Copyright 2000 by the Genetics Society of America

Perspectives
Anecdotal, Historical and Critical Commentaries on Genetics
Edited by James F. Crow and William F. Dove

Thomas H. Jukes (19061999)

James F. Crow
Genetics Department, University of Wisconsin, Madison, Wisconsin 53706

OM Jukes accepted our invitation to write a Perspectives on the early history of molecular evolution, and in August 1999 he sent a rough beginning containing some now-forgotten early history. He planned an extensive revision and continuation, but on November 1 his death intervened. We have decided to publish his early draft, realizing that it was but a start toward the article that he had planned. Tom, along with Jack L. King and Motoo Kimura, formulated the neutral theory of molecular evolution. Earlier, the idea had been foreshadowed by Sueoka (1962) and Freese (1962). They had each suggested mutation pressure of near-neutral changes to account for the much greater diversity of DNA than of amino acid content among bacterial species. Remarkably, they had these insights before the redundancy of the code was recognized. The neutral theory of molecular evolution in eukaryotes started with Kimura (1968). He argued that the rate of protein evolution was too fast to be compatible with Haldanes (1957) cost of natural selection, and therefore most of the changes must be neutral, driven by mutation and random drift. King and Jukes (1969) had independently arrived at the same conclusion and discovered Kimuras paper while writing theirs. They submitted a manuscript to Science, only to have it turned down. One reviewer said it was obviously true and therefore trivial; the other said that it was obviously wrong. King and Jukes appealed, and the second time it was accepted. This time I was a reviewer; if my recommendation was decisive, I am pleased. The King and Jukes approach was quite different from Kimuras and included a number of arguments. It was more convincing, partly because of their marshaling a larger variety of evidence and partly because of growing doubt of the applicability of Haldanes principle. The King-Jukes paper had the intentionally provocative title, Non-Darwinian evolution. The theory pro-

Author e-mail: jfcrow@facstaff.wisc.edu

Genetics 154: 955958 (March 2000)

duced an immediate outcry from traditional students of evolution, undoubtedly abetted by the title. In the ensuing polemics, Kimura played the major role. King died prematurely in 1983 and Jukes wrote mainly about other things, although he did participate in one joint paper (Jukes and Kimura 1984). One of his interests was the evolution of the genetic code ( Jukes 1983). I particularly liked his showing how, in an orderly sequential way, mutation pressure in the codon and anti-codon could produce the unexpected codes in bacteria and mitochondria (Jukes 1985). He also developed a widely used correction for multiple undetected changes in evolutionary base substitutions ( Jukes and Cantor 1969). Kimura became a crusading advocate for the neutral theory and spent the rest of his life on the subject. In one paper after another, he offered further, increasingly convincing evidence. He also developed a solid mathematical theory, much of it carried over from his own earlier work, which turned out to be remarkably well preadapted for use in molecular evolution. His book The Neutral Theory of Molecular Evolution (Kimura 1983) became a landmark. The jury is still out as to the full extent of random changes in determining the course of molecular evolution, but the neutral theory has formed a basis for phylogenetic reconstruction and the molecular clock; it has also become the null hypothesis for numerous selection experiments. Kimura died in 1994 (Crow 1995). Happily, there was never a public rivalry among the three discoverers. King and Kimura were frequent friendly correspondents. Jukes (1991) acknowledged Kimuras great contributions and sent him a reprint with best wishes and thanks. Kimura rightly receives the lions share of the credit, but we should not forget the independent discovery by King and Jukes and the two forerunners, Sueoka and Freese. Jukes was primarily a nutritionist, with a number of solid accomplishments, especially in vitamins. Some of these have been mentioned by Maddox (1999). Jukes was also an outspoken polemicist and did not hesitate to speak clearly and forcefully against what he thought

