by Isaac Asimov
Beadle and his co-workers went on to use this sort of information to study the relation of genes to the chemistry of metabolism. There was a way to show, for instance, that more than one gene is involved in the making of arginine. For simplicity’s sake, let us say there are two—gene A and gene B—responsible for the formation of two different enzymes, both of which are necessary for the synthesis of arginine. Then a mutation of either gene A or gene B will rob Neurospora of the ability to make the amino acid. Suppose we irradiate two batches of Neurospora and produce an arginineless strain in each one. If we are lucky, one mutant may have a defective A gene and a normal B gene; the other, a normal A and defective B. To see if that has happened, let us cross the two mutants at the sexual stage of their life cycle. If the two strains do indeed differ in this way, the recombination of chromosomes may produce some spores whose A and B genes are both normal. In other words, from two mutants that are incapable of making arginine, we will get some offspring that can make it. Sure enough, exactly that sort of thing happened when the experiments were performed.
It was possible to explore the metabolism of Neurospora in finer detail than this. For instance, here were three different mutant strains incapable of making arginine on an ordinary medium. One would grow only if it was supplied with arginine itself. The second would grow if it received either arginine or a very similar compound called citrulline. The third could grow on arginine or citrulline or still another similar compound called ornithine.
What conclusion would you draw from all this? Well, we can guess that these three substances are steps in a sequence of which arginine is the final product. Each requires an enzyme, First, ornithine is formed from some simpler compound with the help of an enzyme; then, another enzyme converts ornithine to citrulline; and finally, a third enzyme converts citrulline to arginine. Now a Neurospora mutant that lacks the enzyme for making ornithine but possesses the other enzymes can get along if it is supplied with ornithine, for from it the spore can make citrulline and then the essential arginine, Of course, it can also grow on citrulline, from which it can make arginine, and on arginine itself. By the same token, we can reason that the second mutant strain lacks the enzyme needed to convert ornithine to citrulline. This strain therefore must be provided with citrulline, from which it can make arginine, or with arginine itself. Finally, we can conclude that the mutant that will grow only on arginine has lost the enzyme (and gene) responsible for converting citrulline to arginine.
By analyzing the behavior of the various mutant strains they were able to isolate, Beadle and his co-workers founded the science of chemical genetics.
They worked out the course of synthesis of many important compounds by organisms, Beadle proposed what has become known as the one-gene-one-enzyme theory—that is, that every gene governs the formation of a single enzyme—a suggestion that is now generally accepted by geneticists, For their pioneering work, Beadle and Tatum shared in the Nobel Prize in medicine and physiology in 1958.
ABNORMAL HEMOGLOBIN
Beadle’s discoveries put biochemists on the qui vive for evidence of gene-controlled changes in proteins-particularly in human mutants, of course. A case turned up, unexpectedly, in connection with the disease called sickle-cell anemia, one of the more than 1600 genetic diseases in human beings.
This disease had first been reported in 1910 by a Chicago physician named James Bryan Herrick. Examining a sample of blood from a black teenage patient under the microscope, Herrick found that the red cells, normally round, had odd, bent shapes, many of them resembling the crescent shape of a sickle, Other physicians began to notice the same peculiar phenomenon, almost always in black patients, Eventually investigators decided that sickle-cell anemia is a hereditary disease, It follows the Mendelian laws of inheritance: apparently there is a sickle-cell gene that, when inherited in double dose from both parents, produces these distorted red cells, Such cells are unable to carry oxygen properly and are exceptionally short-lived, so there is a shortage of red cells in the blood, Those who inherit the double dose tend to die of the disease in childhood, On the. other hand, when a person has only one sickle-cell gene, from one of his parents, the disease does not appear. Sickling of his red cells shows up only when the person is deprived of oxygen to an unusual degree, as at high altitudes, Such people are considered to have the sickle-cell trait; but not the disease,
It was found that about 9 percent of the black people in America have the trait, and 0.25 percent have the disease. In some localities in Central Africa, as much as a quarter of the black population shows the trait. Apparently the sickle-cell gene arose as a mutation in Africa and has been inherited ever since by individuals of African descent. If the disease is fatal, why has the defective gene not died out? Studies in Africa during the 1950s turned up the answer. It seems that people with the sickle-cell trait tend to have greater immunity to malaria than do normal individuals. The sickle cells are somehow inhospitable to the malarial parasite. It is estimated that, in areas infested with malaria, children with the trait have a 25 percent better chance of surviving to childbearing age than have those without the trait. Hence, possessing a single dose of the sickle-cell gene (but not the anemia-causing double dose) confers an advantage. The two opposing tendencies—promotion of the defective gene by the protective effect of the single dose, and elimination of the gene by its fatal effect in double dose-tend to produce an equilibrium that maintains the gene at a certain level in the population.
