The transition from inorganic to organic chemistry in the late 19th century added proteins and nucleic acids, alongside sugars, starches, woods and fats, to the portfolio of components of living organisms. The disciplines of biochemistry and molecular biology emerged in the 20th century with the elucidation of metabolic and biosynthetic pathways and the demonstration that proteins are enzymes. Microbial genetics focused on metabolic enzymes led to the conclusion that genes are synonymous with proteins. Proteins were thought to be the central molecules of life and the active components of the chromosomes that transmitted genetic information; the other main component, DNA, seemed too simple and was thought to act simply as a scaffold. Genetic mapping developed along with the concept of ‘genes’ as discrete entities. Theoretical mathematical biology flourished, influenced by microbial genetics and the ‘lethality’ of mutations. Darwinian evolution and Mendelian inheritance were integrated in the ‘Modern Synthesis’, which asserted that mutations are random and that Lamarckian inheritance of experience does not occur. DNA and RNA were initially confused, but the former was found to be mainly located in the nucleus and the latter in the cytoplasm. DNA was eventually shown to contain the genetic information by transforming the phenotype of bacteria, but this finding was slow to be accepted until the elucidation of the structure of DNA with its evident ability to be replicated according to the rules of base pairing and to encode information in the sequence of its nucleotides.

Sugars and Fats

Sugars, starches, oils and fats derived from plants and animals had of course been known for eons and used for nutrition, cooking and other practical applications.

In 1789, François Poulletier de La Salle and Michel Chevreul described a substance that could be extracted by alcohol from bile stones and named it ‘cholesterine’ (cholesterol) from the Greek ‘chole’, meaning bile and ‘stereos’, meaning solid. ⁷ In 1815, Henri Braconnot classified fats into ‘suifs’ (greases) and ‘huiles’ (fluid oils). ⁸ In the next decade, Chevreul developed a more detailed classification, encompassing greases, tallow, waxes, resins, oils and volatile oils, among others. ⁹ In 1847, Theodore Gobley isolated phospholipids from brains and egg yolks. ¹⁰ The identification of more complex forms, such as glycolipids and sphingolipids, came later as did the generic term ‘lipid’, coined in the 1920s by Gabriel Bertrand from the Greek ‘lipos’ (fat). ¹¹

Also in 1789, Lavoisier determined that sugar is composed of carbon, hydrogen and oxygen and that the fermentation of sugar by yeast produced ethanol and carbon dioxide, long exploited in brewing and baking. ^{1 , 12} In 1833, Anselme Payen and Jean-François Persoz discovered the first enzyme activity (‘distase’), ¹³ and in 1839 Payen coined the term ‘cellulose’ from the French word ‘cellule’ for cell. ^{14 , 15}

In 1857, Claude Bernard isolated and introduced the term ‘glycogen’ for the starch-like substance stored in the livers of mammals. ¹⁶ The term ‘carbohydrate’ (French ‘hydrate de carbone’) also originated around this time to describe high molecular weight chains of simple sugars such as glucose, whose composition could be expressed generally as C_n(H₂O)_n.

Proteins: ‘The Locus of Life’

In 1828, Friedrich Wöhler produced urea from ammonium cyanide, which showed that inorganic molecules can be converted into organic compounds ¹⁷ and demolished the widely held belief that the latter could only be produced through some sort of ‘vitalism’. ¹⁸ In 1839, Gerhardus Mulder described substances (fibrin, egg albumin and gelatin) that contain large amounts of C, H, O and N, and also S and P, which he considered ‘the most essential substances of the animal kingdom’. ^{19 , 20} He shared the results with the chemist Jons Berzelius, who suggested that these substances be called ‘proteins’, from the Greek πρωτειος (‘primarium’ or first). ^{19 , 21 , 22}

Mulder’s work also showed that proteins from animals and plants (which played a ‘principal role in their economy’) shared a similar, but varied, atomic composition. ²⁰ Their molecular nature, however, remained nebulous for decades. ^{23 , 24} Although proteins initially represented more a concept than a defined chemical entity, it became gradually accepted that, as major constituents of all organisms, they were central to the processes of life. Indeed, the ‘colloidal nature’ ^a of the substances of living tissues, at the time popularly described as the ‘protoplasm’, ²⁰ exhibited many of the properties that were associated with proteins, and was in fact explicitly regarded a “proteinaceous substance”. ²⁵

This idea was prevalent among the early proponents of Darwinian evolutionary theory. One of the most prominent, Thomas Huxley, defined the protoplasm in 1868 as the “locus of life” and postulated that the physical basis of life (including heredity) lay in this universal biological substance. ^{20 , 26} Huxley remarked: “It may be truly said, that the acts of all living things are fundamentally one” and that “all protoplasm is proteinaceous.” ²⁷

