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Much of our present understanding of the composition, structure, and function of genes has come from the joining of previously disparate work in microbiology, biochemistry, and genetics, a fusion that marked the emergence of molecular biology in the 1940s.(1) Since the 1940s, investigators seeking to elucidate gene function have turned by-and-large from the morphological and biochemical complexities of the fruit fly, mice, and maize, materials they had used to unravel problems of gene transmission, to simpler microbial and viral materials. To contemporary geneticists and molecular biologists, aware of the infinitely greater complexities of the human organism, it thus seems "remarkable" and "amazing" that, stemming from work on a human hereditary disorder, sickle cell anemia, "the genetically controlled protein system about which most is known is human hemoglobin" (Sutton 1961, p. 50).
In this chapter we will see how "the existence of mutant proteins in humans afflicted with sickle cell anemia proved to be the critical key in the elucidation of the nature of the genetic apparatus" (Handier 1970, p. 664). As in our other case studies, it is a story in which a clinical problem feeds into and affects the course of a stream of biological research, a stream which is fed by many sources and which in turn has numerous branches. It also is a story that relates how much we have learned from what the British physician, Sir Archibald Garrod, called "the lesson of rare maladies," the inherited abnormalities that provide researchers with a "natural experiment." In this case, the natural experiment, sickle cell anemia, led to the concept of "molecular disease" and thence to a frontal assault on the question of how genes control protein structure. And, in delineating how gene mutations alter the structure of the human hemoglobin molecule, researchers have made major strides in resolving one of the oldest and most difficult problems in genetics, the problem of allelism. (Alleles are alternative or variant forms of genes which occur at the same locus or place on two members of a pair of homologous chromosomes.) "The problem of allelism," as Levitan and Montagu explain, "arises whenever two traits are found that affect the same cell, tissue, or organ system. . . [For example] every time a new red blood cell antigen, or blood type is discovered, the question arises as to whether it represents a further complexity of a known system (and thus probably conditioned by a new allele at a previously discovered locus) or whether it is a new system conditioned by still another independent locus. This problem goes to the very core of genetics. We are in effect looking for evidence to delineate the unit factor of genetics, the gene" (Levitan and Montagu 1971, p. 549).
Conceptually, the bearing of sickle cell research on our understanding of the function of genes is indirectly but inextricably linked with the ideas of Sir Archibald Garrod (1857-1910) concerning the biochemical nature of gene action. Garrod, who died the same year that the first clinical report of sickle cell anemia was published, worked in that post-Mendelian era when genetics and biochemistry were pursued largely as separate endeavors. When those fields were joined, his clinically-based concept of inborn errors of metabolism would be rediscovered and reenunciated as the one gene-one enzyme concept, on the basis of research with the bread mold, Neurospora. Then, in the 1950's, once again growing out of a clinical framework, this time provided by sickle cell anemia, GarrGd's ideas would find their modern echo in the one gone-one polypeptide concept of the genetic control of protein structure.
In 1902 Garrod, an Oxtord-trained pathologist, published the first of a series of papers on alkaptonuria, a disorder manifested clinically by a blackening of the urine on exposuro to air. Six years later, in the Royal Society's Croonian Lectures, Garrod summarized his first decade of researches on alkaptonuria and three other human disorders, albinism, cystinuria, and pentosuria, four 'rare maladies" that he tern-wd "irA)orn errors of metabolism." In his lectures, published in 1909 with a second edition in 1923, Garrod "clearly and [END OF PAGE 73] explicitly interpreted [alkaptonurial] and the other 'inborn errors of metabolism' as blocks at specific points in the normal pattern of intermediary metabolism, where some specific enzyme, normally present, was absent because of alteration of the controlling gene" (Glass 1965, p. 231).
Garrod, a physician thoroughly trained in the biological and chemical sciences of his time, drew upon and uniquely joined contemporary researches in protein biochemistry and in the newly rediscovered science of Mendelian genetics. As he pointed out in the first of his 1908 Croonian lectures:
The great strides which recent years have witnessed in the sciences of chemical physiology and pathology, the newly-acquired knowledge of the constitution of proteins and of the part played by enzymes in connexion with the chemical changes brought about within the organism, have profoundly modified our conceptions of the nature of the metabolic processes . . . The conception of metabolism in block is giving place to that of metabolism in compartments. The view is daily gaining ground that each successive step in the building up and breaking down, not merely of proteins, carbohydrates, and fats in general, but even of individual fractions of proteins and of individual sugars, is the work of special enzymes set apart for each particular purpose. (Garrod 1909, pp. 4-6)
In introducing his audience to the anomalies "which may be classed together as inborn errors of metabolism," Garrod first distinguished them from "diseases of metabolism" such as diabetes, gout, and obesity. "The liability to develop diabetes or gout is often inherited, but the diseases themselves are not inherited for they are never congenital. Developing at any period of life, the mischief, once begun, tends to become aggravated as time goes on, but the rate of aggravation differs widely in individual cases and is often conspicuously controlled by appropriate treatment" (Garrod 1909, p. 131). In contrast to these diseases, Garrod explained some of the inborn errors of metabolism "are certainly and all of them are probably, present from birth.' Clinically, he stated,
The chemical error pursues an even course and shows no tendency to become aggravated as time goes on. With one exception they bring influenced by any therapeutic measures at our disposal. Yet they are characterized by wide departures from the normal of the species far more conspicuous than any ordinary individual variations, and one is tempted to regard them as metabolic sports, the chemical analogues of structural malformations. (Garrod 1909, pp, 13-14)
Today's thick textbooks devoted to inborn errors of metabolism bear witness to Garrod's insight in 1908 that "among the complex metabolic processes of which the human body is the seat there is room for an almost countless variety of such [metabolic] sports." But he acknowledged that in his own day there was adequate evidence for classifying only four anomalies as inborn errors of metabolism.
Of the four, alkaptonuria was Garrod's most comprehensively analyzed inborn error of metabolism. For a substantial body of clinical, biochemical, and hereditary information about alkaptonuria existed by 1908, including Garrod's own investigations, which he uniquely synthesized and interpreted. The blackened urine of alkaptonuria was known to be caused by the excretion of homogentisic or "alkapton" acid, a substance formed in the body from tyrosine and phenylalanine. Garrod recognized that "two explanations are possible of the fact that alkaptonurics excrete homogentisic acid, whereas normal persons do not. Either the alkapton acid is a strictly abnormal product formed by a perverted metabolism of tyrosin and phenyl-alanine . . . or it is an intermediate product of normal metabolism which is usually completely destroyed and does not come to excretion, but which in alkaptonuria escapes destruction" (Garrod 1909, p. 66). Garrod then documented his reasons for concluding that the second of these hypotheses was the correct one. Homogentisic acid, he argued, is a product of normal metabolism, but the alkaptonuric, unlike the normal individual, lacks the "power of destroying homogentisic acid when formed - in other words of breaking up the benzene ring of that compound" (Garrod 1909, p. 79). This inability, Garrod correctly speculated, was due to the absence or inactivity of an enzyme that normally catalyzes the breakdown of homogentisic acid.(2) "We may further conceive," he wrote, "that the splitting of the benzene ring in normal metabolism is the work of [END OF PAGE 74] a special enzyme, that in congenital alkaptonuria this enzyme is wanting, whilst in disease its working may be partially or even completely inhibited" (Garrod 1909, p. 80).
Garrod's recognition that the metabolic defect in alkaptonuria represented an inborn or inherited condition, in turn, was based on familial patterns of the disease's occurrence. From familial studies, Garrod found that the four metabolic disorders he was analyzing had "the liability for each of them to occur in several members of a family, most often in collaterals of the same generation, born of normal parents" Garrod 1909, p. 22). An explanation for the inborn error's mode of inheritance within such families was provided by William Bateson, who was responsible for having Mendel's paper translated into English, was the first to demonstrate Mendelian inheritance in animals, and who introduced such terms as "genetics" and "allelomorph." Drawing upon Bateson's 1902 report to the Evolution Committee of the Royal Society on "The Facts of Heredity in The Light of Mendel's Discovery," Garrod concluded that "the mode of incidence of alkaptonuria finds a ready explanation if the anomaly in question be regarded as a rare recessive character in the Mendelian sense" (Garrod 1909, p. 26). In Inborn Errors, he examined at length the Mendelian nature of alkaptonuria as a recessive trait, and also presented evidence for pentosuria, cystinuria, and albinism as metabolic defects due to the recessively inherited absence of a single enzyme. "To the students of heredity," Garrod pointed out a scant eight years after the rediscovery of Mendel's work, "the inborn errors of metabolism offer a promising field of investigation." However, he wisely cautioned those who would enter the arena of medical genetics, "their adequate study from this [genetic] point of view is beset with many difficulties" (Garrod 1909, p. 22).