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T. H. Jukes

was bad science or faulty logic. An example is his strong critique of Paulings advocacy of massive doses of vitamin C. He could be cantankerous, but he was usually right and always honest. Among other things, Tom waged a spirited battle against creationism. His wideranging scientic and social interests are reected in his frequent columns, along with book reviews and letters, in Nature. I counted 72 such contributions in the years 19751980. Each of Toms friends has a favorite remembrance. Here is mine. Tom greatly admired Aldous Huxley, the most imaginative of the Huxleys ( Jukes 1996). We both enjoyed Huxleys masterpiece, Point Counter Point, a brilliant, erudite, witty satire on the excesses of British society in the 1920s. The book abounds in sophistication and esoterica; one needs The Brittanica within reach (which Huxley is rumored to have read). Tom introduced me to Huxleys short stories, particularly Young Archimedes, the tragedy of a mathematical prodigy whose lesser musical talent was mercilessly exploited. And he admired Brave New World. He called attention to Huxleys prescience when he described conditioning testtube embryos for different roles in life; those that were to be rocket engineers were kept in constant rotation so they would have a better sense of balance in space (this was published in 1932!). Tom loved music. In his last years he went to the lab in the morning and spent the afternoons at home listening to records. He admired Huxleys structuring of Point Counter Point after the Bach B minor suite for ute and strings. He also loved the Beethoven String Quartets and marveled at Huxleys use of the heiliger Dankgesang (holy song of thanksgiving) from the A minor quartet at the climax of the book. When Huxley or

the heiliger Dankgesang comes to mind, I shall always think of Tom Jukes. Following is the rough draft as Jukes submitted it, except for a few bibliographic corrections. It complements an earlier paper ( Jukes 1991), and Tom was trying not to duplicate. He planned extensive additions, and obviously he intended to say more about the nearly neutral hypothesis of Ohta. His draft is presented here, not as what Tom would have liked for it to be, but as a nal tribute to an admired colleague.

LITERATURE CITED
Crow, J. F., 1995 Motoo Kimura (19241994). Genetics 140: 15. Freese, E., 1962 On the evolution of the base composition of DNA. J. Theor. Biol. 3: 82101. Haldane, J. B. S., 1957 The cost of natural selection. J. Genet. 55: 511524. Jukes, T. H., 1983 Evolution of the amino acid code, pp. 191207 in Evolution of Genes and Protein, edited by M. Nei and R. K. Koehn. Sinauer Associates, Sunderland, MA. Jukes, T. H., 1985 A change in the genetic code in Mycoplasma capricolum. J. Mol. Evol. 22: 361362. Jukes, T. H., 1991 Early development of the neutral theory. Perspect. Biol. Med. 34: 473485. Jukes, T. H., 1996 The third Huxley. J. Mol. Evol. 42: 481. Jukes, T. H., and C. R. Cantor, 1969 Evolution of protein molecules, pp. 2132 in Mammalian Protein Metabolism, edited by H. N. Munro. Academic Press, New York. Jukes, T. H., and M. Kimura, 1984 Evolutionary constraints and the neutral theory. J. Mol. Evol. 21: 9092. Kimura, M., 1968 Evolutionary rate at the molecular level. Nature 217: 624626. Kimura, M., 1983 The Neutral Theory of Molecular Evolution. Cambridge University Press, Cambridge. King, J. L., and T. H. Jukes, 1969 Non-Darwinian evolution. Science 164: 788798. Maddox, J., 1999 Thomas Hughes Jukes (19061999). Nature 402: 478. Sueoka, N., 1962 On the genetic basis of variation and heterogeneity of DNA base composition. Proc. Natl. Acad. Sci. USA 48: 166169.

The Neutral Theory of Molecular Evolution

Thomas H. Jukes
Space Sciences Laboratory, University of California, Oakland, California 94608

N 1966, I became interested in the amino acid sequences of cytochrome c molecules (Jukes 1966). I noted that these sequences differed in the cytochromes c of various species to an extent that seemed unnecessary from the standpoint of their function. I stated, The changes produced in proteins by mutations will in some cases destroy their essential functions, but in other cases the change allows the protein molecule to continue to serve its purpose. Early indication of neutrality may be found in the publications of E. T. Reichert and A. P. Brown (1909). They compiled the crystallographic structure of vertebrate hemoglobins on a taxonomic basis. They stated

the principle that substances that show differences in crystallographic structure are different chemical substances. In short, if two crystals have identical crystalline structure, the molecules of which they are composed are identical. A report of their studies is shown in Table 1. They commented that an increase in the divergence of crystallographic properties was found to be parallel to the taxonomic separation of various animals. Of much interest is the fact that a sample of blood labeled as that of a baboon was found upon examination of the hemoglobin crystals to be that of a cat, and a subsequent follow-up showed that the mislabeling of the sample vial had occurred (Reichert and Brown 1907). Their