Similarly, in 1871, Charles Darwin cautiously speculated about the role of proteins in the origin of life, while stressing the common ancestry of species:

It is often said that all the conditions for the first production of a living organism are now present, which could ever have been present. But if (and oh! what a big if!) we could conceive in some warm little pond, with all sorts of ammonia and phosphoric salts, light, heat, electricity, &c., present, that a proteine compound [emphasis added] was chemically formed ready to undergo still more complex changes, at the present day such matter would be instantly devoured or absorbed, which would not have been the case before living creatures were formed. ²⁸

During the second half of the 19th century, the diversity of proteins became apparent and the empirical observations made were key to framing the concepts of enzymes and ‘biological specificity’. ^{20 , 29} During this time, Louis Pasteur and others demonstrated the nexus between enzymatic activity and ‘life’ in fermentation, ¹² by then a well-established paradigm of physiological chemistry.

Pasteur also showed, using swan-necked flasks, which allowed air exchange but limited microbial contamination, that life did not arise spontaneously, and noted that organic molecules are chirally left-handed, ^b a fundamental step forward in biochemistry, underpinning, among other things, modern drug development. Also studying fermentation, in 1894 Emil Fischer promoted the importance of stereochemical rules (popularized as the ‘Lock and Key Model’) to explain the interaction of substrate with enzyme, highlighting the central role and exquisite specificity of the “so-called enzymes” in biological processes. ³¹ In 1897, Eduard Buchner demonstrated that yeast extracts ferment alcohol from sugar, showing that biochemical processes do not necessarily require living cells, but are catalyzed by the enzymes formed in cells. ³²

Between 1899 and 1908, Fischer and others, notably Ernest Fourneau, Franz Hofmeister and Albrecht Kossel, made important advances in the understanding of the chemistry of proteins, sugars and nucleic acids, including the description of the peptide bond. The latter was a crucial shift in understanding how animal substances arise, from the traditional view that proteins are acquired from plants to the realization that they are synthesized from a set of constituent parts (amino acids ^c ) incorporated into peptide chains: the ‘peptide theory’, largely promoted by the work of Kossel. ^{20 , 23 , 34 , 35}

It was not until 1926 that James Sumner first purified an enzyme (urease), ³⁶ but proteins were already regarded as the molecules that underlie life processes, the ‘Protein World’. ³⁷ Aleksandr Oparin proposed in 1924 that life originated on Earth through gradual chemical evolution of carbon-based molecules in a “primordial soup”, ³⁸ with John (‘J. B. S.’) Haldane independently advancing a similar theory 5 years later. ^{20 , 38–40} Both suggested in the 1920s that polypeptides were the initial particles of ‘colloidal size’, whereas nucleic acids, which had been discovered 50 years earlier and known to be major components of chromosomes (see below), were not mentioned. ^d The idea that proteins were the primordial molecules was reinforced by the famous experiment by Stanley Miller and Harold Urey in 1953, which demonstrated the formation of amino acids ^e from inorganic molecules in the simulated reducing and highly electrified atmospheric conditions presumed to exist in the primitive Earth. ^{45 , 46}

Biochemistry became a recognized scientific discipline in the 1930s and 1940s with the advent of a better understanding of the structure-function relationships of proteins, driven by physicists using techniques such as X-ray crystallography and isotopic labeling. ²⁰ These advances coincided with the emergence and the coining of the term ‘molecular biology’, ⁴⁷ whose focus is to understand the molecular basis of genetic material and how it determines cellular and organismal phenotypes.

In this sense, molecular biology is distinct from biochemistry, although there is considerable overlap and many biochemists, but perhaps not molecular geneticists or cell and developmental biologists, would assert that the terms are synonymous. ^f In any case both new disciplines were centered on the study of proteins, which heavily influenced the conceptions of genetic information and the mechanisms of heredity and development.

The period from 1930 to 1970 also saw great progress in the characterization of intermediary metabolism, the other heart of biochemistry. ²⁰ The achievements included specification of the glycolytic (fermentation) pathway, the urea/ornithine, citric acid and glyoxylate cycles, ⁴⁹ the pathways for lipid synthesis and degradation, the synthesis of amino acids and complex carbohydrates, and the nucleoside and pentose phosphate pathways for the synthesis of nucleotides and enzymatic cofactors, notably by Hans Krebs, Gustav Embden, Otto Meyerhof (who also discovered the universal energy currency, adenosine triphosphate, ATP), Jakub Karol Parnas, Otto Warburg, Horace Barker, David Green, Peter Mitchell, Ephraim Racker and Salih Wakil, among many others. ^{20 , 50–52}