When Garrod delivered his Croonian Lectures in 1908, the prevailing concept of gene action offered by the nascent science of genetics was "one gene-one character." As Bentley Glass observes, Garrod's pioneering foray into biochemical genetics represented two major steps forward from this early understanding of gene action: "from recognition of the relation of the specific altered gene to the particular blocked step in the metabolic pattern, and thence to the lack of the specific enzyme governing that pattern" (Glass 1965, p. 233). But Garrod might well have echoed Gregor Mendel's bitter words, "Meine Zeit wird schon kommen" [my time will surely come] , for the import of the British physician's work, like that of the Austrian monk's, would be unrecognized by geneticists, and by biochemists, for several decades.
For those interested in understanding the processes of biomedical discovery, innovation, and diffusion, the question of why work such as Garrod's was "profoundly neglected" during his lifetime is as interesting and significant as the content of the work itself. As with other instances of scientific and clinical discoveries that underwent a long latency period before they were "rediscovered," there are multiple explanations of why Garrod's biochemical -genetic concept of gene action lay fallow for many years. One partial explanation, advanced by Garrod's principal rediscoverer, George Beadle, invokes the familiar argument that "the time was not ripe." Because Garrod was ahead of his time, Beadle argued, neither biochemists nor geneticists would take his concept seriously. Further, Beadle wrote, "I strongly suspect that an important common component of the unfavorable climate for receptiveness in these two instances [Mendel and Garrod] is the persistent feeling that any simple concept in biology must be wrong" (Beadle 1966, p. 31).
Other, interrelated explanations have been presented by geneticist and historian Bentley Glass, who also has studied the reception of Mendel's work. Glass argues that Garrod's lectures on human heredity must have been known to geneticists, as was Mendel's 1865 paper in its time. But Garrod, a physician, was presenting studies in biochemical genetics in an era when genetics and biochemistry were separate fields of inquiry. As late as 1946, Beadle still felt the need to urge the fusion that Garrod had personified.
If the maximum possible understanding of what the organism is and what it does is to be obtained, it is clear that our approach must be made from many sides. . the chemist cannot understand what the organism does chemically without considering genes. Therefore, the methods of genetics, which are biological - not chemical, must supplement those of chemistry. In the same way, a biologist would be blind if he were to ignore chemistry in his attempts to understand how the organism is built and how it functions. Biochemical genetics represents an approach in which the cooperation of two disciplines is essential. (Beadle 1946, p. 53) [END OF PAGE 75]
Futher, as Garrod recognized, research into clincal inborn metabolic disorders presented many promem!, for the geneticist, Thus, "there was a common attitude among geneticists who worked with Drosophila or maize or other experimental organisms that human heredity was refractory to analysis and that little basic insight could be gained from studies of hereditary abnormalities in an organism that could not be bred at will" (Glass 1965, P. 232).
Compounding the problems of receptivity created by a physician working in human biochemical genetics, Glass continues, was the relative lack of direct interest in enzymes among geneticists. There were relevant Lines of research pursued by a small group of geneticists, particularly on the topics of flower pigmentation and the fermentation of sugars by yeasts, and by 1940 these researches had "established that many biochemical reactions are, in fact, controlled in specific ways by genes" (Beadle and Tatum 1941, p. 499). But how this genetic control is exerted was unclear, and there was debate as to whether enzymes were genes themselves or merely products of genes, An answer had been proffered by Garrod in 1908, but most of those working through the early 1940s were either unaware of Inborn Errors or, if they had read it, failed to see the signifi- cance of Garrod's concept.(3)
For multiple reasons, then, it was not until the mid1940's that Garrod's concept of gene action - one gene/one metabolic block/one enzyme deficiency would be "rediscovered" and clearly enunciated as the one gene-one enzyme hypothesis. The name linked most closely with this rediscovery is George W. Beadle, who in the 1930s and 1940s conducted Nobel Prize winning researches on how genes act on development. Beadle's first researches, conducted with Boris Ephrussi, were carried out on eye pigments of Drosophila. The hypothesis that he and Ephrussi formulated to explain their initially unexpected experimental results, Beadle later reflected, "was a scheme closely similar to that proposed by Garrod for alkaptonuria . . . But at the time we were oblivious of Garrod's work, partly because geneticists were not in the habit of referring to it, and partly through failure of ourselves to explore the literature. Garrod's book was available in many libraries" (Beadle 1958, p. 592).
In 1937, Beadle, now working at Stanford, was joined by a young biochemist, Edward L. Tatum. They found that "isolating the eye-pigment precursors of Drosophila was a slow and discouraging job," and "realized this was likely to be so in most cases of attempting to identify the chemical disturbances underlying inherited abnormalities; it would be no more than good fortune if any particular example chosen for investigation should prove to be simple chemically" (Beadle 1958, p. 594).
Thus, Beadle and Tatum realized, the biochemical study of gene function required a new approach and new biological material, something less morphologically complex than the fruit fly or other diploid organisms that had been suitable for defining the laws of genetic transmission. The new approach they selected, Beadle feels, was an "obvious one," but at the time it represented a "bold step" of "inverting the experimental attack on the problem [of gene action] . Instead of waiting for the accumulation of information necessary to understand the biochemical basis of the complex morphological mutants of the higher forms, attention was turned to simpler microbial material" (Handler 1970, p. 25).(4)
The organism chosen by Beadle and Tatum was the now famous bread mold, Neurospora crassa (see Beadle 1964; Tatum 1964; and Beadle and Tatum 1941). "it is sometimes thought," Beadle said in his 1958 Nobel Prize address, "that the Neurospora work was responsible for the 'one gene-one enzyme' hypothesis - the concept that genes in general have single primary functions, aside from serving an essential role in their own replication, and that in many cases this function is to direct specificities of enzymatically active proteins. The fact is that it was the other way around - the hypothesis was clearly responsible for the new approach" (Beadle 1958, pp. 596-597). For, as Beadle and others understood in retrospect, the one gene-one enzyme concept had been gradually albeit implicitly envolving in the work of many investigators since it was formulated by Garrod in 1908. Once he was aware of Garrod's work, Beadle realized that:
In this long and roundabout way, first in Drosophila and then in Neurospora, we had rediscovered what Gerrod had seen so clearly so many years before. By now we knew of his work and we were aware that we had added little if anything new in principle. We were working with a more favorable organism and were able to produce, almost at will, inborn errors of metabolism for almost any chemical reaction whose product we could supply through the medium. Thuswewereable to demonstrate that what Garrod had shown for a few genes and a few chemical reactions in man, was true for many genes and many reactions in Neurospora. (Beadle 1958, p. 526) [END OF PAGE 76]
At the time, no one could have foreseen the connection between Garrod's work, or the later researches of Beadle and Tatum, and the publication of a paper by Chicago physician James B. Herrick on "Peculiar and elongated sickle-shaped red blood corpuscles in a case of severe anemia." "This case is reported because of the unusual blood findings, no duplicate of which I have ever seen described," Herrick wrote in 1910. He had first seen his patient, a 20 year old black who had been raised in the West Indies, when he came to Herrick's hospital in 1904 with a cough, fever, weakness, dizziness, a headache and nasal discharge, a yellow tinge to the whites of his eyes, and a history of skin ulcerations. The physician's examination revealed one striking finding: a large number of red blood cells which, in contrast to normal red cells, had a nucleus and were oddly shaped.
Nucleated reds were numerous, 74 being seen in a count of 200 leukocytes, there being about 5,000 to the c.mm. The shape of the reds was very irregular, but what especially attracted attention was the large number of thin, elongated, sickle-shaped and crescent-shaped forms. . They were not seen in specimens of blood taken at the same time from other individuals and prepared under exactly similar conditions. They were surely not artifacts, nor were they any form of parasite . . . in the fresh specimen where there was a slight current in the blood before it had become entirely quiet, all of the red corpuscles, the elongated forms as well as those of ordinary forms, seemed to be unusually pliable and flexible, bending and twisting in a remarkable manner as they bumped against each other or crowded through a narrow space and seeming almost rubber-like in their elastic resumption of the former shape. One received the impression that the flattened red discs might by reason of unusual pliability be rolled up as it were into a long narrow bundle. Once or twice I saw a corpuscle of ordinary form turn in such a way as to be seen on edge, when its appearance was suggestive of these peculiar forms. (Herrick 1910, pp. 518-519)
After a month of treatment (rest, nourishment, and iron for his anemia) the patient was discharged. His condition was improved but the odd-shaped and nucleated red cells were still present. Herrick kept track of the patient until early 1907, recording his hospitalizations for problems such as "muscular rheumatism" and "bilious attacks." Clinically, Herrick acknowledged, "no conclusion can be drawn from this case." He and a colleague had unsuccessfully attempted to reproduce the types of red cells seen in the patient, which suggested to Herrick "that some unrecognized change in the composition of the corpuscle itself may be the determining factor." However, Herrick concluded, "the question of diagnosis must remain an open one unless reports of other similar cases with the same peculiar blood-picture shall clear up this feature" (Herrick 1910, P. 521).
