Mutations and Evolution

Darwin's theory of natural selection, which was published practically simultaneously with the establisment of the ATLANTIC, opened new worlds to science. Dr. Evelyn Witkin, a brilliant young biologist at State University of New York College of Medicine in New York City, tells how genetics, a branch of science not yet born in Darwin's day, has carried forward our understanding of evolution.

When Charles Darwin began his voyage around the world aboard H.M.S. Beagle, he shared with his contemporaries the almost unquestioned belief that every species of plant and animal then inhabiting the earth had originated in a separate act of creation. No other way had ever been found to explain the exquisite adaptations of structure and behavior by which each form of life seems so perfectly designed for its place in nature. By the end of the live-year journey, an altogether new and startling idea had begun to develop in the mind of the young naturalist. Today, less than a century after the publication of The Origin of Species, the theory of evolution has long been accepted as a fact of life.

The brilliance of Darwin's insight lay in his integration of two simple and seemingly unrelated biological truths, and in his projection of their inevitable consequences on a vast scale of time. One was that the individual members of a species are not all precisely alike, the differences among them tending to be inherited. The other, somewhat less obvious, was that the infinite expansion of populations is checked by limitations in the availability of food, and by other restrictive conditions of life. It follows directly, reasoned Darwin, that any inheritable trait that enhances the survival and fertility of an individual will he "naturally selected" — that is, will be transmitted to a larger fraction of the population in each succeeding generation. In this way, by the gradual accumulation of adaptive variations, the species now existing have evolved from earlier and more primitive progenitors, and owe their intricate mechanisms of adjustment not to purposeful planning but to the impassive operation of natural laws.

In the great upheaval of scientific thought that followed the announcement of the theory of evolution, the phenomena of heredity and variation were suddenly thrust into the forefront of biology. Almost' nothing was known of the way in which hereditary differences arise, and of the mechanisms of their transmission, but Darwin foresaw the development of a "grand and almost untrodden field of inquiry" in which the causes of variation and the laws of heredity would be discovered. Even as Darwin called upon the future to solve the mysteries of inheritance, Gregor Mendel was laying the foundation for the new science of genetics. Genetics has contributed richly to the synthesis of facts and ideas from almost every branch of the natural sciences that has been built upon Darwinism. As the diverse and intricate mechanisms of evolution have come to be understood, it has grown increasingly certain that the raw materials upon which they depend are the mutations of genes.

The hereditary endowment of a plant or animal is now known to be determined by a very special kind of material found primarily in the threadlike chromosomes that may be seen under the microscope in the nucleus of the cell. The invisible elements of which this material is composed, the genes, were once regarded as discrete particles strung along the chromosome like beads. Recent evidence has modified this concept considerably, and many geneticists now think of genes as chemically differentiated regions of the chromosome, not necessarily separated one from another by definite boundaries, but each having a distinctive structural pattern from which it derives a highly specific role in the metabolism of the cell.

Every cell in the body contains a set of chromosomes and genes, descended directly by a long line of cell divisions from the set originally constituted in the egg cell at fertilization. The human embryo develops into a person, rather than into, a tree or an elephant or a monstrosity, because the material carried in its chromosomes, its constellation of genes, initiates and guides a marvelously coordinated sequence of reactions that leads inevitably, under normal conditions, to the differentiation and growth of a human being.

Throughout the life of the individual, the genes, continue to exert their control over the complex chemistry of the cells and tissues of the body. As older tissue is gradually replaced by new tissue in the mature person, the food that is consumed is converted quite specifically into more of the very same individual, even though an identical diet, fed to a dog, would be transformed into more dog. We are a long way from understanding just how genes direct the manifold activities of living systems, but we know with growing certainty that the range of possible responses of any cell or organism to the conditions it may encounter is largely gene-determined.