Nucleic Acids and Chromosomes

The history of nucleic acids ^g began with Friedrich Miescher’s discovery in 1869 of “nuclein”. This was a substance distinct from proteins, isolated from the nuclei of pus cells and characterized by resistance to protease digestion, a high content of phosphorus and the absence of sulfur. ^54–56 Miescher made this discovery in the laboratory of Felix Hoppe-Seyler, who had the experiments repeated and 2 years later published Miescher’s paper together with others describing the isolation of nuclein from various sources. Miescher floated the idea that nuclein might be the genetic material, presumably because it was enriched in sperm, but vacillated on the issue. ⁵⁷ Nuclein was later found to have an acidic nature and was named ‘nucleic acid’ (German ‘nucleïnsäure’) by his student Richard Altmann in 1889. ^{56 , 58 , 59}

Although Miescher’s nuclein was later shown to be deoxyribonucleic acid (DNA), the ‘nuclein’ isolated from yeast by Hoppe-Seyler was not (mainly) DNA but the first description of what would later become known as ribonucleic acid (RNA). ⁵⁵

In 1882, Walther Flemming described fibrous structures in the nucleus and their separation in mitosis, which he visualized by staining and termed ‘chromatin’ (stainable material). ^60–62 Along with Edouard Van Beneden, he also described centrosomes ^{63 , 64} (see Chapter 15), a term introduced by Theodor Boveri in 1888. ^{65 , 66} Later that year, Heinrich Waldeyer termed the nuclear fibers ‘chromosomes’ (stainable bodies) (Figure 2.1). ^{62 , 67}

Figure 2.1

Drawings of chromosome segregation during mitosis by Walther Flemming.

In the years leading up to the turn of the 20th century, Albrecht Kossel showed that nucleic acids are an inherent component of chromatin and identified their constituent nucleoside bases: the ‘purines’ ^h guanine (G) and adenine (A), which have a double ring structure; and the ‘pyrimidines’ cytosine (C), thymine (T) and (what would be later recognized as its RNA counterpart) uracil (U), which have a single ring structure. ^{54 , 56 , 68 , 69}

Chromosomes as the Mediators of Genetic Inheritance

Gregor Mendel’s principles of inheritance involving binary combinations of simple genetic traits were re-discovered around 1900 by the botanists Hugo DeVries, Carl Correns, and brothers Armin and Erich von Tschermak-Seysenegg, ^70–73 and promulgated by William Bateson, who translated Mendel’s 1865 paper in 1901. ⁷⁴ Bateson also coined the word ‘genetics’ (from the Greek gennō, γεννώ; ‘to give birth’) and many other genetic terms that are still in use, such as ‘homozygote’ and ‘heterozygote’. ⁷⁵

Around the turn of the century, Carl Rabi and Boveri ⁱ described chromosome territories, nuclear transplantation in sea urchins and abnormal chromosomes in cancer, leading them to conclude that developmental differentiation is a consequence of regulatory structures within the hereditary material. ^76–79

Based on these and other observations, William Sutton proposed in 1903 that chromosomes “constitute the physical basis of the Mendelian law of heredity” and that “one of the most characteristic features of chromatin is a large percentage content of highly complex and variable chemical compounds, the nucleo-proteids”, ⁸⁰ which had been first described by William Halliburton in 1895. ⁸¹ Shortly thereafter, Wilhelm Johannsen coined the terms ‘gene’, ‘genotype’ and ‘phenotype’, although he did not speculate about the nature of the gene. ^{82 , 83}

Direct evidence for the Chromosome Theory of Inheritance was first provided by the discovery of ‘sex chromosomes’ independently by Nettie Stevens and Edmund Wilson in 1905. ^{84 , 85} In 1914, chromosomes were conclusively demonstrated to be the entities that carry genes ^j by Calvin Bridges ^{86 , 87} working in the laboratory of Thomas Hunt Morgan, alongside Alfred Sturtevant and Hermann Muller, using the powerful fruitfly (Drosophila melanogaster) genetic system ^k that they had developed. ⁸⁸

The previously conceptual genes had found a physical home, a ‘locus’. Morgan, Bridges, Muller and Sturtevant proposed that genes are linearly arranged on chromosomes like beads on a string and suggested that new combinations arise by ‘crossing-over’ (which was observed microscopically) and exchange of genetic material between pairs of chromosomes at meiosis, termed ‘recombination’. ^88–91 On the other hand, Muller’s studies identified many loci (‘allelomorphs’) ⁹² that later turned to encode regulatory elements, including transposon insertions (Chapter 10) (Figure 2.2).

Figure 2.2

Morgan's diagram of Drosophila melanogaster chromosome linkage groups and genetic maps determined by recombination frequency, which “cannot be represented in space in any other way than by a series of points arranged in a line like beads on a (more...)