Other reports were forthcoming, which confirmed and elaborated upon Herrick's clinical findings and, began to explore the possible etiology of the newly recognized disorder. A second puzzling case of severe anemia with sickle-shaped cells was reported in 1911 by Washburn, and a third in 1915 by two physicians from Washington University Medical School in St. Louis. "It will be seen that there is a striking similarity in both the blood-picture and the clinical history of our case with those of Herrick and Washburn," wrote Drs. Jerome Cook and Jerome Meyer. The three patients "were of Negro blood" and had "peculiar. . recurring leg ulcer," severe anemia, and a "peculiar discoloration of the sclerae" (the coating of the eyeball). "A glance at the blood slides leaves no doubt as to the identical character of the elongated and sickle-shaped red cells" (Cook and Meyer 1915, pp. 650, 651).
As had Herrick and Washburn, Cook and Meyer ruled out syphilis or a parasitic infection as causative agents of the strange disorder. They were able to suggest another possible explanation for the disease, however, based on investigations they had conducted with Dr. Victor Emmel, a colleague "who has made extensive studies of blood development and morphology."
The possibility of an inherited anomaly suggested itself. The fact that the three other children of the family had suffered from severe anemia encouraged investigation in this direction. Examination of the father's blood showed 4,500,000 red cells, 11,100 white cells, and 86 per cent hemoglobin... the stained smear showed none of the peculiarities of the daughter's blood. Dr. Emmel, however, found some resemblance in the behavior of the blood. When preparations of the fresh blood were [END OF PAGE 77] temperature, the microscope revealed long, snarp projections and elongations from many of the red cells in the blood of both the father and the daughter. Dr. Emmel has not found similar appearances in the blood of any other person. (Cook and Meyer 1915, p. 649)
In his observations of the father's blood, Emmel had seen the phenomenon of sickling in the otherwise norMal-looking red blood cells of carriers, a phenomenon that the physicians correctly interpreted as suggesting A hereditary disorder. Emmel's primary interest in this case was to explain the sickling process, and in a 1917 paper he reported in more detail on his studies of blood samples from Cook and Meyer's patient and her father. Based on these studies, and his knowledge of the development of the blood cells, he proposed a logically consistent hypothesis about the mechanism of red cell sickling. Knowing that undeveloped red blood cells within the bone marrow are spherical in shape, Emmel proposed that sicklinq is "in part due to an accentuated or abnormal activity of the same factor which in normal hernatogenesis are involved in the transformation of the original spherical erythrocyte [red blood cell] into a biconcave disc-shaped form" (Emmel 1917, p. 598).
While Emmel's hypothesis would be proved incorrect, it directed other investigators toward a fruitful line of research into the nature of the sickling process. In a major paper of 1927, E. V. Hahn and E. B. Gillispie reviewed the growing body of clinical literature on 11 sickle cell anemia" - a term introduced by Mason in 1922 - and reported an their own experimental study of sickle cell formation. Hahn and Gillispie set up a gas chamber apparatus under the microscope, along with red blood cell preparations from a patient with sickle cell anemia, and treated the cells with different gases. When oxygen or carbon monoxide was added to the preparation, the sickle cells immediately returned to the normal discoid shape. When carbon dioxide was added, it took only four minutes for all the normal discoid cells to sickle. In addition to this experiment, they also observed the red blood cells at various partial pressures of oxygen, and found that the normally shaped cells of a person with sickle cell trait sickled when oxygen tension fell below 45 millimeters, less than a third of normal oxygen.
The gas chamber experiment simulated different conditions of hemoglobin, the substance in red blood cells that in an oxygenated state combines with oxygen and transports it throughout the body, and in a deoxygenated state acts to remove carbon dioxide. Based on their study, Hahn and Gillispie were the first to suggest the role of hemoglobin in sickle cell anemia, which they too viewed as a hereditary disorder.
The red corpuscles of persons with the "sickle cell trait" are transformed into sickle cells in vitro as a result of asphyxia. The transformation takes place when the oxygen tension falls below a partial pressure of 45 mm. of mercury. Oxygen and carbon monoxide induce restoration of the discoid form.
All of the facts relating to sickle cells are consistent with a hypothesis that the sickle form is stable when the hemoglobin is dissociated, and that the discoid form is stable when the hemoglobin is combined...
Reasons exist for believing that the only specific cause for active sickle cell anemia is the unique hereditary anomaly of the red corpuscles which predisposes to it. (Hahn and Gillispie 1927, p. 254) (5)
While Cook, Meyer, and Emmel had proposed in 1915 that sickle cell anemia was hereditary, it would be more than 30 years before the correct mode of inheritance was worked out. In the interim, investigators often grouped together persons with sickle cell anemia and those who were only carriers of the disease, and the literature presented an understandably confusing array of terminology. Thus, for example, Hahn and Gillispie attempted to replace the "awkward" terms "sickle cell," "latent sickle cell anemia" (clinically healthy but with the inherited trait) and "active sickle cell anemia," with terms derived from the Greek word for "sickle": "drepanocyte," "drepanocytemia," and "drepanocytic-anemia" (Hahn and Gillispie 1927, p. 254).
A first attempt to explain the inheritance of sickle cell anemia in Mendelian terms was made in 1923 by J. G. Huck and W. H. Taliaferro. Based on their study of two families, and in the absence of differentiation between the trait and the disease, Huck and Taliaferro concluded that "sickle cell anemia in man is an inherited condition and behaves as a single Mendelian character which is dominant over the normal condition and which is not sex-linked" (Taliaferro and Huck 1923, p. 597; see also Huck 1923). [END OF PAGE 78]
Taliaferro and Huck's explanation of sickle cell anemia as due to the inheritance of a single dominant "character" or gene was widely accepted until the late 1940's, when it was disproven in the light of a more sophisticated understanding of homozygous and heterozygous modes of inheritance.(6) In the interim, researchers also sought to resolve the confusion between sickle cell anemia and sickle cell trait and to determine whether the trait and the disease were limited to the black population. A trio of investigators from University of Tennessee's Department of Clinical Pathology and the Nutritional Division of the University of Florida's Experimental Station clarified many of these questions about sickled cells through their survey of "normal and hospital negroes and white people" in the early 1930s. Based on their studies of over 8,000 individuals, and on other surveys reported in the literature, Diggs, Ahmann, and Bibb found that the incidence of the sickle cell trait among negroes was 7.3 percent, and that 1 in 40 of those with the trait also would exhibit sickle cell anemia. "The sickle cell trait," they reported, "has not been demonstrated in recorded surveys of white people, and the only reasonably proved instances of the sickle cell trait in families with unmixed blood have been limited to those of the Mediterranean stock" (Diggs et al. 1933, p. 777). Those exhibiting the sickle cell trait without severe anemia, Diggs and his colleagues emphasized, should not be considered as ill, for their observation showed that "the trait is compatible with long life" and "the incidence [of the trait] in hospital cases has not been proved to be higher than in healthy individuals." Thus, they concluded, "the importance of the sickle cell trait appears to be limited to the relatively small group who in addition to the trait have sickle cell anemia" (Diggs et al. 1933, p. 777). But why some of those who inherit the trait, "thought to be transmitted as a dominant Mendelian characteristic," also develop severe anemia remained a mystery "due to factors unknown" (Diggs et al. 1933, p. 769).
Why some persons had sickled cells but not severe anemia became clearer genetically in the late 1940's, when James V. Neel from the University of Michigan's Heredity Clinic demonstrated that sickled cells were not inherited as a dominant Mendelian characteristic. On the basis of Taliaferro and Huck's hypothesis, Neel pointed out in a 1949 paper, "the inference was that this [dominant] gene was more strongly expressed in some individuals (sickle cell anemia) than in others (sicklemia [or sickle cell traitl )" (Neel 1949, p. 64). In a 1947 paper reviewing the chemical detection of genetic carriers, however, Neel had suggested another mode of inheritance: "There is present in the colored population a certain factor which when heterzygous may have no discernible effects but usually results in sickling, and when homozygous tends to result in sickle cell anemia" (Neel 1947,p. 129).
Neel's interpretation of the inheritance of sickle cell anemia was generated by his study of another hereditary blood disorder, thalassemia or Cooley's anemia, a condition which would become significantly linked with sickle cell research in the 1950s. By the mid-1940s, clinicians and geneticists knew that thalassemia was found relatively frequently among persons living in, or descended from residents of, the Mediterranean countries, and that the condition could occur in either a severe or relatively mild form. In 1944, Neel and W. W. Valentine reported on their investigation of 34 parents, siblings, or immediate collaterals of three patients with thalassemia, and one with mild hereditary anemia. From their genetic and hematologic study, they suggested that the mild familial anemia was due to a heterozygous or carrier state, while severe thalassemia resulted from a homozygous inheritance from two carriers. The mild and severe forms of the disorder, they proposed, could be designated as thalassemia minor and thalassemia major (Valentine and Neel 1944).(7)
Reasoning from his analysis of thalassemia, Neel recognized that there was a relatively simple way to determine whether his hypothesis about the inheritance of sickle cell anemia was correct. "it the homozygousheterozygous hypothesis is correct, then both the parents of any patient with sickle cell anemia should always sickle . . . If, on the other hand, the disease is due to a dominant gene with variable expression, only one parent need sickle, although occasionally, due to the chance marriage of two sicklers, both parents may sickle." (Neel 1949, p. 64) Thus far, Neel reported, he had tested 42 parents of 29 patients with sickle cell anemia for the occurrence of sickling. Although only 13 couples were tested, the results were convincing: the blood of all 42 parents sickled, "This is the result expected from the homozgous-heterozygous hypothesis. On the other hand, the probability of the occurrence of such a number of positive parents under the variable dominant hypothesis is (0.76542 )42 or 0.000013" (Neel 1949, p. 65).(8)
Reviewing survey data from the U. S. and Africa on the incidence of sickling, Neel briefly discussed the subject [END OF PAGE 79] of gene frequency in sickle cell trait and disease. He then closed his short paper by noting that, given this new understanding of the genetic transmission of sickled cells, one could both accurately predict the disease's occurrence and, if so desired, greatly reduce its incidence.