In regions where malaria is not an acute problem, the gene does tend to die out. In America, the incidence of sickle-cell genes among blacks may have started as high as 25 percent. Even allowing for a reduction to an estimated 15 percent by admixture with non-black individuals, the present incidence of only 9 percent shows that the gene is dwindling away. In all probability it will continue to do so, If Africa is freed of malaria, the gene will presumably dwindle there, too.
The biochemical significance of the sickle-cell gene suddenly came into prominence in 1949 when Linus Pauling and his co-workers at California Institute of Technology (where Beadle also was working) showed that the gene affects the hemoglobin of the red blood cells: persons with a double dose of the sickle-cell gene are unable to make normal hemoglobin, Pauling proved this by means of the technique called electrophoresis, a method that uses an electric current to separate proteins by virtue of differences in the net electric charge on the various protein molecules. (The electrophoretic technique was developed by the Swedish chemist Arne Wilhelm Kaurin Tiselius, who received the Nobel Prize in chemistry in 1948 for this valuable contribution.) Pauling, by electrophoretic analysis, found that patients with sickle-cell anemia had an abnormal hemoglobin (named hemoglobin S), which could be separated from normal hemoglobin. The normal kind was given the name hemoglobin A (for “adult”) to distinguish it from a hemoglobin in fetuses, called hemoglobin F.
Since 1949, biochemists have discovered other abnormal hemoglobins besides the sickle-cell one, and they are lettered from hemoglobin C to hemoglobin M. Apparently, the gene responsible for the manufacture of hemoglobin has been mutated into many defective alleles, each giving rise to a hemoglobin that is inferior for carrying out the functions of the molecule in ordinary circumstances but perhaps helpful in some unusual condition. Thus, just as hemoglobin S in a single dose improves resistance to malaria, so hemoglobin C in a single dose improves the ability of the body to get along on marginal quantities of iron.
Since the various abnormal hemoglobins differ in electric charge, they must differ somehow in the arrangement of amino acids in the peptide chain, for the amino-acid make-up is responsible for the charge pattern of the molecule. The differences must be very small, because the abnormal hemoglobins all function as hemoglobin after a fashion. The hope of locating the difference in a huge molecule of some 600 amino acids was correspondingly small. Nevertheless, the German-American biochemist Vernon Martin Ingram and co-workers tackled the problem of the chemistry of the abnormal hemoglobins.
/> They first broke down hemoglobin A, hemoglobin S, and hemoglobin C into peptides of various sizes by digesting them with a protein-splitting enzyme. Then they separated the fragments of each hemoglobin by paper electrophoresis—that is, using the electric current to convey the molecules along a moistened piece of filter paper instead of through a solution. (We can think of this as a kind of electrified paper chromatography.) When the investigators had done this with each of the three hemoglobins, they found that the only difference among them was that a single peptide turned up in a different place in each case.
