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This book relates how, between 1954 and 1961, the biologist Seymour Benzer mapped the fine structure of the rII region of the genome of the bacterial virus known as phage T4. Benzer’s accomplishments are widely recognized as a tipping point in mid-twentieth-century molecular biology when the nature of the gene was recast in molecular terms. More often than any other individual, he is considered to have led geneticists from the classical gene into the molecular age.
Drawing on Benzer’s remarkably complete record of his experiments, his correspondence, and published sources, this book reconstructs how the former physicist initiated his work in phage biology and achieved his landmark investigation. The account of Benzer’s creativity as a researcher is a fascinating story that also reveals intriguing aspects common to the scientific enterprise.
The formation of genetics during the first decades of the twentieth century, following the rediscovery of the long-overlooked paper by Gregor Mendel that provided its fundamental principles, has been the subject of many historical accounts. In this chapter I do not attempt to recapitulate this complex history but only draw attention to certain features of its development that were particularly relevant to what was viewed by mid-century as "classical genetics."
Mendel explained the results of his experiments on the hybridization of pea plants by assuming the presence in the germ cells of Anlagen that give life to the individuals that display the particular Merkmale by reference to which he differentiated them. When two plants whose Anlagen produced different Merkmale, such as green or yellow seed coats, were mated, the effects of only one of them, which he called the dominant one, were visible in the hybrid progeny. That in the next generation both green- and yellow-coated seeds appeared in definite ratios he attributed to the other, or "recessive," Anlage having remained unaltered during theirassociation, the two types then segregating independently during the formation of the germ cells. At the time there were no structures identified within ordinary or germ cells with which the Anlage could be associated.
Mendel's term Anlage was later translated in the English literature as "factor," and Merkmale as "character." Whereas the paired German words were suggestive of the relation between an inner predisposition and an outward sign, the words factor and character lacked these connotations. Because the factors remained abstract entities without known properties of their own, early geneticists often associated them so closely with the "unit characters" they were supposed to produce that some geneticists nearly obliterated the distinction between factor and character. In an article titled "What Are 'Factors' in Mendelian Explanations?" the American embryologist Thomas Hunt Morgan protested in 1909 against the facile references by some Mendelians to these "hypothetical" factors as the "actual characters themselves." Taking as his illustration the factors for tallness and shortness in Mendel's peas, Morgan asserted that the assumption that two factors can coexist in countless generations of cells without "having produced any influence on each other," then "turn their backs on each other and go their several ways," was a "purely preformation idea" and was only one among various possible explanations. Until all alternate possibilities were examined, it would "be at least judicious to hold the segregation hypothesis, as currently interpreted-a purely formal procedure." While adopting this procedure, it was of "capital importance," according to Morgan, to keep in mind that "the egg need not contain the characters of the adult, nor need the sperm. Each contains a particular material which in the course of the development produces in some unknown way the character of the adult. Tallness, for example, need not be thought of as represented by that character in the egg, but the material in the egg is such that placed in a favorable medium it continues to develop until a tall plant results. Similarly for shortness."
In the same year Wilhelm Johannsen, who had bred "pure lines" of the bean plant Phaseolus, concluded that the concept of unit character did not express the complexity of the relation between the observable characters of his plants and whatever physiological units were involved in forming their overall character. Reviewing the words that others had used to define particulate units of inheritance, Johannsen chose Hugo de Vries's term pangene but dropped the first syllable to free the term from its historical associations with particular theories. "The word 'gene,'" he wrote, "is completely free from any hypothesis; it expresses only the evident fact that, in any case, many characteristics of the organism are specified in the gametes by means of special conditions, foundations, and determiners which are present." Elof Carlson has commented that "Johannson's gene was undefined" and, therefore, free to "take on or discard definition." Only gradually, however, did gene displace factor or unit character as the preferred term in the explanation of the results of genetic experiments.