In a genetic situation such as appears to obtain here, where the heterozygote, who may be termed the genetic carrier of the disease, may be readily distinguished from normal and from the homozygote, it is possible to predict with a high degree of accuracy which marriages should result in homozygous individuals - in this case, children with sickle cell anemia. Since (homozygous) individuals with sickle cell anemia either die young or, if they reach maturity, have a greatly lowered fertility, the vast majority of cases of the disease are the issue of marriages between two (heterozygous) persons with the sickle cell trait. In the absence of marriage between individuals whose erythrocytes exhibit the sickling phenomenon, the frequency of the homozygote would greatly decrease, and sickle cell anemia would tend to disappear, with only a very rare case arising as a result of mutation in a normal individual married to a person homozygous or heterozygous for the sickling gene. (Neel 1949, pp. 65-66)
The chain of researches that would unravel the puzzling question of how red blood cells sickle, and in so doing greatly clarity our understanding of how genes control protein structure, was set in motion on a spring night in 1945. At the time, chemist Linus Pauling was serving as a member of the Medical Advisory Committee that was assisting Vannevar Bush in the preparation of a report to the President on the state and direction of science at the close of World War 11. "One evening," Pauling later recalled, "Dr. William B. Castle, Professor of Medicine in Harvard University, mentioned to the other members of the committee the disease sicklecell anemia, with which he had had some experience. He told about the discovery of the disease by Dr. J. B. Herrick in 19 10, and described the characteristic change in shape of the red corpuscles and the effect of oxygen in preventing the sickling and of carbon dioxide in accelerating it" (Pauling 1955, p. 216).
Castle's remarks immediately caught Pauling's attention, for in 1935, after studying the structure of relatively simple inorganic and organic molecules for a decade, Pauling had become interested in the homoblobin molecule.(9) Beginning with an analysis of the structural origin of hemoglobin's oxygen equilibrium curve, Pauling and his colleagues at the California Institute of Technology went on to examine the denaturation of hemoglobin and other proteins, and the magnetic properties of hemoglobin and its derivatives. For Pauling, long interested in understanding how molecular structure determines chemical properties, the study of magnetic properties was "especially fruitful in providing information about the nature of the bonds formed by the iron atoms in hemoglobin with the neighboring atoms of . . . the globin, and attached molecules such as the oxygen molecule" (Pauling 1955, p. 216).
Pauling thus was well prepared to respond to Castle's discussion of sickle-cell anemia, particularly his description of Hahn and Gillispie's 1927 experiments on the effects of oxygen and carbon dioxide on sickling. Pauling suggested to Castle that "the action of carbon dioxide was to accelerate the dissociation of oxygen from oxyhemoglobin. . ., and I pointed out that the relation of sickling to the presence of oxygen clearly indicated that the hemoglobin molecules in the red cell are involved in the phenomenon of sickling." (Pauling 1955, pp. 216-217)
In 1927 Hahn and Gillispie had recognized that hemoglobin somehow is involved in sickling. Now, in 1945, Pauling's agile mind quickly formulated an idea about the nature of that involvement, an idea triggered in part by his work on the nature of antigen-antibody relationships.(10) "I thought that it was possible that the patients with sickle-cell anemia manufacture an abnormal sort of hemoglobin molecule, such that the molecules are self -complementary, and stick to one another, forming long rods, which then line up side by side to produce a needle-like crystal, which, as it continues to grow, becomes longer than the diameter of the red cell, and thus deforms the red cell into an elongated shape. It was necessary to assume that molecules of sickle-cell oxyhemoglobin, as well as those of normal adult human hemoglobin and oxyhemoglobin, do not have this property of self -complementariness" (Pauling 1960, pp. 2-3).
Pauling, a chemist, recognized the wisdom of collaborating with a physician to explore his new interest in sickle cell anemia. That collaborator, Dr. Harvey Itano, arrived at Cal Tech in the fall of 1946 on a three year American Chemical Society Precloctoral Fellowship in [END OF PAGE 80] Chemistry. Itano had received his M. D. from St. Louis University School of Medicine in 1945, where one of his professors, Dr. Edward Doisy, suggested that he pursue his interest in chemistry by working with Pauling. Pauling agreed to Doisy's plan, and in correspondence with Itano Pauling "suggested that he investigate the hemolobin from the red cells of sickle-cell anemia patients, in order to see whether it was different from normal adult human hemoglobin" (Pauling 1955, p. 217).
Itano began his hemoglobin research in the fall of 1946 by replicating Hahn and Gillispie's finding that ,18ickling is prevented by both oxygen and carbon mon.oxide. He then found that a series of other hemoglobin derivatives similarly prevent sickling and developed a diagnostic test enabling physicians to rapidly differentiate between sickle cell trait and sickle cell anemia Otano and Pauling, 1949).
Then, with Pauling's guidance, Itano joined forces with two postdoctoral fellows, S. J. Singer and A. C. Wells, to determine whether there were significant differences in the physical and chemical properties of hemoglobins from normal adults and from persons with sicklemia (sickle cell trait) and sickle cell anemia.
Four months after J. V. Neel's paper in Science, documenting his hypothesis about the homozygous inheritance of sickle cell anemia Pauling, Itano, Singer, and Wells reported the results o; their physical -chemical researches in the same major journal. They found, initially, that most of the properties of hemoglobin from the blood of normal individuals and sickle cell anemia patients were identical. But then a striking and fundamental difference was found, by the painstaking measurement of the different hemoglobins' electrophoretic mobility.(11)
At the time of their November 1949 report, Pauling's group had done eiectrophoresis studies, each taking 6 to 20 hours, on hemoglobins from 15 persons with sickle cell anemia, 8 with sickle call trait, and 7 with normal blood. "The results," they announced, "indicate that a significant difference exists between the electrophoretic mobilities of hemoglobin derived from erythrocytes of normal individuals and from those of sickle cell anemic individuals" (Pauling et al. 1949, p. 544), Electrophoresis of hemoglobin from sickle cell trait carriers also was found to have a characteristic pattern, one that looked like the pattern produced by a roughly equal mixture of normal hemoglobin and sickle cell anemia hemoglobin. At least electrophoretically, therefore, "the two components in sicklemia hemoglobin are identifiable with sickle cell anemia hemoglobin and with normal hemoglobin" (Pauling et al. 1949, p. 546).
Based on a further series of ongoing physical-chemical tests, the Cal Tech researchers reported it seemed probable that the difference between normal and sickle cell hemoglobins resided in the globin portion of the molecule. Their experimental observations, Pauling and his colleagues judged, thus supported the picture of the sickling process that Pauling had first envisaged while talking to William Castle in 1945.
We can picture the mechanism of the sickling process in the following way [the investigators wrote in 19491. It is likely that it is the globins rather than the hemes of the two hemoglobins that are different. Let us propose that there is a surface region on the globin of the sickle cell anemia hemoglobin molecule which is absent in the normal molecule and which has a configuration complementary to a different region of the surface of the hemoglobin molecule. This situation would be somewhat analogous to that which very probably exists in antigen-antibody reactions. The fact that sickling occurs only when the partial pressures of oxygen and carbon monoxide are low suggests that one of these sites is very near to the iron atom of one or more of the hemes, and that when the iron atom is combined with either one of these gases, the complementariness of the two structures is considerably diminished. Under the appropriate conditions, then, the sickle cell anemia hemoglobin molecules might be capable of interacting with one another at these sites sufficiently to cause at least a partial alignment of the molecules within the cell, resulting in the erythrocyte's becoming birefringent, and the cell membrane's being distorted to accommodate the now relatively rigid structures within its confines. The addition of oxygen or carbon monoxide to the cell might reverse these effects by disrupting some of the weak bonds between the hemoglobin molecules in favor of the bonds formed between gas molecules and iron atoms of the hemes. (Pauling et al. 1949, pp. 546-547)
At the time their Science paper was published, Pauling, Itano, and their associates recognized that "some of the details of this picture of the sickling process are as yet conjectural," awaiting future experimental confirmation or disproval. But if the proposal was correct, they [END OF PAGE 81] realized that it "supplies a direct link between the existence of 'defective' hemoglobin moiecules and the pathological consequences of sickle cell disease" (Pauling at al. 1949, p. 547).