They proceeded to break down and analyze this peptide. Eventually they learned that it was composed of nine amino acids, and that the arrangement of these nine was exactly the same in all three hemoglobins except at one position. The respective arrangements were:
Hemoglobin A: His-Val-Leu-Leu-Thr-Pro-Glu-Glu-Lys
Hemoglobin S: His-Val-Leu-Leu-Thr-Pro-Val-Glu-Lys
Hemoglobin C: His-Val-Leu-Leu-Thr-Pro-Lys-Glu-Lys
As far as could be told, the only difference among the three hemoglobins lay in that single amino acid in the seventh position in the peptide: it was glutamic acid in hemoglobin A, valine in hemoglobin S, and lysine in hemoglobin C. Since glutamic acid gives rise to a negative charge, lysine to a positive charge, and valine to no charge at all, it is not surprising that the three proteins behave differently in electrophoresis. Their charge pattern is different.
But why should so slight a change in the molecule result in so drastic a change in the red cell? Well, the normal red cell is one-third hemoglobin A.
The hemoglobin A molecules are packed so tight in the cell that they barely have room for free movement. In short, they are on the point of precipitating out of solution. Part of the influence that determines whether a protein is to precipitate out is the nature of its charge. If all the proteins have the same net charge, they repel one another and keep from precipitating. The greater the charge (that is, the repulsion), the less likely the proteins are to precipitate. In hemoglobin S the intermolecular repulsion may be slightly less than in hemoglobin A, and hemoglobin S is correspondingly less soluble and more likely to precipitate. When a sickle cell is paired with a normal gene, the latter may form enough hemoglobin A to keep the hemoglobin S in solution, though it is a near squeak. But when both of the genes are sickle-cell mutants, they will produce only hemoglobin S. This molecule cannot remain in solution. It precipitates out into crystals, which distort and weaken the red cell.
This theory would explain why the change of just one amino acid in each half of a molecule made up of nearly 600 is sufficient to produce a serious disease and the near-certainty of an early death.
METABOLIC ABNORMALITY
Albinism and sickle-cell anemia are not the only human defects that have been traced to the absence of a single enzyme or the mutation of a single gene. There is phenylketonuria, a hereditary defect of metabolism, which often causes mental retardation and results from the lack of an enzyme needed to convert the amino acid phenylalanine to tyrosine. There is galactosemia, a disorder causing eye cataracts and damage to the brain and liver, which has been traced to the absence of an enzyme required to convert a galactose phosphate to a glucose phosphate. There is a defect, involving the lack of one or another of the enzymes that control the breakdown of glycogen (a kind of starch) and its conversion to glucose, which results in abnormal accumulations of glycogen in the liver and elsewhere and usually leads to early death. These are examples of inborn errors of metabolism, a congenital lack of the capacity to form some more or less vital enzyme found in normal human beings. This concept was first introduced to medicine by the English physician Archibald Edward Garrod in 1908, but it lay disregarded for a generation until, in the mid-1930s, the English geneticist John Burdon Sanderson Haldane brought the matter to the attention of scientists once more.
Such disorders are generally governed by a recessive allele of the gene that produces the enzyme involved. When only one of a pair of genes is defective, the normal one can carry on, and the individual is usually capable of leading a normal life (as in the case of possessor of the sickle-cell trait). Trouble generally comes only when two parents happen to have the same unfortunate gene and have the further bad luck of combining those two in a fertilized egg. Their child, then, is the victim of a kind of Russian roulette. Probably all of us carry our load of abnormal, defective, even dangerous genes, usually masked by normal ones. You can understand why the human geneticists are so concerned about radiation or anything else that may increase the mutation rate and add to the load.
Nucleic Acids
The really remarkable thing about heredity is not these spectacular, comparatively rare aberrations, but the fact that, by and large, inheritance runs so strictly true to form. Generation after generation, millennium after millennium, the genes go on reproducing themselves in exactly the same form and generating exactly the same enzymes, with only an occasional accidental variation of the blueprint. They rarely fail by so much as the introduction of a single wrong amino acid in a large protein molecule. How do they manage to make true copies of themselves over and over again with such astounding faithfulness?
The answer must lie in the chemistry of the long strings of genes that we call chromosomes. One major portion of the chromosomes, about half of its mass, is made up of proteins. This is no surprise. As the twentieth century wore on, biochemists expected any complex bodily function to involve proteins. Proteins seemed to be the complex molecules of the body, the only ones complex enough to represent the versatility and sensitivity of life.