Analysis of the physical relationships between genetic markers (such as eye color or developmental mutations) relied on measuring the frequency of their co-inheritance in genetic crosses. Co-segregation of markers occurs in 50% of the progeny if the markers are on different chromosomes (or far apart on the same chromosome), but at a higher frequency if the loci are located near each other and separated only by occasional recombination events.

These inheritance patterns gave rise to the concepts of gene ‘linkage’ ^l and ‘linkage groups’ (i.e., genes connected on the same chromosome), and ‘genetic distance’ (the frequency of recombination between linked genes, measured in ‘centimorgans’, or cM ^m ). It also led to the description of phenotypic differences due to ‘cis’ interactions between genes located nearby on one chromosome; ‘trans’ interactions between genes located distally or on different chromosomes; homozygous and heterozygous variants, where the same or different variants (‘alleles’) are present on the parental chromosomes; ‘recessive’ mutations, where one copy of the ‘wildtype’ allele is sufficient for function and masks the presence of the damaged variant; ‘dominant’ mutations, where the mutant allele overrides the ‘normal’ gene; and partial ‘penetrance’ where the frequency of a phenotype differs from normal Mendelian ratios, due to ‘haploinsufficiency’ or the influence of ‘modifier’ genes.

The genetic maps resulting from such crosses led to the conclusion that genes are discrete objects with exclusive borders – a perception that reflects the low resolution of these early studies (and many others to this day). It also led to the idea of the ‘gene for …’, not just physical traits like eye color or, later, human genetic disorders such as cystic fibrosis (Chapter 11), but ultimately also for psychosocial traits, as if all genetic influences could be viewed in binary terms (genes -> traits), which underpinned the subsequent ‘one gene – one enzyme (protein) – one function’ assumption in biochemistry. ^{94 , 95}

The view of genes as particulate entities was reinforced by the work of Nikolay Timoféeff-Ressovsky, who attempted to measure the “radius” of genes, ⁹⁶ which heavily influenced Max Delbrück, who co-authored their ‘Classical Green Pamphlet’ in 1935, ⁹⁷ which was “the starting point” for Erwin Schrödinger’s subsequent ruminations on the physical nature of genetic information (see below) and the “keystone in the formation of molecular genetics”. ⁹⁸

However, Muller had shown by X-ray mutagenesis in 1930 that rearrangements that moved active genes near to heterochromatic regions (Chapter 4) of Drosophila chromosomes resulted in changes in the pattern of expression of these genes, ⁹² called ‘position effect variegation’ (PEV). ⁹⁹ This was also observed by Barbara McClintock and others in maize, in that case with transposition underlying PEV (Chapter 5).

These observations led Muller and Richard Goldschmidt to challenge the conception of genes as distinct entities. ^100–102 Nonetheless, the view of the gene as a discrete unit persisted even in the face of later discoveries that many genes (i.e., DNA segments that express RNA products) overlap or reside within other genes, ^103–110 which has led to difficulties in genome annotation and a barrier to understanding the genome as an information continuum (Chapters 13–16).

Goldschmidt also coined the term ‘phenocopy’ to describe morphological changes in Drosophila that could be induced by imposition of stress during development, insisting on this basis, to little avail, that gene function had to be considered in developmental context ¹¹¹ (Chapter 5). Goldschmidt was considered a heretic by the neo-Darwinists. ^{102 , 112}

The Modern Synthesis

During the first four decades of the 20th century, evolutionary biologists and geneticists, notably Bateson, Haldane, R. A. (Ronald) Fisher, Sewall Wright, Ernst Mayr, G. Ledyard Stebbins, Theodosius Dobzhansky ⁿ and Julian Huxley, among other theoreticians of the time – collectively referred to as the ‘neo-Darwinists’ – brought together Mendelian inheritance, Darwinian gradualism and selection, and statistical genetics into what is known as the ‘Modern Synthesis’, after the title of Huxley’s 1942 book. ¹¹⁵ These pioneers of theoretical population genetics introduced important concepts such as the relevance of population size, genetic drift and the strength of selection, in parallel developing statistical methods and models that have found widespread applications to this day. ^{116 , 117}

A tenet was that all evolutionary phenomena and species diversity can be explained in a way consistent with known genetic mechanisms, ^o with the unifying theme that natural and artificial selection operates on heritable variation arising by random mutation. ^p Biometric population approaches (which showed continuous trait variation) and Mendelian inheritance were reconciled by Fisher and others by invoking polymorphic multi-factorial traits and the ‘infinitesimal model’ of variation and selection. ^{113 , 115 , 122–124}

A corollary of the supposition that mutation occurs randomly is that experience cannot influence the characteristics of the next generation, contrary to the proposal of Jean-Baptiste Lamarck a century before Darwin. ^q As evidence against ‘Lamarckian evolution’, evolutionary biologists recalled the 19th-century work of August Weismann, ¹²⁷ who asserted that somatic cells only received a subset of the information contained in the germline, and that information does not flow in reverse from somatic to germ cells and (therefore) cannot be transmitted to the next generation. ^r The latter came to be known as the ‘Weismann Barrier’, ¹²⁸ but was challenged in the 1940s and 1950s by Conrad Waddington (the father of epigenetics, Chapter 14), who provided evidence of the inheritance (‘genetic assimilation’) of characteristics acquired in response to environmental perturbation ^129–131 (Chapter 5).