With this statement, Pauling introduced his fundamentally important concept of sickle cell anemia as a genetically transmitted molecular disease. Discussing the genetics Of sickle cell disease, Pauling and his co-inveStigators briefly reviewed Taliaferro and Huck's dominant gene theory, and Neel's recent report of his study supporting a heterozygous-homozygous mode of inheritance. Then, as investigators are prone to do in the competitive world of research, Pauling's group added a priority claim.(12) "Our results had caused us to draw this inference before Neel's paper was published. The existence of normal hemoglobin and sickle cell anemia hemoglobin in roughly equal proportions in sicklemia hemoglobin preparations is obviously in complete accord "pith this hypothesis" (Pauling et al. 1949, p. 547).
Pauling, Itano, Singer, and Wells then reached the core of their paper: an explanation of the probable relationship between the genetics of sickle cell anemia and the mechanism of sickling.
In fact, if the mechanism proposed above to ac- count for the sickling process is correct, we can identify the gene responsible for the sickling process with one of an alternative pair of alleles capable through some seri . es of reactions of intro- ducing the modification into the hemoglobin mol-ecule that distinguishes sickle cell anemia hemo- globin from the normal protein.
The results of our investigation are compatible with a direct quantitative effect of this gene pair; in the chromosomes of a single nucleus of a normal adult somatic cell there is a complete absence of the sickle cell gene, while two doses of its allele are present; in the sickiemia somatic cell there exists one dose of each allele; and in the sickle cell anemia somatic cell there are two doses of the sickle cell gene, and a complete absence of its normal allele. Correspondingly, the erythrocytes of these individuals contain 100 percent normal hemoglobin, 40 percent sickle cell anemia hemoglobin and 60 percent normal hemoglobin, and 100 percent sickle cell anemia hemoglobin, respectively. This investigation reveals, therefore, a clear case of a change produced in a protein molecule by an allelic change in a single gene involved in synthesis. (Pauling et al. 1949, p. 547; italics added)
With this statement, Pauling's group affirmed the soundness of Dr. James Herrick's first hypothesis about the cause of the "peculiar" sickle-shaped red blood cells he had first observed under his microscope in 1904: "that some unrecognized change in the composition of the corpuscle itself may be the determining factor." Pauling and Itano's investigations, part of a sequence of inquiries into the nature of sickle cell anemia that began with Herrick's first case report in 1910, also forged new links between physical chemistry, human and basic genetics, and clinical medicine. That fusion would profoundly alter our understanding of the nature of gene action, and resolve many of the puzzling questions about allelism that we noted at the beginning of this chapter. For, in a line of inquiry that at the time seemed far removed from Beadle, Ephrussi, and Tatum's basic genetic researches with Drosophila and Neurospora, the work of Pauling and his colleagues soon would intersect with and significantly modify the one gene-one enzyme hypothesis.
While Pauling et al.'s 1949 report provided strong empirical evidence for the concept of a molecular disease, it left many questions unresolved and, not surprisingly, provided a major catalyst for further study. The torrent of researches on normal and abnormal hemoglobins since 1949 is suggested by the fact that as of 1976 some 300 abnormal hemoglobins, in addition to hemoglobin S (sickle-cell hemoglobin), had been discovered, and the precise chemical nature of 259 of these variants had been established (Neel 1976, p. 57).
In the remainder of this chapter, our focus will be on three interrelated lines of work through the early 1960s that marked the coming of age of "molecular pathology" and that, joining with other areas of genetic research, produced a new understanding of how genes control the structure of proteins.(13) At the center of these investigations were two critical questions posed by the work of Pauling's group: what is the exact biochemical nature of the difference between normal and sickle cell hemoglobin and where in the hemoglobin molecule is this difference localized? [END OF PAGE 82]
One of the first areas of study to bear fruit after 1949 was the mechanism of the sickling process. In his first conversation about sickle cell anemia with William Castle in 1945, and again in his 1949 Science paper, Pauling had suggested a mechanism for the sickling process involving the combination of complementary sites on adjacent hemoglobin molecules, analogous to the presumed interactions between antigens and antibodies. With this hypothesis, the known effects of oxygen and carbon monoxide in reversing sickling were explained by a disruption of the weak bonds between the hemoglobin molecules by the bonds formed between the gas molecules and iron atoms of heme.
The essentials of this picture of the sickling process were substantiated by 1951 through several lines of investigation, including further experiments by Pauling (St. George and Pauling 1951). The principal confirmation of Pauling's view of the sickling mechanism came from studies conducted in Boston, Massachusetts and Cambridge, England. (Fittingly, Dr. William Castle directed the work of Dr. John Harris in Boston, in the Thorriclike Memorial Laboratories of Boston City Hospital.) In a 1950 paper, Harris reported on his study with the polarizing microscope of various concentrations of sickle-cell hemoglobin in oxygenated and deoxygenated states. As Pauling had hypothesized, Harris found that the cleoxygenated hemoglobin formed spindle-shaped bodies arranged as tactoids. This formation, Harris explained, "is evidence of a specific arrangement or linkage of the individual molecules with the formation of long chains of hemoglobin elements. . ." (Harris 1950, p. 199).
At first independent of Harris' work, although the two groups later corresponded about their findings, comparable studies were initiated at the University of Cambridge's Cavendish Laboratory by noted protein crystallographer F. M. Perutz. Perutz had spent some 10 years collecting x-ray diffraction data from hemoglobin crystals in an effort to map the protein's threedimensional structure, when in 1950 his attention was directed to sickle cell anemia by Dr, F. Eirich, from the Polytechnic Institute in Brooklyn, New York. Then, following up on a suggestion by Dr. C. A. Stetson of the Rockefeller Institute, Perutz and Mitchison reported in 1950 that their solubility studies of normal and sickle cell hemoglobin, using a polarizing microscope, indicated that "the reduced [deoxygenated] hemoglobin in sickle cells is in a crystalline state" (Perutz and Mitchison 1950, p. 678). Several months later Perutz reported on further studies, using x-ray diffraction to see whether there were any obvious structural differences between sickle-cell anemia and normal oxyhemoglobin molecules. But the nature of the molecular differences between normal and sickle cell hemoglobin was not to be revealed by x-ray diffraction. "As to the structural differences between normal and sickle-cell anemia haemoglobin, our crystallographic results provide no clue, but merely serve to emphasize the close similarity between the two proteins." To Perutz and his collaborators, "this was a most surprising result, considering that normally two related but slightly different proteins - for example the haemoglobins of closely related animal species - have totally different crystal structures" (Perutz, Liquori, and Eirich 1951, pp. 931, 929).(14)
Another line of attack on the problem of localizing the structural basis for sickled cells was undertaken by the eminent hemoglobin researcher W. A. Schroeder. Working with A. M. Kay and A. C. Wells, Schroeder's approach to the molecular disease puzzle was to see whether there were differences in the amino acid content of hemoglobins from blacks with and without sickle cell anemia. Their results, like Perutz's x-ray diffraction studies, were largely negative, for comparative analysis indicated no significant differences in the basic and acidic amino acid content of normal adult and sickle cell hemoglobin. Slight differences, however, were detected in the two hemoglobins' content of four uncharged amino acids. Perhaps, Schroeder suggested, these differences might affect the folding or coiling of the polypeptide chains so as to change the acid or base content of other amino acid groups, and thus indirectly alter the electrophoretic properties of the hemoglobins (Schroeder, Kay, and Wells 1950).
Schroeder's work, like other researches at the time, proceeded on the basis of Pauling's suggestion that the structural differences between hemoglobins A (normal) and S (sickle-cell) lay in the globin portion of the molecule. The correctness of this assumption was demonstrated in 1952 by Havinga and Itano in Pauling's laboratory. By separating the heme from the globin and subjecting the globin portions to electrophoresis, they found that the globins had the same differences in electrophoretic mobility as did the whole hemoglobin A and S molecules (Havinga and Itano 1952). In a series of concurrent experiments, Havinga ruled out a variety of possible reasons for differences between hemoglobins S and A, including optical rotation, phosphorus [END OF PAGE 83] content and the number of terminal acid residues (Havings 1953).