As is well known, Morgan overcame his general reluctance to follow the formal procedures of Mendelism as the result of a chance observation. As he reported it in 1910, "in a pedigree culture of Drosophila which had been running for nearly a year through a considerable number of generations, a male appeared with white eyes. The normal flies have brilliant red eyes." Breeding this male, to which he referred without comment as a "mutant," with its sisters produced almost entirely red-eyed offspring, but in the generation bred from the hybrids there were red-eyed males and females and white-eyed males. Morgan accounted for these results by the "hypothesis" that the white-eyed male carried the "'factor' for white eyes" and was heterozygous for a "sex factor." The outcome, he wrote, "is Mendelian in the sense that there are three reds to one white," but the whites were confined to the male sex. Further crosses verified his hypothesis. That Morgan continued to be wary of the idea of "factors," as commonly used, is indicated by the fact that he placed quotation marks around the word. He apparently had no comparable qualms about adopting the word mutation to designate a fly that had arisen spontaneously with a character distinctly different from those of its forebears. That word had been coined by Hugo de Vries to describe the apparent sudden origin of a new species of evening primrose in a single generation. Morgan's new usage associated mutations instead with discontinuous but relatively small modifications arising in an existing species or variety.
During the following two years Morgan and the young students who worked with him in the "fly room" at Columbia University found many more spontaneous mutations, including five eye colors and nine wing modifications, and analyzed them according to Mendelian principles. Some but not all of them turned out to be sex-linked. Those that were not, Morgan concluded in 1911, contained the factor for that character in "another part of the hereditary mechanism," perhaps in another chromosome. These rather vague connections between factors, chromosomes, and sex linkage became much clearer later that year, after Morgan encountered another departure from strictly Mendelian principles. When he bred flies differing in two sex-linked characters (white-eyed with long wings and red-eyed with rudimentary wings), he found that the two did not always appear together in the offspring, although they had to be on the same chromosome, according to his theory of sex linkage. Later he noticed that they nevertheless remained together more frequently in the second generation than would be predicted according to the "law" of independent segregation.
In order to explain these anomalies, Morgan referred to a cytological "chiasmatype" theory published in 1909 by the Belgian Frans Alfons Janssens. Having observed that when homologous chromosomes pair together during the reduction division they twist around each other, and that when they then separate they do so in a single plane, Janssens inferred that this process would result in an exchange of material between the chromosomes. Connecting the cytological behavior of chromosomes with the genetic behavior of the factors, Morgan reasoned, "If the materials that represent these factors are contained in the chromosomes, and if those that 'couple' [are] near together in a linear series, then when the parental pairs (in the heterozygote) conjugate like regions will stand opposed." Citing Janssens's evidence for the manner in which the chromosomes couple and separate, Morgan concluded that "in consequence, the original materials will, for short distances, be more likely to fall on the same side of the split, while remoter regions will be as likely to fall on the same side as the last, as on the opposite side. In consequence, we find coupling in certain characters, and little or no evidence of coupling in other characters, the difference depending on the linear distance apart of the chromosomal materials that represent the factors."
Morgan contrasted his explanation with that of the leading Mendelian of the time, William Bateson, who had accounted for similar observations in chickens by assuming that certain allelomorphic pairs couple with each other and others repel but without being able to offer any underlying mechanism for the process. Morgan believed that his own explanation could account for Bateson's results as well as those seen in Drosophila. They are a "simple mechanical result of the location of the materials in the chromosome, and of the method of union of the chromosomes.... Instead of random segregation in Mendel's sense we have associations of factors that are located near together in the chromosomes. Cytology furnishes the mechanism that the experimental evidence demands."
Morgan made this proposal nine years after Theodor Boveri and William Sutton had independently concluded that the strong parallels between the behavior of chromosomes and that of Mendelian factors suggested that the chromosomes were the physical bearers of the genetic factors. As late as 1910 Morgan had been among those who remained uncommitted with regard to this question. His explanation of coupling now moved him to become a strong advocate for the chromosome theory.
In 1912 Morgan made his first attempt to "analyze the constitution of the chromosome." It was based on the results of his experiments on two classes of sex-linked characters: eye color and wing length. The two principle conclusions to which his studies had led him were that "sex-limited inheritance is explicable on the assumption that one of the material factors of a sex-limited character is carried by the same chromosomes that carry the material factor for femaleness" and that "the 'association' of certain characters in inheritance is due to the proximity in the chromosomes of the chemical substances (factors) that are essential for the production of these characters." The eye colors that Morgan discussed were the red wild type and the mutants vermilion, pink, and orange. Influenced by Bateson's idea that mutants result from the loss of the factor present in the wild type (known as the "presence and absence theory"), Morgan introduced a symbolism that assumed each of the three mutant eye colors to result from the loss of one of the three factors necessary to produce normal red eyes. This view probably appealed to Morgan in part because it fit with his more general conviction that there is no simple one-to-one relation between factors and characters but that the latter are the products of complex interactions between multiple factors during development. He similarly attributed the mutant short wings to the loss of a factor for normal wings.
