid sequences

of proteins because with these methodsit is easier and more accurate to se-quence the DNA coding for proteinsthan to sequence the proteins directly.The techniques are also used to deter-mine the intervening sequences that oc-cur in eukaryotic genes and the se-quences that occur in control regions ofbacterial DNA. By using these methods,molecular biologists hope to learn whichsequences control gene expression inhigher organisms and how they do so."DNA sequences are the basic, under-lying structures [of molecular biology].There is nothing more primitive. Yourquestions are ultimately posed there,"says Gilbert.Sanger and Gilbert are about as dif-

ferent as two scientists can be, and theycame upon their sequencing methods byentirely different paths. Sanger is quiet,modest, self-effacing; Gilbert is muchmore flamboyant. Ted Friedman of theUniversity of California at San Diego,who spent a sabbatical year with Sanger,says, "If you talk to Sanger and do notknow who he is, you would think he isthe lab caretaker. If you allow him to, hewill melt into the woodwork." GeorgeBrownlee of Oxford University, who un-til recently was at Cambridge with Sang-er, adds, "Sanger certainly doesn't givehimself airs. But in my view, he ranksamong the great scientists of our time."According to Friedman, Sanger's out-

standing feature is his "uncanny beliefand knowledge that sequencing can bedetermined by very simple methods." Inthe 1950's, Sanger studied protein se-quencing at a time when no one knewwhether all proteins of a particular type

Walter Gilbert

SCIENCE, VOL. 210

have the same sequences. His first NobelPrize was awarded for this work. Thenhe attacked the problem of RNA se-quencing, developing the widely usedfingerprinting method. About 10 yearsago, he set out to sequence DNA, eventhough this problem, too, was consid-ered intractable.

Sanger's method evolved graduallyfrom more than one line of attack on theproblem. In the early 1970's, he discov-ered the plus-minus sequencing method,a direct precursor of the method he usestoday. In the plus-minus method the ob-ject is to obtain a set of nested segmentsof the DNA to be sequenced. The firstsegment consists of the first nucleotide,the second consists of the first two nucle-otides, the third of the first three nucle-otides, and so on. These segments areconstructed in such a way that the identi-ty of the last nucleotide of each nestedsegment is known. Once obtained, thenested segments can be separated ac-cording to size by electrophoresis on anultrathin polyacrylamide gel. The sepa-rated fragments can be detected becauseeach is isotopically labeled.The key to the plus-minus method is

obtaining the nested segments. Sangerconstructs them by synthesizing them.He separates the two strands of the DNAto be sequenced and then makes partialcopies of one of those strands. To ensurethat the partial copies include all nestedsegments and that the terminal nucle-otide of each segment is known, Sangermakes the copies under conditions inwhich one nucleotide is limited in quanti-ty. For example, he provides limitedquantities of adenine so that the DNAcopying will eventually stop because oflack of adenine. Then all of the copiesmade will end just before an adenine,and the next nucleotide of each of the re-sulting segments is adenine. In a similarway, Sanger synthesizes segments end-ing before each of the other three DNAnucleotides.

Sanger has since improved the plus-minus method to make it more efficient.Instead of supplying limited quantities ofeach nucleotide, he supplies derivativesof the nucleotides that cause DNA syn-thesis to stop.

In contrast to Sanger, Gilbert did notdeliberately set out to sequence DNA. Ahighly visible, active scientist who runs alarge laboratory, Gilbert has worked on awide variety of problems in the past 20years, ranging from how bacterial genesare organized and expressed to gene con-trol in higher organisms and genetic engi-neering. He is also chairman of the boardand cochairman of the board of directorsof the gene splicing firm Biogen.21 NOVEMBER 1980

Gilbert, working with Allan Maxam,who is now at Harvard Medical School'sSidney Farber Cancer Institute, cameupon a DNA sequencing technique al-most by chance. Gilbert recalls that oneday in early 1975, Andrei Mirzabekov, ofthe USSR Academy of Sciences, ap-peared in his office and urged him to try anew approach to studying how proteinsrecognize specific sequences of DNA.Gilbert had long been interested in thelac repressor protein of E. coli, whichbinds to the lac operon segment ofDNA,and, in fact, it was Gilbert who isolatedthe lac repressor and operator. Mirzabe-kov and his colleagues had been probingprotein-DNA interactions with dimethylsulfate, a reagent that methylates theDNA nucleotides adenine and guanine.After reacting with dimethyl sulfate,DNA breaks easily at these bases.

Gilbert decided to expose lac operonDNA to dimethyl sulfate and then break

A k >G 4C 4T|

Part of the sequencing pattern obtainedfroma piece of DNA about 130 base pairs inlength. The letters at the top of the columns,A, G, C, and T (adenine, guanine, cyto-sine, and thymine) indicate which base waspreferentially cleaved by chemicals. The dark-est band in each column represents the basemissing from the end of the initial segments,with the exception of cytosine (C). All darkbands in the C column represent cytosineseven if bands also appear in the T column atthat position. Bands that appear in the Tcolumn but not in the C column represent thy-mines (T). To read the sequence of the DNA,read off the base represented by each band,starting from the bottom of the columns.[Source: Walter Gilbert and Allan Maxam]

the DNA at adenines and guanines. Forcomparison, he would bind lac repressorto the operon and repeat the experiment.The adenines and guanines that reactedwith the repressor should be protectedfrom the dimethyl sulfate, and so theDNA should not break there. Since thesequence of lac operon DNA was known(it had been copied into RNA and theRNA sequenced), it would be possible tolearn where the repressor binds on thisDNA.

After these experiments, Gilbert andseveral of his associates discovered asecond lac operon of unknown se-quence. Maxam repeated the dimethylsulfate experiments with this new lac op-eron and the lac repressor. When he andGilbert saw the results, they realized thatthey had the beginning of a DNA se-quencing method. By using dimethyl sul-fate and adjusting the reaction condi-tions, they could break DNA at eitheradenines or guanines. Now if they couldfind a way to break DNA preferentiallyat thymines or cytosines, they could gen-erate nested segments whose terminalnucleotides were known. With this idea,Maxam set to work to develop ways ofbreaking DNA at thymines or cytosines.He recalled that under appropriate chem-ical conditions, hydrazine preferentiallyweakens DNA at one or the other ofthese nucleotides. After a summer ofwork, Maxam succeeded in perfectingthe chemical method of sequencingDNA.The difference between the Sanger

method and the Maxam-Gilbert methodis that Sanger generates nested segmentsby synthesizing them and Maxam andGilbert generate the segments by break-ing the DNA at specific bases. Bothmethods are currently used, and re-searchers experienced with both say thatthe choice between them depends in parton the length ofDNA to to be sequencedand in part on the personal preferencesof the investigator. Tom Maniatis of theCalifomia Institute of Technology, forexample, uses Sanger's method for verylong sequences of DNA because it isfaster. For shorter sequences, one or afew genes long, the two methods arecomparable in speed, but Maniatis pre-fers the Maxam-Gilbert method because"Allan has established the protocol socompletely that anyone who tries themethod is successful."The full ramifications of recombinant

DNA technology and DNA sequencingmethods are not yet known. But thesetechniques are changing molecular biolo-gists' perceptions of what can be learnedabout the genes of higher organisms.

-GINA BARI KOLATA889