The effort to locate the structural difference that accounted for normal and abnormal hemoglobin molacules was further complicated between 1950 and 1953 as clinical geneticists, led by James V. Neel and Harvey Itano, discovered three more abnormal varieties of adult human hemoglobin, named in alphabetical order hemoglobins C, D, and E. These newly recognized abnormal hemoglobins, the researchers quickly found, could manifest themselves alone, or in synergistic interaction with sickle cell anemia, or with thalassernia (see note 7). Still another disease, they found, could result if an individual simultaneously had the sickle cell and the thalassernia minor traits. Thus, by 1953, Itano was able to review ten distinct hereditary clinical states associated with abnormal hemoglobin metabolism (Itano 1953; see also reviews by Itano 1955, Pauling 1955). For both clinical and basic researchers in genetics, biochemistry, and physical chemistry, there was now an almost bewildering array of molecular hemoglobin disorders with which to work. In terms of the central questions raised by the molecular disease concept, that of where and how the hemoglobin molecules differed structurally, researchers focused principally on hemoglobins A, S, and C. It structural differences could be pinpointed researchers knew they would gain a critical insight into "a fundamental question in biochemical genetics:" "whether or not the abnormal hemoglobin genes are alleles, that is, whether mutations resulting in the alteration of hemoglobin metabolism takes place at one or more than one chromosomal locus" (Itano 1955, p. 293). In 1949, we recall, Pauling et al. had postulated that the genes for hemoglobins A and S were allelic, and, with the discovery of hemoglobin C in 1950 by Itano and Neel, a series of studies soon "furnished strong biochemical and genetic evidence that the genes for hemoglobins S and C are allelic or closely linked" (Itano 1955, p. 293).
Through the early 1950s, the varied attempts to locate the structural difference between normal and sickle cell hemoglobin, beyond implicating the globin portion of the molecules, had served primarily to rule out a series of possibilities. The one viable hypothesis seemed to be Schroeder et al.'s suggestion that the molecules might be composed of the same polypeptide chains, but folded in different ways. "The interesting possibility exists," said Pauling in his 1955 Harvey lecture, "that the gene responsible for the sickle-cell abnormality is one that determines the nature of the folding of polypeptide chains, rather than their composition" (Pauling 1955, p. 222).
A little over a year after Pauling's Harvey lecture, the baffling problem of the specific chemical difference between hemoglobins A and S began to be resolved by the researches of a young protein chemist, Vernon M. Ingram, working in Perutz's laboratory. Although only two decades have elapsed since Ingram's first paper, the strides that have been made in analyzing the sequential and spatial structure of proteins make it difficult to appreciate today the problems facing protein chemists in the mid-1950's and the corresponding elegance of Ingram's work. Proteins are extremely complex macromolecules, and in the early 1950s little was known about the exact sequences in which given amino acids form given proteins, much less about the relationships between sequencing and the three-dimensional shapes of a protein's polypepticle chains (see note 14). After years of arduous work, biochemist Frederick Sanger finished thefirst complete sequence analysisof a protein in 1953, an achievement that "contributed decisively to the proof of the peptide theory" - the long debated theory that protein molecules are built of chains of amino acids bound together by pepticle bonds (Fruton 1972, p. 148). The protein he studied was the hormone insulin, a relatively small protein whose longest polypeptide chain contained only 51 amino acids. Complex as the sequence analysis of insulin's structure proved to be, hemoglobin presented a far more formidable task, for, with a molecular weight of about 65,000, researchers knew it would probably contain about 600 amino acid residues.
Vernon Ingram arrived at the Cavendish Laboratory in 1952, to join in Perutz's x-ray crystallography studies of hemoglobin crystals. Then, Ingram recalls, "about the same time as the Watson-Crick breakthrough in the same lab, I went off in a slightly different direction from Perutz and began to study the relationship between the heme group and its adjacent polypepticle chains" (Ingram, personal communication). Ingram's interest in sickle cell hemoglobin was aroused in 1955 by the arrival at Cavendish of an Oxford physician, A. C. Allison. Allison was one of a number of clinicians then investigating both the incidence of sickle cell trait and disease in Africa, and the hypothesis proposed by Brain in 1952 that the evolutionary significance of hemoglobin S might lie in its conferring protection against malarial parasites (Brain 1952; Allison 1954; Pauling 1955). Allison also became interested in the formation of hemoglobin [END OF PAGE 84] crystals in sickle cells, and came to the Cavendish to do fiber x-ray crystallography studies. Given the state of fiber x-ray techniques at the time, Ingram remembers, Allison "didn't get any significant x-ray pictures," and departed leaving behind some of his sickle cell hemoglobin samples. But Allison's visit to the Cavendish would prove highly significant in other respects, for it "was the quite fortuitous appearance of those sickle cell samples" which triggered Ingram's idea of looking at their protein chemistry (Ingram, personal communication). It was not, of course, completely fortuitous. For Ingram, working "in a lab which was passionately interested in anything to do with hemoglobin," was familiar with the research of Pauling's group and with the subsequent attempts to determine the difference between hemoglobins A and S.
When Ingram decided that he, too, would seek to uncover how the protein globins of normal and sickle cell hemoglobins differed, he knew from previous investigations that the difference, whatever it was, must be a small one. He knew, too, that the hemoglobin molecule was too large for the type of complete sequence analysis of insulin that Sanger had performed. Faced with this reality, Ingram devised a short cut, utilizing a combination of methods that enabled him to obtain and analyze relatively small pepticle fragments from the whole molecule. In the mid-1950s, one of the newest and most reliable ways of separating pepticles and amino acids was by means of the enzyme trypsin, which split polypeptide chains by specifically degrading the chemical bonds formed by the carboxyl groups of two amino acids, lysine and arginine. Other studies of hemoglobin's amino acid composition had indicated that there were some 60 of these particular carboxyl groups in hemoglobin A and S molecules. But, because Perutz's x-ray diffraction analysis had indicated that each hemoglobin molecule was composed of two identical "half molecules," Ingram anticipated that "the number of pepticles obtained by the action of the trypsin should be about thirty, an average chain-length of ten amino-acids. Small differences in the two proteins [hemoglobin A and S], he reasoned, will result in small changes in one or more of these peptides" (Ingram 1956, p. 793).
To try to detect these small differences, Ingram ingeniously combined paper electrophoresis and the paper chromotagraphy methods that he had watched Sanger use. By this combination he created a twodimensional method that enabled him to comparatively "fingerprint" the hemoglobin S and A fragments he obtained from the tryspin digest. The fingerprints revealed approximately 30 pepticle spots, confirming Perutz's view that the human hemoglobin molecule consists of two identical half molecules. As he also had expected, Ingram saw that the normal and sickle cell globins contained the same number of peptides. But the fingerprints did what other attempts to locate a difference between the S and A globins had been unable to do - they revealed a small difference in the largely identical molecules. As Ingram reported in 1956, ". . . there is one peptide spot clearly visible in the digest of haernoglobin S which is not obvious in the haemoglobin A 'finger print" (Ingram 1956, p. 793).
Prior to the development of Ingram's fingerprinting method, it had been impossible to decide whether the small but critical difference between hemoglobins A and S resided in the amino acid sequences of the polypepticle chains, or in the ways those chains were folded. But by means of the trypsin digest and fingerprinting, as Ingram concluded in his short paper:
One can now answer at least partly the question put earlier, and say that there is a difference in the amino-acid sequence in one small part of one of the polypepticle chains. This is particularly interesting in view of the genetic evidence that the formation of haernoglobin S is due to a mutation in a single gene. It remains to be seen exactly how large a portion of the chains is affected and how the sequences differ. (Ingram 1956, p. 294)
Ingram's experiment thus had opened a new vista in biochemical genetics and protein chemistry, providing the way to show with detailed precision the relationship between a gene and the protein under its control. Less than a year after his first paper, Ingram published a second short report in which he answered his 1956 question of "how large a portion of the chains is affected and how the sequences differ."
I have now found that out of nearly 300 aminoacids in the two proteins, only one is different; one of the glutamic acid residues of normal haemoglobin is replaced by a valine residue in sickle cell anaernia haernoglobin. The latter is an abnormal protein which is inherited in a strictly Mendelian manner; it is now possible to show, for the first time, the effect of a single gene mutation as a change in one amino-acid of the haemoglobin polypeptide chain for the manufacture of which that gene is responsible. (Ingram 1957, p. 326) [END OF PAGE 85]
Vernon Ingram had detected the basis for the shown, caused by the smallest possible alteration in the large hemoglobin molecule: the substitution of a single amino acid, glutarnine, by another, valine. The technique of protein fingerprinting, as Ingram realized, had simply and decisively answered one of the most intriguing questions in the explosive new field of molecular genetics, that of precisely how a gene mutation affects the specific structure of a protein.
What, then, was the nature of the mutation within the hemoglobin gene that produced this small but signif icant alteration in the structure of the hemoglobin molecule? Presumably, Ingram wrote in 1957, it is an equally small change in the hemoglobin gene. "it is not known, but it may well be that this involves a replacement of no more than a single base-pair in the chain of the deoxyribonucleic acid of the gene" (Ingram 1957, p. 327). This hypothesis about a human gene and its protein, Ingram pointed out, accorded well with the new knowledge of genetic subunits being gained from the standard organisms of molecular geneticists, Neurospora, Aspergillus, and bacteriophage.