Dealing with the fact that in several of his experimental crosses the number of mutants found departed radically from the expected Mendelian ratios, Morgan gave three types of explanation. Some of these "disturbances" probably resulted from mutants' being less fertile or less viable than normal flies. "In some cases," however, "the disturbance can be traced directly to the principle of 'association.' By that I mean that during segregation certain factors are more likely to remain together than to separate, not because of any attraction between them, but because they lie near together in the chromosome."
This was as far as Morgan carried the analysis. Although he stated that "such associations will be more or less common according to the nearness of the associating factors in the chromosome," he did not attempt to relate the differing degrees of disturbance that he had observed quantitatively to the degree of "nearness" of the factors.
Because Morgan had earlier expressed skepticism about the chromosome theory of inheritance, some historians have inferred that he remained more reluctant than his students to accept that view and was only gradually forced to it by the accumulating evidence that they produced largely between 1911 and 1915. His own writings from this period suggest, on the contrary, an eagerness to pursue the consequences of the view that the factors were material particles embedded in the chromosomes. Not only in the passages quoted above but throughout this 1912 paper, Morgan referred to factors, which he placed in parentheses, as materials and chemical substances. His theory of eye color in particular seemed to lend itself to a chemical interpretation. Although he acknowledged that he had no facts to offer concerning the chemical nature of the three colors, he appears to have believed that it would be feasible to investigate factors in terms of their chemical properties. He was not, as often portrayed, simply content with an abstract notion of these factors.
When Morgan discussed his theory of "associative inheritance" with the students in his lab, one of them, Alfred H. Sturtevant, immediately thought beyond Morgan's semiquantitative notion of the relation of the interchange of factors between homologous chromosomes to the nearness of their association on the chromosomes. One could use the relative frequency with which two factors separated, Sturtevant saw, to plot their distances from each other on the chromosome. Staying up all night and neglecting his undergraduate homework, he was able to construct such a diagram, which included several sex-linked factors on the basis of experimental results already obtained in the lab.
Following up this quick preliminary effort, Sturtevant studied the frequency of the interchange between homologous chromosomes, which he called "crossing over," among six sex-linked factors. What Morgan had called "association" Sturtevant called "linkage," defining a unit of distance between factors as "a portion of the chromosome of such length that, on the average, one cross-over will occur in it out of every 100 gametes formed. That is, percent of cross-overs is used as an index of distance." If the "hypothesis" were correct, then the sum of the distances calculated between any two factors and a third one situated between them should be equal to the distance between the outside two. Sturtevant found this relation to hold accurately for factors between which the distances were short. For longer distances, the observed distance between the outside factors was somewhat less than that calculated from the sum of the distances of the factors from an intermediate one. Sturtevant explained this discrepancy by assuming that "double crossing over" could in some cases cause the two more distant factors to end up on the same chromosome. "In mapping out the distances between the various factors," therefore, Sturtevant relied "so far as possible on the percent of cross-overs between adjacent points."
In the paper in which he submitted these results for publication in November 1912, Sturtevant labeled the line on which he depicted these distances a "diagram" (fig. 1.1). He assumed that the order in which the factors were shown represented their actual order on the chromosome but acknowledged that "there is no way of knowing whether or not these distances as drawn represent the actual relative distances apart of the factors." If the chromosomes were not of uniform strength, then a break was more likely to come at a weak place than at a stronger one. Such considerations, he thought, "will not detract from its value as a diagram." In his summary, he concluded that his results "form a new argument in favor of the chromosome view of inheritance, since they strongly indicate that the factors are arranged in a linear series, at least mathematically." We may note that, although he referred casually to "mapping out" the distances, Sturtevant did not label the result a genetic map. In the common usage of the time, a map was a two-dimensional representation of a portion of the surface of the earth. Sturtevant's spare linear diagram did not immediately evoke the image of such a map.
Excerpted from Reconceiving the Gene by Frederic Lawrence Holmes Copyright © 2006 by Yale University. Excerpted by permission.
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