The ideas introduced by the Modern Synthesis had a lasting influence on the conceptual landscape of molecular biology and the interpretations of experimental observations. These included the later gene-centric models of genome variation, ‘fitness’ and evolution, and the invocation of ‘junk’ DNA (Chapter 7), as well as the concept that the sequences of important genes are ‘conserved’ during evolution, which has frequently been assumed in efforts to discriminate functional and non-functional regions of genomes (Chapter 11).

There was considerable cross-fertilization in the 1940s and 1950s between theoretical evolutionary biologists and microbial geneticists, notably Delbrück ^s and Joshua and Esther Lederberg, assessing lethal/competence phenotypes in bacteria and their viruses, which would lend support to the spontaneous and random nature of mutations as opposed to ‘pre-adaptations’ or ‘directed mutations’ ¹³⁴ – a debate at the time.

Equally if not more importantly for the understanding of genetic programming in the following decades, the emphasis on lethal loss-of-function mutations overlooked the differences between essential genes and variations in regulatory (non-protein-coding) sequences that may have no effect on the viability of plants and animals but are the major drivers of quantitative trait variation, adaptive evolution, survival and reproductive success (Chapters 7 and 11).

These assumptions – that inheritance occurs entirely through Mendelian genes and mutations occur randomly – became entrenched before virtually anything was known about the nature of genetic variations and their phenotypic impact, especially in relation to the ‘complex traits’ that have large environmental components, and resulted, in part, in the lack of appreciation of Barbara McClintock’s later work on mobile genetic elements (Chapter 5). They also underpinned the dichotomy of the classical and historical ‘Nature versus Nurture’ ^t debates that raged for decades, not realizing or even considering that such complex phenotypes may be the integrated outcomes of overlapping genetic and epigenetic processes.

The concept of genetic determinism overflowed into sociological and political arenas, notably the common and at the time fashionable idea ^u (notably, extending from observations of selective breeding in agriculture and of domesticated animals by Darwin and others) that there are superior and inferior human characteristics (especially intelligence) that vary between individuals and ‘races’ ¹³⁷ and might be improved by selective breeding (promoted by Francis Galton, who coined the term ‘eugenics’) or, worse, by sterilization, as occurred in the USA and by genocide in Nazi Germany. Genetics was also politicized in Stalinist Russia by Trofim Lysenko, who favored Lamarckian evolution in line with communist ideology, which led to the abolishment of population genetics and the persecution of geneticists and other scientists in the USSR. ^{138 , 139}

Distinguishing DNA and RNA

During the same period, research on nucleic acids was surprisingly limited, despite the finding that they are a major component of chromosomes, the repositories of genetic information. ¹⁴⁰ Indeed, nucleic acids barely feature in the history of biochemistry before 1940, ²⁰ a consequence of the prevailing view that proteins have greater chemical diversity than the assumed monotony of nucleic acids, thought to be merely structural or metabolic entities.

This view was strengthened by the ‘tetranucleotide hypothesis’ put forward in 1909 by the chemist Phoebus Levene, who identified ribose sugars in ‘yeast nucleic acid’ ^{141 , 142} and deoxyribose sugars in calf thymus gland. ^143–145 Levene proposed that phosphate-sugar-base units formed circular tetramers containing each of the four ‘nucleotides’, based on the perception that they are present in equimolar proportions. ^{54 , 142 , 145–147}

Levene’s work in identifying the five common nucleotides (A, G, C, T/U) and the phosphodiester bonds between them was a major advance, ^{145 , 147} but the tetranucleotide hypothesis implied that nucleic acids could not carry complex information. However, during the 1920s and 1930s, Einar Hammarsten showed that the molecule later recognized as DNA has a very high molecular weight, using a gentler method for its isolation that avoided harsh treatment with alkali. ¹⁴⁸ Robert Feulgen (who discovered the eponymous DNA stain, see below), as well as Hammarsten and his student Erik Jorpes, then demonstrated that nucleic acid (RNA) from pancreas contained more guanine than other bases. ^{54 , 149}

These observations did not fit with a simple equimolar tetranucleotide structure (although Jorpes tried to rationalize the guanine excess within a pentanucleotide structure ¹⁴⁹ ), but were not recognized and Levene’s hypothesis held sway for decades. ^{140 , 145 , 147} In addition, there was a perception that the different types of nucleic acids do not occur in all organisms, which argued against universal biological roles. One form, the ‘thymonucleic acid’ or ‘zoonucleic acid’ had been isolated from animal glands, pus cell nuclei and sperm and was initially thought to be present exclusively in animals; the other, known as ‘pentose nucleic acid’, ‘zymonucleic acid’, ‘yeast nucleic acid’ or (plant) ‘phytonucleic acid’, was thought to be absent from animal cells until the early 1940s. ^{54 , 150 , 151} Thus, there was confusion about the distribution and functions of what later became known as DNA and RNA.