Prespectives TABLE 1

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Crystallographic comparison of reduced hemoglobins of species in Felidae contrasted with a few other species of carnivora Axial ratio a:b:c 0.9742:1:0.3707 0.9742:1:0.3839 0.9657:1:0.3667 0.9489:1:0.3931 0.9656:1:0.3939 0.9605:1:0.3944 0.9869:1:0.3914 0.6745:1:0.2863 0.6494:1:0.2894 1.2239:1:1.1429 1.2131:1:1.1970

Specic name Felidae Felis leo Felis tigris Felis bengalensis Felis pardalis Felis domestica Lynx canadensis Lynx rufus Canidae Canis familiaris Vulpes fulvus Ursidae Ursus americanus Otariidae Phoca vitulina

Common name Lion Tiger Leopard Ocelot Domestic cat Lynx Wildcat Dog Red fox Black bear Harbor seal

From Reichert and Brown (1909).

Thomas H. Jukes, courtesy of Carol Fegte.

amino acid. These results would imply an absolute specicity for the mechanisms responsible for protein synthesis and this should be taken into account when considering such mechanisms (Sanger 1952).

monograph remains as the earliest landmark in the history of molecular evolution. Hemin crystals obtained from different species were always the same, so that the differences were due to the globin portion of the molecule. It is now known that the differences are due to amino acid substitutions throughout the polypeptide chains of the globins. These substitutions are the result of single-base changes in the DNA strands of the hemoglobin genes. The concept that each protein from each species of animal was a single chemical substance at the molecular level was implicit for the hemoglobins in the report by Reichert and Brown. It was again stated in 1952 by Sanger as a result of his studies of the amino acid sequence in insulin:
It has frequently been suggested that proteins may not be pure entities but may consist of mixtures of closely related substances with no absolute unique structure. The chemical results so far obtained suggest that this is not the case and that a protein is really a single chemical substance, each molecule of one protein being identical with every other molecule of the same protein. Thus it was possible to assign a unique structure to the phenylalanyl chains of insulin. Each position in the chain was occupied by only one amino acid and there was no evidence that any of them could be occupied by a different residue. Whether this is true for other proteins is not certain but it seems probable that it is. The N-terminal residues of several pure proteins have been determined . . . and this position is always found to be occupied by a single unique

Further consideration of these ideas led to the writing and publication of an article entitled Non-Darwinian evolution, by Jack King and myself, in 1969. In retrospect, it might have been better to entitle the article Non-adaptive evolution, because non-Darwinian probably raised the hackles of admirers of Charles Darwin. (It is amusing to remember that Darwin himself raised a storm of indignation among his contemporaries.) In the meantime, Kimura had published a short note in Nature (Kimura 1968) in which he pointed out that the rate of random xation of neutral mutation in evolution, per species per generation, is equal to the rate of occurrence of neutral mutation per species per generation and is independent of population size. Kimura became assiduously concerned with the neutral theory and published a textbook on it (Kimura 1983). The theory postulates that nucleotide substitutions inherently take place in DNA as a result of point mutations followed by random genetic drift. In the absence of selection constraints, the substitution rate reaches the maximum value set by the mutation rate, i.e., about 5 10 9 substitutions per site per year, or at a lower rate when constraints are imposed by natural selection (King and Jukes 1969). Deleterious mutations have long been familiar; for example, the effects of X rays are known to produce such mutations. Benecial mutations are quite rare, but are of great importance. For example, mutation changes improve the function of hemoglobins. The lamprey, a

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T. H. Jukes Synonymous substitutions are not strictly neutral, but because of their minute effect, random drift predominates such that the rate of substitution is only slightly less than the completely neutral prediction. It was concluded that the strictly neutral theory has not held up as well as the nearly neutral theory, yet remains invaluable as a null hypothesis for detecting selection. On the other hand, the main difference between the nearly neutral and the traditional selection theories is that the former predicts rapid evolution in small populations, whereas the latter predicts rapid evolution in large populations.