All the members of our species have in common the basic genetic make-up that sets us apart from other forms of life. Nevertheless, no two individuals, with the exception of identical twins, have exactly the same heredity, which is another way of saying that every person possesses a unique pattern of chromosomal genes. Differences in skin pigmentation, eye and hair color, stature, and facial features are familiar hereditary traits by which individuals and groups of individuals differ from one another. These and the host of other inherited variations, from fingerprint patterns to blood types, are manifestations of the differences that exist in the structure and arrangement of the genic material.

Some hereditary variations, such as eye color, are known to depend upon differences in the state of a single gene. This does not imply that one gene, all by itself, is responsible for the formation of blue or brown pigment in the iris of the eye. It means that a change in this particular gene can alter the integrated functioning of the whole gene system so as to result in the production of a different kind of pigment. Other characteristics, such as height, depend upon the states of a relatively large number of genes.

Genes do not exist in a vacuum. They are always present in an environment that must be taken into account in understanding how they work. The environment within the cell and within the organism, and the more unpredictable environment outside, are intimately bound up with the functioning of genes and have varying degrees of influence upon the ultimate expression of heredity. A trait or characteristic is not, in itself, inherited. That which is determined by genes is the capacity to produce certain traits under certain conditions.

In the case of eye color, this distinction may seem unimportant, since an individual having the genetic constitution for blue eyes will have blue eyes under any environmental conditions.' Its meaning becomes evident, however, when we consider inherited characteristics that are more directly responsive to environmental variables. The Himalayan rabbit is a case in point. This rabbit has a pattern of white fur, with black fur at the extremities (ears, tips of paws, tail), and this pattern is passed along from generation to generation. If a patch of white fur from the back of such a rabbit is shaved off, and the new fur allowed to grow back while the animal is kept in a cool place, it will grow in black instead of white. Thus it is not the pattern itself that is inherited, but the capacity to produce black pigment at low temperatures and not at higher temperatures. Since the temperature at the extremities is normally lower than that of the rest of the body, the typical Himalayan pattern is obtained. Similarly, although stature is basically under the control of genes, it can be influenced significantly by nutritional factors.

Genes are remarkable not only for the way they direct the intricate pathways of metabolism and development. They have in addition, unique properties that give them special importance in biology, as the raw materials not only of evolution but probably of life itself. Genes have the ability to organize material from their surroundings into precise copies of their own molecular configurations, and they exercise this power every time a cell divides.

They are also capable of undergoing structural changes, or mutations; and once such a change has occurred, it is incorporated into the copies that the gene makes of itself. A single unit having these properties, and having also the ability to aggregate with other such units, would possess the essential features of a living being, capable of unlimited evolution through the natural selection of variant forms and combinations most efficient in reproducing themselves. Many biologists believe that life may have originated with the accidental formation of "naked genes," organic molecules able to duplicate their own structure, and their variations in structure, from materials available in the environment.

Although the chemical nature of genes is not yet known with certainty, one of the most important recent advances in genetics is the evidence that their definitive properties can be accounted for by the theoretical structure and behavior of the molecules of compounds known as desoxyribonucleic acids, or DNA. Chromosomes contain large amounts of DNA. Its molecules are very big, as molecules go, built up in long chains from only four kinds of simple chemical building blocks. The order in which these units occur, and the number of repetitions of similar groupings, are thought to be the basis of the specific activity of different regions of the chromosome — in other words, of genes. The study of the properties of these molecules provides a way to explain the mechanism by which genes duplicate themselves and reproduce the variations that they may undergo.

Mutations, as has already been suggested, are considered to be changes, on the molecular level, in the structure or organization of genes. A mutation in any gene is likely to be reflected in a modification of its contribution to the delicately interwoven pattern of control exercised by the whole constellation of genes, and may be detected by its effect on some physical or metabolic characteristic of the organism.

Mutations, in nature, are rather rare events, occurring usually with frequencies of from one in a thousand to one in a billion gene duplications. They have an extremely wide range of effects, from fatal disturbances of normal development to perceptible reductions of life expectancy, from striking changes in appearance to slight alterations of metabolism that can he detected only with sensitive laboratory instruments.