It was also shown during the same period that DNA and RNA have differences in their nucleoside base composition (with DNA containing adenine, guanine, cytosine, thymine, whereas RNA contains adenine, guanine, cytosine and uracil, the demethylated analog of thymine) and in the sugars in their sugar-phosphate backbone, deoxyribose in DNA and ribose in RNA, hence ‘deoxyribonucleic acid’ and ‘ribonucleic acid’. ¹⁵²

Erwin Chargaff, who later observed the purine-pyrimidine equivalences that underpinned the base pairing critical to the elucidation of the structure of DNA and the templating of messenger RNA, pointed out in 1950 that

Although only two nucleic acids, the deoxyribose nucleic acid of calf thymus and the ribose nucleic acid of yeast, had been examined analytically in some detail, all conclusions derived from the study of these substances were immediately extended to the entire realm of nature; a jump of a boldness that should astound a circus acrobat. ¹⁵³

In 1924, Feulgen and Heinrich Rossenbeck developed a sensitive histochemical reaction that intensely stained DNA. ¹⁵⁴ It demonstrated that DNA is present in the nucleus, but not the cytoplasm, ^v of plants and animals, and constituted important evidence for DNA as the possible genetic material. ¹⁵⁵ Jean Brachet, perhaps the most important of the early RNA biochemists, developed new methods to differentiate DNA and RNA in the 1930s and early 1940s, using basic dyes and cytochemical staining, later combined with ribonuclease treatments (Figure 2.3). ^156–160 His work showed that both DNA and RNA are universal constituents of animal and plant cells and that RNA, unlike DNA, is mainly located in the cytoplasm. Later, it was shown in bacteria that, while the DNA composition varies between different species, both DNA and RNA are always present. ^{161 , 162}

Figure 2.3

Jean Brachet's photographs of the perturbation of amphibian morphogenesis by RNase treatment. (Reproduced from Brachet, with permission from Elsevier under Creative Commons 4.0 license.)

Although DNA was linked with chromosomes, the function of RNA was for a long time a mystery. It was speculated that RNA might serve as an energy repository or a cytoplasmic precursor converted into DNA during cell division (the ‘conversion hypothesis’). ¹⁶⁰ It was also unclear what form RNA might take, since the 2′ hydroxyl group of ribose represented a potential bifurcation point, such that RNAs could exist as branched molecules. Only later, in the 1950s, was this idea dispensed with, when studies by Alexander Todd and others demonstrated that RNAs are linear polymers ^w whose nucleotides, as in DNA, are linked by 3′-5′ phosphodiester bonds. ^{165 , 166}

Confirmation of the role of RNA as an information molecule emerged from the study of plant and animal viruses that contained RNA but not DNA. In 1937, Frederick Bawden and Norman Pirie identified RNA in tobacco mosaic virus (TMV), ¹⁶⁷ despite the previous suggestion by Wendell Stanley, who had crystallized TMV, that protein was the active ‘auto-catalytic’ agent of the virus. ¹⁶⁸ Later experiments revealed that the TMV and other plant viruses, as well as foot-and-mouth disease virus and other animal viruses, contained RNA as the sole nucleic acid, which indicated that RNA must act the template for viral replication and protein synthesis. ^{169 , 170}

One Gene-One Protein and the ‘Nature of Mutations'

The association between ‘genes’ and proteins dates back to 1902, when Archibald Garrod provided evidence for the inheritance of human disorders and phenotypes that reflected enzymatic deficiencies, such as alkaptonuria. ¹⁷¹ The causes of the enzyme deficiencies in such ‘inborn errors of metabolism’ (which included other congenital disorders, such as albinism) were still only vaguely defined, ¹⁷² as they preceded the conceptual terms ‘gene’ and ‘genome’. ^x

Indeed, even George Beadle and Edward Tatum’s influential 1941 ‘one gene – one enzyme’ principle, ¹⁷⁴ based on studies of mutants of the filamentous fungus Neurospora crassa (red bread mold) (Figure 2.4), ^y was not well accepted before the nature of hereditary material was understood. ¹⁷⁸ The phrase subsequently morphed into ‘one gene – one protein’ when it was realized that not all proteins are enzymes, and even this changed when it was found much later that, in the higher eukaryotes, one gene can produce variations of the same protein by alternative splicing (Chapter 7).

