The Neutral Theory of Molecular Evolution

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

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 fixation 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 x 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. Beneficial mutations are quite rare, but are of great importance. For example, mutation changes improve the function of hemoglobins. The lamprey, a "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 fibrinogen in the formation of fibrin, 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

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.

She also said

In the beginning of 1970s, I thought that the borderline mutations should be important, whose behaviors were influenced 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 influenced by both selection and random drift. In this article, theoretical studies of such "nearly neutral" mutations are reviewed (OHTA 1992 ).

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	LITERATURE CITED

TOP 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 38:1895-1902.

JUKES, T. H., 1966 Molecules and Evolution. Columbia University Press, New York.

KIMURA, M., 1968  Evolutionary rate at the molecular level. Nature 217:624-625[Medline].

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 164:788-798[Free Full Text].

OHTA, T., 1973  Slightly deleterious substitutions in evolution. Nature 246:96-98[Medline].

OHTA, T., 1992  The nearly neutral theory of molecular evolution. Annu. Rev. Ecol. Syst. 23:263-286.

OHTA, T., 1996  The current significance and standing of neutral and nearly neutral theories. Bioessays 18:673-677[Medline].

REICHERT, E. T. and A. P. BROWN, 1907  The crystallography of hemoglobins. Proc. Soc. Exp. Biol. Med. 5:66-68.

REICHERT, E. T., and A. P. BROWN, 1909 The differentiation and specificity of corresponding proteins and other vital substances in relation to biological classification 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. 7:1-67.

SUEOKA, N., 1961  Compositional correlation between deoxyribonucleic acid and proteins. Cold Spring Harbor Symp. Quant. Biol. 26:35-43[Medline].