The results presented in this communication are certainly what one would expect on the basis of the widely accepted hypothesis of gene action; the sequence of base-pairs along the chain of nucieic acid provides the information which determines the sequence of amino-acids in the polypeptide chain for which the particular gene, or length of nucleic acid, is responsible. A substitution in nucleic acids leads to a substitution in the polypeptide. (Ingram 1957, pp. 327-328)
Less than a month after Ingram's 1957 paper, another important report issued from PaUling's laboratory. Perutz's x-ray crystallography studies and Ingram's fingerprint had led them to conclude that the hemoglobin molecule consisted of two identical "half molecules." Now, on the basis of complex quantitative chemical analysis, H. L. Rhinesmith, W. A. Schroeder, and Pauling had succeeded in identifying the number and kind of polypeptide chains in normal adult hemoglobin. The globin, they determined, consists of two sets of polypeptide chains, or a total of four chains forming two identical half molecules (Rhinesmith, Schroeder, and Pauling 1957). With this report, there was now a solid line of evidence, derived from a complex human protein, that confirmed and greatly refined the concept of gene action generated in 1908 by Sir Archibold Garrod's study of inborn errors of metabolism - that of a one-to-one relationship between genes and enzymes.
That concept, as we have seen, had been rediscovered and reenunciated as the one gene-one enzyme hypothesis by George Beadle and his associates in the 1930's early 1940's, through their biochemical genetic researches with Drosophilia and Neurospora. Ingram recalls that while he was at the Cavendish laboratory from 1952-1958 the one gene-one enzyme hypothesis was "well accepted" by himself and his colleagues, so that he promptly saw the "sickle cell hemoglobin business" as providing a "direct illustration" of the correctness of Beadle et al.'s formulation (Ingram, persornal communication). But acceptance of the one gene-one enzyme hypothesis was far from universal among geneticists from the early 1940's through the mid-1950's, in part because, like Garrod's explanation, it seemed too simple an account of gene action.(16)
There were, of course, other reasons besides it's simplicity for the slow acceptance of the one gene-one enzyme concept and all that it implied.
It would take some years to understand the relationship between mutations and enzymes, primarily because so little was known concerning the structure of proteins at the time there was so much discussion of the one gene-one enzymes concept. We may have difficulty in appreciating the problem unless we keep in mind that the concepts were formulated without any clear idea of the properties of proteins which conferred specificity . . . Although proteins were known to be composed of polypeptide chains which were assumed to be folded in specific ways, there was no clear concept of what determined the sequence of amino acids or whether the sequence was related to the three-dimensional shape. Did genes control the specificity of enzymes and if so how? If a mutation changed an enzyme activity was it because the protein was changed or produced in smaller amounts? Would the mutation of one gene change more than one protein? Could genes mutate in a variety of ways?
Without a clear concept of the nature of the gene or of its supposed product, a specific proteien, little progress could be made. Geneticists could study the phenotypes produced by mutation as had been done for years. by Neurospora, which could be grown in a simple, completely defined medThe improvement pro-[END OF PAGE 86]videdium, was that the change in phenotype could be defined much more precisely in chemical terms. In an attempt to simplify the problem, the question was put in the following form. Do any genes have a single primary function and if so what proportion of mutations produces changes of this nature? (Taylor 1965, pp. 4-5)
With this precis of the questions rampant in genetics through the early 1950s, the impact of the researches on sickle cell hemoglobin is apparent. By 1957, principally through the researches of Ingram and of Paul ing's group, geneticists knew that a mutant hemoglobin gene produced a disease by altering the presence of a single amino acid in the polypepticle chain of the hemoglobin molecule. The one gene-one enzyme concept of gene action had been essentially correct. But it had been too simple, because enzymes are proteins, but not all proteins are enzymes. Principally through research from many quarters on the nature of hemoglobin, it was gradually recognized that structural proteins, too, might be the direct products of gene action, and the one gene-one enzyme hypothesis was recast in terms of one gene-one protein. Then, the trail of researches into the nature of sickle cells, that began in 1910 with Herrick's first clinical report, revealed the specificity of gene action within a protein, and one gene-one protein was recast as one gene-one polypepticle. "Thus," wrote Bentley Glass on the centennial of Gregor Mendel's publication, "geneticists were led to the conclusion that, as predicted from the Watson-Crick model of the DNA molecule, the sequence of nucleotides in the DNA molecule specifies the sequence of amino acids in the polypeptides" (Glass 1965, p. 234).
Another core aspect of gene action elucidated by the study of abnormal hemoglobins was allelism. As we have seen, Ingram's fingerprints of hemoglobins A and S provided strong indirect support for the supposition advanced initially by Pauling at al. in 1949: that the mutant gene responsible for sickle cell hemoglobin is an allele or variant of the normal hemoglobin gene, occupying the same chromosomal locus. As we also mentioned, the rapid discovery of other abnormal hemoglobins, beginning with the identification of hemoglobin C in 1950, raised new questions about allelism: were hemoglobins C, D, E, and so on, like hemoglobin S, due to another allele at the same locus, or did it represent a new mutational system at an independent locus? The first answers to this fundamentally important question, once again, were provided by Ingram's fingerprints.
In 1951, shortly before Ingram moved from Cambridge, England to MIT in Cambridge, Massachusetts, he and J. A. Hunt published a short note in Nature: "Allelomorphism and the Chemical Differences of the Human Hemoglobins A, S and C." By preparing fingerprints of hemoglobin C and comparing them with their analysis of the A and S fingerprints, Hunt and Ingram had found that C was caused by a different amino acid substitution at the same site in the polypeptide chain as the A and S variation. While hemoglobin S was produced by the substitution of valine for glutamine, hemoglobin C occurred when the same glutamic acid residue was replaced by lysine.
"These results," wrote Hunt and Ingram, "have interesting genetic implications."
They are the first steps in a search for a correlation between the linear fine structure of a gene such as that determined recently by Benzer and Pontecorvo, and the linear structure of the polypeptide chain of the protein the synthesis of which that gene controls...
Our results also shed light on the position of these two mutations on the haemoglobin gene or genes. Genetic evidence shows that the haemoglobin S and C mutations are allelic or occur at linked sites; but the statistical evidence is not sufficiently strong to distinguish between these two possibilities. If we assume a direct relationship between the internal (linear) structure of the gene, and the linear arrangement of the amino-acid residues in the haemoglobin molecule, then the finding that the same amino-acid residue, gfutamic acid, is altered in both mutations implies that they are indeed allelic mutations and occupy the same site on the gene. Thus if these ideas are correct the chemical investigation of proteins such as the human haemoglobins can provide a powerful, though indirect, tool to help the geneticist in mapping the positions on the gene where certain mutations occur.
In order to obtain sufficient statistical data and a sufficient number of mutants, such a programme must eventually turn to proteins from microorganisms. However, it is hoped that the abnormal human haemoglobins will provide a few more [END OF PAGE 87] useful examples of the effects of gene mutations on protein structure. (Hunt and Ingram 1958, pp. 1062-1063; see also Hunt and Ingram 1960).
Hunt and Ingram's modest hope that abnormal human hemoglobins would provide "a few more useful examples" of the effects of gene mutations on protein structure has been amply realized in the years since 1958. The study of inherited hemoglobin abnormalities has provided the clearest evidence that similar traits are often determined by multiple alleles of a single locus. A 1971 text, for example, noted that over 175 "hereditary abnormal hemoglobin" diagnoses had been made, and had been traced to variations or alleles in only five loci. "Remarkably," the authors wrote, "one of the clearest demonstrations" of the existence and effects of multiple alleles "has come from a human character: the variations in the polypeptide chains that make up the globin portion of the hemoglobin molecule." (Levitan and Montagu 1971, p. 551)
The study of abnormal hemoglobins, which now number some 300, is a continuously unfolding one, involving interactions between many areas of clinical and basic research. We have looked at one portion of that study, the trails of inquiry that led from the discovery of strangely elongated and sickle-shaped red blood cells to new knowledge of how normal and mutant genes govern the structure of proteins. This particular path from clinical to basic knowledge, of course, was neither straight not simple. The definition and elucidation of sickle cell anemia as a molecular disease built upon decades' of prior researches into the nature of hemoglobin, and, as we have seen, human and basic geneticists, crystallographers, physical chemists and biochemists, were among those whose often collaborative researches converged to unravel the genetic principles contained in normal and abnormal hemoglobin molecules, and to link their findings with those from other areas of molecular genetics.
To Dr. Vernon Ingram, reflecting in 1976 on the history of hemoglobin research in which he has played such a central role, it is not surprising that "the study of human or animal diseases will frequently and unpredectably lead to fundamental insights into basic mechanisms in biology." But to Ingram, what happened in the arena of molecular genetics was and is surprising. "The coming field in the molecular biology of genetics in the middle 1950s was bacterial and viral genetics," Ingram said. So if somebody say in 1955 had predicted from which field would come the first demonstration that one gene difference is related to results in a specific localized difference in a protein, they would have said from bacterial or viral materials. We were as surprised as anyone else that it in fact happened to come from a human protein" (Ingram, personal communication).