Figure 2.4

George Beadles' ‘lantern slide' explaining the procedure he and Edward Tatum used to isolate metabolic mutants of the filamentous fungus N. crassa, showing complementation and Mendelian segregation of the affected gene. (Reproduced from Horowitz (more...)

Beadle and Tatums’ work, nevertheless, united genetics and biochemistry. ^{94 , 181} It also popularized the use of microbes as model organisms. ¹⁸² In 1946, Tatum and one of his students, Joshua Lederberg (with whom he and Beadle later shared the Nobel Prize) ^z demonstrated genetic recombination in the enteric bacterium Escherichia coli, ¹⁸⁴ which consequently became widely used as a model organism. Moreover, most genetic studies were focused on lethal, conditionally lethal (such as in auxotrophy ^aa or temperature-sensitivity) and/or phenotypically severe mutations, especially in haploid cells (bacteria and haploid Neurospora and yeast), which are overwhelmingly biased to protein-coding sequences (Chapter 7).

At the time, proteins were thought to comprise genes, rather than be the products of them. DNA was considered to have only peripheral functions, such as serving as an “intra-nuclear buffer”, ¹⁶⁰ or acting as a scaffold during gene replication. The latter was championed by the small community of scientists then engaged in nucleic acids research, such as Hammarsten and Torbjörn Caspersson. ¹⁴⁰ Caspersson, whose work was also instrumental in establishing the involvement of RNA in protein synthesis, proposed a metabolic relationship between nucleic acids and “gene reproduction”, noting that “synthesis of nucleic acid is closely connected with gene reproduction”, and that “it may be that the property of a protein which allows it to reproduce itself is its ability to synthesize nucleic acid”. ¹⁸⁵ However, his conception was that the “structure-forming properties” of DNA (alluding to the high molecular weight DNA polymers earlier demonstrated by Hammarsten) was simply auxiliary to the basic proteins of the nucleus. ^{140 , 185}

Research on genetic material up until World War II, therefore, concentrated on proteins as the prime candidate for the genetic material, the “protein version of the central dogma”. ⁵⁴ Even the first reported infectious agents of bacteria, characterized as “filter-passing viruses” (later termed bacteriophages or ‘phages’ for short, soon to play a major role in the understanding of genes, Chapter 3), were considered to be “enzymes with power of growth”. ¹⁸⁶

DNA Is the Genetic Material

In 1944, the physicist Erwin Schrödinger wrote a book entitled What Is Life?, in which he made the logical deduction that the genetic material would be comprised of an “aperiodic crystal” – that is, a molecule of regular structure with information embedded in its fine-scale variations – a “miniature code”, the first use of the term ‘code’ in relation to biology. ¹⁸⁷

Schrödinger’s prediction was borne out in the same year ^bb by Oswald Avery, Colin MacLeod and Maclyn McCarty, who built on the studies of bacterial ‘transformation’ by Fred Griffith in the late 1920s ¹⁸⁹ and showed that the change from a benign to a virulent form of the bacterium Streptococcus pneumoniae could be effected by DNA but not by protein. ^{54 , 190} Their finding was confirmed in E. coli a year later by André Boivin. ¹⁹¹

However, such was the entrenchment of prior expectations ¹⁴⁰ that it took almost 10 years for the conclusion to be widely accepted, ¹⁹² and “even Avery himself was reluctant to accept it until he had buttressed his experiments with the most rigorous controls”. ¹⁴⁴ Arguing that a small amount of contaminating proteins could be present in Avery’s preparations, not only Hammarsten, but also other eminent scientists, especially Alfred Mirsky, were unconvinced that DNA was the genetic material. ^{140 , 144 , 193} James Watson said, in retrospect, that (unfortunately) “most people didn’t take him [Avery] seriously”. ¹⁹⁴ Moreover, as Gunther Stent observed later, ¹⁹² Avery’s finding was “premature”. ^cc General acceptance only came after the 1952 experiments by Alfred Hershey and Martha Chase that showed the uptake of ³² P-labeled DNA, but not ³⁵ S-labeled proteins, into bacteria after infection with bacteriophage T4, ^{190 , 195} and the elucidation of the structure of DNA in 1953.