Mutations in man are responsible for the kinds of hereditary differences we have already discussed, and can produce, as well, such effects as early fetal death, stillbirth, diseases such as hemophilia and sickle cell anemia, color blindness and harelip. It seems quite possible that cancer, leukemia, and other malignant diseases may originate by the occurrence of mutations in body cells other than the reproductive cells.

Although the overall frequency of mutations can be increased considerably by exposure to radiations and a variety of chemicals, there is ordinarily no relation between environmental conditions and the kinds of mutations that occur. Mutations of all sorts arise in natural populations, with low but regular frequencies, in a way that is best explained by considering them to be the consequences of accidental molecular rearrangements, occurring more or less at random in the genetic material. X rays and other kinds of high-energy radiations increase the probability that these accidents or mutations will occur, but we do not know with certainty the causes of so-called "spontaneous" mutations. Natural radiations, such as cosmic rays, undoubtedly cause a fraction of them, but it has been estimated that the intensity of natural radiations is not sufficient to account for all the mutations that occur in plant and animal populations.

Darwin believed that the inheritable variations upon which natural selection acts are caused directly by the influence of the conditions of life upon the organism, or by the effects of use and disuse of particular body parts. Although he appreciated the difficulty of explaining how the environment can provoke appropriately adaptive modifications, and how such changes can be incorporated into the reproductive cells so as to be inherited, it seemed at that time even more difficult to imagine that they could arise by chance. How, then, does modern genetics propose that the orderliness of evolution can follow from accidental variations in the molecular structure of genes, occurring without relation to the demands of the environment?

We do not need to rely upon speculation to answer this question. The study of evolution has moved into the laboratory, and while it is not possible to duplicate here the kinds of changes that have required millions of years in nature, the elementary steps of evolution can be analyzed. For this purpose, the use of bacteria presents many advantages. This is particularly true since the mechanisms of heredity and variation, wherever studied in the plant and animal kingdoms, seem to be fundamentally alike. Genes and mutations are much the same, in their basic behavior, whether they are investigated in fruit flies, in maize plants, in man, or in microorganisms.

The bacterium Escherichia coli, a rod-shaped, one-celled organism normally found in the human intestinal tract, is widely used in research on heredity. It divides every twenty minutes under optimal conditions, and a single cell, placed in one cubic centimeter of culture medium, will produce overnight as many descendants as the human population of the earth. The recent discovery of a sexual process in this organism, as well as in some other kinds of bacteria, has made it possible to interbreed different strains and to apply many of the classical methods of genetic analysis that were developed in the study of higher forms. Escherichia coli is an ideal vehicle for the experimental study of "microevolution."

In the laboratory, a strain of this bacterium can be maintained almost indefinitely, under constant conditions, without undergoing any appreciable change in its characteristics. When the environment under which the bacteria are grown is changed, however, in a way that is somehow detrimental to the population, it will often adapt itself rapidly and effectively to the new conditions.

A good example of the way in which a bacterial culture may adapt to an unfavorable environment is the reaction of Escherichia coli to streptomycin. Most strains of this bacterium are sensitive to streptomycin, and are unable to multiply in the presence of even very small amounts of the antibiotic. Sensitivity to streptomycin is an inherited trait and is transmitted, unchanged, through countless generations. If a high concentration of streptomycin is added to the culture tube in which a sensitive strain is growing, the outcome depends upon the size of the population at the time. If the number of bacteria in the tube when the antibiotic is added is relatively small (a hundred or a thousand), multiplication will stop at once, and no further growth will take place in the tube, no matter how long it is incubated. If the population is large (a hundred million bacteria or more), the addition of streptomycin will arrest multiplication sharply, but incubation of the tube for a few days will almost always result in the ultimate appearance of a fully grown culture containing tens of billions of bacteria. When the bacteria in this culture are tested, they prove to be completely resistant to streptomycin, and are able to multiply vigorously in its presence. Further, we find that resistance to streptomycin is a stable, hereditary characteristic, transmitted indefinitely to the descendants of these bacteria.