And yet, a major portion of chromosomal proteins belonged to a class called histone, whose molecules are rather small for a protein and (worse yet) made up of a surprisingly simple mix of amino acids. They did not seem nearly complicated enough to be responsible for the delicacies and intricacies of genetics. To be sure, there were nonhistone protein components that were made up of much larger and more complex molecules, but they amounted to but a minor portion of the whole.
Nevertheless, biochemists were stuck with the proteins. Surely, the mechanism of heredity could involve nothing else. About half the chromosome consisted of material that was not protein at all, but it did not seem possible that anything that was not protein would suit. Still, it is to this nonprotein constituent of chromosomes that we must turn.
GENERAL STRUCTURE
In 1869, a Swiss biochemist named Friedrich Miescher, while breaking down the protein of cells with pepsin, discovered that the pepsin did not break up the cell nucleus. The nucleus shrank a bit, but remained intact. By chemical analysis, Miescher then found that the cell nucleus consisted largely of a phosphorus-containing substance whose properties did not at all resemble protein. He called the substance nuclein. It was renamed nucleic acid twenty years later when it was found to be strongly acid.
Miescher devoted himself to a study of this new material and eventually discovered sperm cells (which consist almost entirely of nuclear material) to be particularly rich in nucleic acid. Meanwhile, the German chemist Felix Hoppe-Seyler, in whose laboratories Miescher had made his first discovery, and who had personally confirmed the young man’s work before allowing it to be published, isolated nucleic acid from yeast cells. This seemed different in properties from Miescher’s material, so Miescher’s variety was named thymus nucleic acid (because it could be obtained with particular ease from the thymus gland of animals), and Hoppe-Seyler’s, naturally, was called yeast nucleic acid. Since thymus nucleic acid was at first derived only from animal cells and yeast nucleic acid only from plant cells, it was thought for a while that this might represent a general chemical distinction between animals and plants.
The German biochemist Albrecht Kossel, another pupil of Hoppe-Seyler, was the first to make a systematic investigation of the structure of the nucleic-acid molecule. By careful hydrolysis, he isolated from it a series of nitrogen-containing compounds, which he named adenine, guanine, cytosine, and thymine. Their formulas are
now known to be:
The double-ring formation in the first two compounds is called the purine ring, and the single ring in the other two is the pyrimidine ring. Therefore, adenine and guanine are referred to as purines, and cytosine and thymine are pyrimidines.
For these researches, which started a fruitful train of discoveries, Kossel received the Nobel Prize in medicine and physiology in 1910.
In 1911, the Russian-born American biochemist Phoebus Aaron Theodore Levene, a pupil of Kossel, carried the investigation a stage further. Kossel had discovered, in 1891, that nucleic acids contain carbohydrate, but now Levene showed that the nucleic acids contain five-carbon sugar molecules. (This was, at the time, an unusual finding: the best-known sugars, such as glucose, contain six carbons.) Levene followed this discovery by showing that the two varieties of nucleic acid differ in the nature of the five-carbon sugar. Yeast nucleic acid contains ribose, while thymus nucleic acid contains a sugar that is very much like ribose except for the absence of one oxygen atom, and so was called deoxyribose. Their formulas are:
In consequence, the two varieties of nucleic acid came to be called ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
Besides the difference in their sugars, the two nucleic acids also differ in one of the pyrimidines. RNA has uracil in place of thymine. Uracil is very like thymine, however, as you can see from the formula:
By 1934, Levene was able to show that the nucleic acids could be broken down to fragments that contain a purine or a pyrimidine, either the ribose or the deoxyribose sugar, and a phosphate group. This combination is called a nucleotide. Levene proposed that the nucleic-acid molecule is built up of nucleotides as a protein is built up of amino acids. His quantitative studies suggested to him that the molecule consists of just four nucleotide units, one containing adenine, one guanine, one cytosine, and one either thymine (in DNA) or uracil (in RNA).