Today, two decades after that "surprising" event, the study of hemoglobin abnormalities continues to weave between and impact upon clinical and basic research. "The study of the abnormal hemoglobins," James V. Neel wrote in a volume of bicentennial essays on advances in American medicine, "has not only produced a paradigm for 'molecular' diseases in general, but has yielded insights into the fine details of chromosome structure in mammals which would not otherwise be available" (Neel 1976, p. 60). [END OF PAGE 88]
(1) The explosive growth of knowledge generated by molecular biology has revealed Mendel's "characters" or units of heredity to be segments of DNA molecules. Through the intermediary activities of RNA, these DNA segments or genes serve as indirect patterns for the reproduction of polypepticles, chains of amino acids which are, or combine to form, proteins. "Thus, overall, the gene (DNA) clearly specifies the protein. But without proteins, DNA cannot be made; without RNA, proteins cannot be made. Molecular biology is a drama with these as the three major actors, each owes its existence to the others" (Handler 1970, p. 15).
(2) In the second edition of Inborn Errors (1923), Garrod reported that evidence for the correctness of his hypothesis had been provided in 1914 by Gross, who reported finding in normal blood plasma but not in the plasma of alkaptonurics an enzyme that oxidized homogentisic acid. Gross' finding, however, was never replicated, and it was not until 1958 that this hypothesis was confirmed by LaDu and his associates, who demonstrated the absence of the enzyme in liver biopsy specimens from an alkaptonuric patient (Glass 1965, p. 231).
(3) This failure is amply illustrated by the 1942 book, New Paths in Genetics, authored by the eminent J. B. S. Haldane. For, as Glass points out:
. . . Haldane, of all geneticists of his time, was best trained in biochemistry and most open to see its genetic significance. Yet the book is singularly lacking in analyses of possible gene-enzyme relationships. Garrod's work of 1923 is cited for its example of human metabolic abnormalities inherited as simple recessives; the relationship of phenylketonuria and alkaptonuria to blockage of specific steps in amino acid metabolism is implicitly, but not explicitly, recognized, but the enzymatic relationship is scarcely hinted at. (Glass 1965, p. 232)
Unawareness of Garrod's work, in turn, is exemplified by Beadle and Tatum's opening sentence in their now classic 1941 paper, "Genetic Control of Biochemical Reactions in Neurospora."
From the standpoint of physiological genetics the development and functioning of an organism consist essentially of an integrated system of chemical reactions controlled in some manner by genes. It is entirely tenable to suppose that these genes which are themselves a part of the system, control or regulate specific reactions in the system either by acting directly as enzymes or by determining the specificities of enzymes. I (Beadle and Tatum 1941, p. 499)
In their first reference Beadle and Tatum then note that "the possibility that genes may act through the mediation of enzymes has been suggested by several authors," and they refer the reader to publications by Troland (1917), Wright (1927), and Haldane (1937) for discussion and further references.
(4) "The idea was simple," George Beadle has stated.
"Select an organism like a fungus that has simple nutritional requirements. This will mean it can carry out many reactions by which amino acids and vitamins are made. Induce mutations by radiation or other mutagenic agents. Allow meiosis to take place so as to produce spores that are genetically homogeneous. Grow these on a medium supplemented with an array of vitamins and amino acids. Test them by vegetative transfer to a medium with no supplement. Those that have lost the ability to grow on the minimal medium will have lost the ability to synthesize one or more of the substances present in the supplemented medium. The growth requirements of the deficient strain would then be readily ascertained by a systematic series of tests on partially supplemented media.
"In addition to the above specifications we wanted an organism well-suited to genetic studies, preferably one on which the basic genetic work had already been done." (Beadle 1964, p. 594)
(5) Hahn and Gillispie's gas chamber experiments fell into disrepute during the 1930's because other investigators had trouble replicating their findings. In 1940, however, I. S. Sherman reported that, using similar methods, he could distinguish experimentally between sickle cell trait and sickle cell anemia, based on the amount of oxygen tension needed to produce sickling (Sherman 1940).
(6) The term "homozygous" means that an organism has inherited identical genes at a given locus, while "heterozygous" refers to the possession of different alleles at a given locus. Thus, in the case of sickle cell hemoglobin, a homozygous person, who has sickle cell disease, has inherited the sickle cell hemoglobin gene (hemoglobin c), from both parents; the person with sickle cell trait, who is only a carrier of the disease, is heterozygous, having a normal or hemoglobin A gene and its alleiie hemoglobin S gene at the same locus.
(7) The thalassemias, too, provide an excellent illustration of the ways that a clinical disorder can generate and interact with fundamental research. Much like sickle cell anemia, study of the thalassemias has produced more detailed knowledge both of "molecular diseases," and of the mechanisms involved in the genetic control of protein structure. Thus, as a 1976 review article noted, "the thalassemia literature abounds with syndromes that mix classical and molecular genetics with clinical hematology" (Orkin and Nathan 1976, p. 710). From this sometimes confusing mix of inquiries, researchers have found that thalassemia syndromes are caused by deficient production of specific polypeptide chains. Unlike sickle cell anemia, the globin chain in thalassemias, when it is not absent, is structurally normal. Genetically, therefore,
the abnormality in the thalassemias must involve either the globin genes themselves or regulatory elements closely linked to them. Newer technics in cellular and [END OF PAGE 89] molecular biology have been applied to the study of each phase of gene expression from the DNA, to the transcribed messenger RNA's (mRNA's), to protein synthesis utilizing globin mRNA's as templates to define the molecular defects in the thalassemia syndromes.
In one sense the thalassemias are all quite similar in that the defect in globin-chain synthesis can always be traced at least to the level of the globin MRNA's. (Orkin and Nathan 1976, p. 711)
(8) As occurs frequently in science, recognition of the correct mode of inheritance of sickle cell anemia was a case of "simultaneous discovery," for which history has largely credited only one discoverer. In 1949, independent of Neel's work, a British physician named E. A. Beet, who had been studying the incidence of sickle cell trait and disease in East Africa also recognized the heterozygous-homozygous mode of inheritance (Beet 1949).
(9) The human red blood cell or erythrocyte is nearly all hemoglobin, 95% of the dry weight of the cell. Due to the high concentration of hemoglobin in the cell, and the corresponding lack of such usual cellular components as a nucleus, hemoglobin is easily isolated and had been a favorite subject for protein research since the second half of the 19th century. When Pauling turned his attention to the hemoglobin molecule in 1935, chemists knew that the molecule consisted of four heme or ironcontaining groups with which oxygen combines to form oxyhemoglobin, and globin, a bulky protein in which the heme is "imbedded." At the time, little was known about the detailed structure of the globin portion of the molecule.
(10) For a discussion of antigen-antibody theory and research, including Pauling's own work on this topic, see Chapter 6.
(11) Pauling and his colleagues, in 1949, were able to utilize a highly sophisticated form of electrophoresis, a technique which permits the measurement of the physical properties of molecules such as hemoglobin, recording characteristic patterns that enable one to identify different molecules or chemical substances. Electrophoresis, for example, can distinguish two different proteins as follows: the proteins are placed in an electric field, with a positive and negative side; proteins have positive and negative surface charges, which causes positively charged proteins to migrate to the negative pole of the electrophoretic field, at a rate depending on the strength of the positive charge. Conversely, a negatively charged protein moves to the positive pole of the field. Hemoglobin electrophoresis had first been performed in 1939, by B. D. Davis and E. J. Cohn.
(12) As documented by historians and sociologists of science, priority disputes are a frequent and characteristic accompaniment of research, for reasons that are understandable in terms of the values and the rewards of scientific research. For the classic sociologic study of priority disputes, see Merton 1957.
(13) Among the many and significant areas of hemoglobin research that we will not touch upon are work on the evolution of proteins, the differences between fetal and adult hemoglobins, and chromosomal relationships of hemoglobin loci. For a succint but comprehensive review and bibliography of these and other aspects of hemoglobin research, see Neel 1976.
(14)The difficulties can be appreciated
when one realizes that "proteins are immensely complex macro-molecules
since they are polymers built up from 20 different building blocks (the amino
acids). Thus the organic chemist must determine both how the amino acids are
linked together and what their order is within a given linear polypeptide chain.
Likewise, the biochemist wishes to know both how the backbone linkages are connected
and what trick is used to order the amino acids during synthesis
. . .Aside from the question of sequence, there is also the problem of how polypepticle
chains assume their final 3-D configurations. The correct functioning of almost
all proteins depends not only upon possession of the correct amino acid sequence
but also upon their exact arrangement in space" (Watson 1965, pp. 169-170).
[No footnote #15 present in original text]
(16) George Beadle remembers that:
"In 1945 I held a Sigma Xi National Lectureship under which I gave lecures on biochemical genetics and the one gene-one enzyme interpretation. I was much impressed with the resistance to this notion, especially in agricultural colleges where workers were familiar with the genetics of such characteristics as egg production, and milk production in dairy cattle. They were sure gene action could not be generally described in the simple way we had postulated. It seems to me the status of concept dropped to an all-time low at the Cold Spring Harbor Symposium of 1951. In rereading the volume on those meetings, I have the impression that the number whose faith in one gene-one enzyme remained steadfast could be counted on the fingers of one hand - with a couple of fingers left over" (Beadle 1966, p. 30).[END OF PAGE 90]
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