Thus, by exposing a large population of streptomycin-sensitive bacteria to a high concentration of the antibiotic, the emergence of a genetically resistant strain can be brought about. This, indeed, is a strikingly adaptive change, and at first sight it may seem to substantiate the old idea that the environment can cause useful modifications that are then inherited. The careful study of the events leading to the appearance of a streptomycin-resistant strain proves without doubt that this is not so.

It can be readily demonstrated, first of all, that the adaptation to streptomycin does not come about by the mass conversion of the entire sensitive population, but rather is the result of the selective overgrowth of the culture by a few individuals that are able to multiply in its presence, while the division of the rest of the population is inhibited. It is for this reason that adaptation occurs only when the exposed population is large enough to contain at least one such individual. The critical question is this: how did these rare individuals acquire the properties that enabled them and their descendants to multiply in the presence of streptomycin?

This question has deep roots in biological controversy. It recalls, in a new form, the arguments over Lamarck's idea that modifications of the individual caused by environment can be inherited by descendants. Although Lamarckism has long since been disproved to the satisfaction of most biologists by repeated demonstrations that such inheritance just doesn't happen, the idea has persisted in bacteriology until very recently that microorganisms are somehow quite different from other plants and animals, and that permanent hereditary changes of an adaptive kind can be produced in bacteria directly as a result of the action of the conditions of life.

Two alternative hypotheses can be considered in planning experiments to determine the true origin of streptomycin-resistant variants. The first is that a small number of initially sensitive bacteria were modified as a direct result of the action of streptomycin, thereby acquiring permanent resistance. This would be an example of an adaptive hereditary change caused by the environment, as Darwin envisaged the origin of most hereditary variations. The second possibility is that the resistant individuals had already acquired the properties necessary for resistance before coming into contact with streptomycin, as a result of a mutation during the normal division of the sensitive population. In this case, the role of the antibiotic would be entirely passive, providing conditions that favor selectively the multiplication of those rare individuals present in the population that are already equipped, by virtue of the previous occurrence of a chance rearrangement of a particular gene, to withstand its inhibitory action.

During the past fifteen years, a great many experiments have been designed and conducted in a number of laboratories for the purpose of determining which of these hypotheses is correct. They have established beyond doubt that the second one is right, and that streptomycin-resistant variants originate by mutation, at a very low rate, during the growth of sensitive strains that have never been exposed to streptomycin. The proof depends upon the demonstration that the very first generation of resistant individuals in a culture, to which streptomycin has just been added already consists of related family groups, or clones, in just the way that would be predicted if their resistance were the consequence of a hereditary change that had taken place some generations back.

The development of resistance to streptomycin illustrates the way in which mutations provide the basis for adaptive changes in bacterial populations. Actually, any culture of Escherichia coli, apparently quite homogeneous when hundreds or even thousands of bacteria are compared, contains within it rare variants that differ from the predominant type in one or more of countless ways. When a suitable selective environment is provided, it can be shown that a culture contains mutants resistant to many antibiotics, to the action of radiation, to all sorts of chemicals that inhibit particular steps in metabolism — mutants that differ from the standard type in the sugars they can ferment, in their rate of growth, in the complexity of their nutritional requirements, in their antigenic properties, and in almost any characteristic for which a method of detection can be found.

In every case that has been carefully studied, these differences are found to originate without any contact with the conditions under which they happen to be advantageous, and their rates of occurrence are ordinarily not increased by such contact. This is true not only in bacterial cultures, where mutations can be demonstrated rapidly and dramatically. Natural populations of other plants and animals, including man, are known to contain mutations of many kinds that occur with no apparent causal relation to the conditions of growth.

Thus, in a way that Darwin could not have surmised, chance, through mutation, plays a most important part in evolution. It would be difficult indeed to imagine how a species could long survive, or progress in evolution, if it were dependent for its flexibility upon variations directly caused by the conditions of life. Quite aside from the fact that modifications produced in this way are not inherited, except in very special cases, it would require the intervention of some purposive and prescient agent to guarantee that previously unencountered conditions could typically provoke in the organism just those responses that are required to enhance adjustment.