primitive organism, has a single hemoglobin chain, but mammals have a tetrameric hemoglobin that increases their function of oxygen transport from the lungs to the tissues. We can see the reduced hemoglobins in our own blue veins as they are on their way to the lungs for reoxygenation. Once the neutral theory had been stated, examples of its effect became evident. For example, in the genetic code, some base pair changes are without effect on protein structure: ACC and ACG both are codons for threonine, and to change from ACC to ACG would therefore be neutral. Of the 349 possible single base changes in the 61 amino acid-specifying codons, 134 are substitutions to synonymous codons. These should be neutral with respect to natural selection. Directional mutation pressure should, therefore, give rise to many neutral mutations. In 1961, before the genetic code had been discovered, Sueoka noted amino acid differences between AT-rich and GC-rich bacterial species (Sueoka 1961). Once the neutral theory had been stated, many examples of neutral changes came to light. Cox and Yanofsky (1967) studied a strain of Escherichia coli containing the Treffers mutation gene, which produces a trend toward a DNA of a higher GC content than that in the original stock. Thousands of such mutations accumulated in the laboratory cultures without markedly impairing the motility of the mutated strains. In other organisms (or viruses), such as bacteriophage T4, the bias may be in the opposite direction. In mammalian hemoglobins, most changes in residues occurring on the outside of the molecule appear to be selectively neutral. In contrast, harmful changes are produced when they occur in the interior of the molecule. The neutrality of the change is therefore dependent on its location (King and Jukes 1969). During blood clotting, two peptide fragments are removed enzymatically from brinogen in the formation of brin, the blood-clotting protein. Fibrinopeptide A, one of those fragments, shows a rapid rate of evolutionary change. One can infer that these are neutral changes, since this fragment is discarded. From these considerations, we concluded that the genome becomes virtually saturated with such changes that are not eliminated by natural selection. We conclude that most proteins contain regions where substitutions of amino acids can be made without producing appreciable changes in protein function (King and Jukes 1969). Ohta (1996) challenged the neutral hypothesis of evolution by pointing out

She also said

In the beginning of 1970s, I thought that the borderline mutations should be important, whose behaviors were inuenced by both random genetic drift and selection. These are called slightly deleterious or nearly neutral mutations and the theory proposing the importance of this class was published in 1973 (Ohta 1973).

Earlier she concluded

The . . . revision is to clarify the interaction of natural selection and random drift at the molecular level. Natural selection cannot be so simple as to be all or nothing. There are numerous types of mutations whose behavior is inuenced by both selection and random drift. In this article, theoretical studies of such nearly neutral mutations are reviewed (Ohta 1992).

LITERATURE CITED
Cox, E. C., and C. Yanofsky, 1967 Altered base ratios in the DNA of an Escherichia coli mutator strain. Proc. Natl. Acad. Sci. USA 38: 18951902. Jukes, T. H., 1966 Molecules and Evolution. Columbia University Press, New York. Kimura, M., 1968 Evolutionary rate at the molecular level. Nature 217: 624625. Kimura, M., 1983 The Neutral Theory of Molecular Evolution. Cambridge University Press, New York. King, J. L., and T. H. Jukes, 1969 Non-Darwinian evolution. Science 164: 788798. Ohta, T., 1973 Slightly deleterious substitutions in evolution. Nature 246: 9698. Ohta, T., 1992 The nearly neutral theory of molecular evolution. Annu. Rev. Ecol. Syst. 23: 263286. Ohta, T., 1996 The current signicance and standing of neutral and nearly neutral theories. Bioessays 18: 673677. Reichert, E. T., and A. P. Brown, 1907 The crystallography of hemoglobins. Proc. Soc. Exp. Biol. Med. 5: 6668. Reichert, E. T., and A. P. Brown, 1909 The differentiation and specicity of corresponding proteins and other vital substances in relation to biological classication and organic evolution: the crystallography of hemoglobin. Carnegie Institution of Washington, Pub. No. 116. Sanger, F., 1952 The arrangement of amino acids in proteins. Adv. Protein. Chem. 7: 167. Sueoka, N., 1961 Compositional correlation between deoxyribonucleic acid and proteins. Cold Spring Harbor Symp. Quant. Biol. 26: 3543.