Of course, the occurrence of a diversity of mutations in populations of bacteria and other organisms does not necessarily equip them to meet successfully every environmental challenge. Some strains of bacteria, for instance; are unable to adapt to streptomycin, since their spectrum of mutations does not include the particular modification of metabolism that is required for streptomycin resistance. Furthermore, since there are limits to the range of conditions that can support life, any sufficiently drastic changes, such as those that would take place in the center of a hydrogen bomb explosion, are not likely to prove conducive to the survival of any living thing.

Even within the range of more tolerable conditions, the suddenness of change is sometimes more decisive than its magnitude. For example, the bacterium Escherichia coli can be made resistant to streptomycin, penicillin, and chloromycetin, if the mutants resistant to each of these antibiotics are selected sequentially, but such a triply resistant strain cannot be obtained if the sensitive strain is exposed simultaneously to all three agents. This is explained by the negligible probability that any one individual in a finite population will have undergone mutation in three particular genes, each of which mutates very infrequently and independently of the others.

Observations of this kind, incidentally, although originally made in laboratories of genetics,, have found important applications in medical practice. Many people who have used antibiotics to combat infection have had the experience of dramatic relief of symptoms, only to be followed within a few days by a recurrence, this time failing to respond to the same antibiotic. Sometimes this can be explained by selection of a variant, present in the infecting population of bacteria, that is resistant to the antibiotic and that has its chance to multiply once the sensitive population is eliminated by the first round of treatment. In some cases, a physician will recommend the use of a combination of two or more unrelated antibiotics simultaneously, knowing that mutants resistant to more than one such drug are much less likely to be present. While the use of combinations of antibiotics is not always feasible for medical reasons, under certain conditions it has effectively prevented the occurrence of relapses caused by selection of resistant variants.

There is, of course, much more involved in the complicated saga of evolution than the simple picture of mutation and selection that accounts for bacterial adaptation to streptomycin. Nevertheless, the continuity of life from its first stirrings, and its steady progress toward higher levels of organization, has depended, and continues to depend, upon the reservoir of adaptive responsiveness that is provided initially by the mutations of genes.

Why, it may be asked, if mutations are the source of evolutionary progress, do we hear so much about the genetic dangers of radioactive fall-out, overexposure of the reproductive organs to clinical radiations, and the heightened radiation levels of the atomic age? We know that radiations increase considerably the frequency with which mutations of all sorts occur. Mutations, in themselves, are neither good nor bad. Streptomycin resistance is good for Escherichia coli in the presence of streptomycin, but when the antibiotic is removed, many of the resistant mutants are unable to grow, some of them actually requiring streptomycin for growth. Similarly, radiation-resistant mutants are at a distinct advantage in the presence of ultraviolet light or X rays, yet, in competition with the sensitive form when no radiation is present, they die out rapidly. At any stage in the history of a species, under natural conditions, the mutations that are occurring have undoubtedly occurred before, and most of those that are advantageous under the conditions then prevailing have already been established as part of the predominant gene complex. Thus most mutations are bound to be harmful in some way; the most frequently occurring mutations in the fruit fly are known to be those having lethal effects. Increased mutation rates as a result of exposure to unnatural amounts of radiation, therefore, are likely to be injurious, not only to the individual progeny of particular people, but to the vigor of mankind.

While the genetic hazards of radiation are of most immediate concern, there are more positive implications of the new knowledge of genetics and evolution for the future of humanity. The degree of control that has been achieved over environmental forces, and over the constitutional infirmities that would otherwise reduce the chances of survival and procreation of a significant segment of mankind, has already weakened the hitherto unchallenged power of natural selection. If man should one day choose to put to use the far greater power of his conscious and purposeful intervention, his biological future will be shaped by. his own hands. There are still undreamed-of possibilities in the multipotent clay that is his to mold.