Levene’s tetranucleotide structure was finally and convincingly refuted by Chargaff’s demonstrations in the late 1940s that DNA “formed extremely viscous solutions in water” (confirming Hammarsten’s earlier observations), implying a structure much larger than a tetranucleotide, and by paper chromatography ^dd that its constituent bases were not present in equimolar proportions. ¹⁵³ Instead, Chargaff showed that the pyrimidine (T, C) and purine bases (A, G) are present in equal amount in DNA, and that A and T, as well as G and C, occurred in the same proportions. ^ee That is, %G=%C and %A=%T, and that the ratios of these pairs of nucleotides (%G+C/%A+T) were the same in different tissues of the same organism, but varied between organisms. ^207–209

The Double Helix – Icon of the Coming Age

Chargaff’s data was crucial for Watson and Crick’s interpretation of the X-ray diffraction patterns of DNA fibers obtained by Rosalind Franklin ^ff and Raymond Gosling, building on work by others, ^gg notably Bill Astbury and Florence Bell who more than a decade earlier had determined the planar structure of, and the ~3.4 angstrom spacing between, the bases (stacked like “a pile of plates”), ^210–213 which led to the elucidation of its double-helical structure. ^214–216

As is well known, and was enormously compelling at the time, ²¹⁷ although it took a few years for its significance to be widely appreciated, ²¹⁸ this structure is governed by nucleotide base (purine-pyrimidine) pairing rules, which immediately suggested a mechanism for the duplication of genetic information. ²¹⁴ This was subsequently demonstrated in 1958 by Matthew Meselson and Franklin Stahl using labeling with heavy isotopes of nitrogen to distinguish the template from newly synthesized DNA strands in buoyant density gradients. ²¹⁹ An enzyme mediating the synthesis of new, complementary strands was discovered by Arthur Kornberg and colleagues in 1956 and termed DNA-dependent DNA polymerase. ^220–222

Not so well known is that John Masson Gulland, Denis Jordan and colleagues had shown in 1947 that DNA is held together by hydrogen bonds, ²²³ and that their PhD student James Creeth had, in his 1948 PhD thesis, proposed a model for the structure of DNA comprising two chains with a sugar-phosphate backbone on the exterior and hydrogen-bonded bases between the nucleotide bases of opposite chains in the interior (Figure 2.5). ^{224 , 225}

Figure 2.5

(a) Photograph 51 of B-DNA. X-ray diffraction photograph of a DNA fiber at high humidity (Franklin and Gosling, 1953). Interpretation of the helical-X and layer lines added in blue. (b) Watson-Crick model of B-DNA. (Adopted from Watson and Crick, with (more...)

Acceptance of the DNA as the genetic material and the significance of its double-helical structure in genetic inheritance and gene expression came only after concerns about the ability of its strands to unwind had been resolved by the Meselson and Stahl experiment, and a plausible mechanism for the expression of genetic information (i.e., RNA-templated protein synthesis) had been established ^{218 , 227} (Chapter 3).

A subtle but important feature of the structure of DNA ^hh is that its strands are antiparallel, with both strands going from 5′ to 3′ (with respect to the phosphate linkages that connect the sugar-phosphate backbone) but in the opposite direction to each other, with DNA replication, RNA transcription and protein synthesis all proceeding from 5′ to 3′ in relation to the sugar-phosphate linkages. The linear arrangement of the bases along the backbone of the helix was fundamental to the logical deductions and experimental approaches to deciphering the genetic code.

The principles of copying/reading by base pairing and the directionality of the information were critical for understanding the synthesis and roles of RNA. Similarly, the subsequent demonstration by Julius Marmur, Paul Doty and colleagues that complementary strands of DNA (and RNA transcribed from the DNA) could recognize each other by base pairing ^{233 , 234} played an important part in the identification of messenger RNA and in the first analyses of genomic sequence composition and complexity (Chapter 3).

Further Reading

1. Cairns J. (2008) The foundations of molecular biology: A 50th anniversary. Current Biology 18: R234–36. [PubMed: 18364220]
2. Cobb M. (2014) Oswald Avery, DNA, and the transformation of biology. Current Biology 24: R55–R60. [PubMed: 24456972]
3. Crow J.F. (2005) Hermann Joseph Muller, evolutionist. Nature Reviews Genetics 6: 941–945. [PubMed: 16341074]
4. Harman O.S. (2005) Cyril Dean Darlington: The man who ‘invented’ the chromosome. Nature Reviews Genetics 6: 79–85. [PubMed: 15630424]
5. Horder T.J. (2006) Gavin Rylands de Beer: How embryology foreshadowed the dilemmas of the genome. Nature Reviews Genetics 7: 892–898. [PubMed: 17047688]
6. Hunter G.K. (2000) Vital Forces: The Discovery of the Molecular Basis of Life (Academic Press, New York).
7. Huxley J. (1942) Evolution: The Modern Synthesis (George Allen & Unwin Ltd, New York).
8. Kohler R.E. (1975) The history of biochemistry: A survey. Journal of the History of Biology 8: 275–318. [PubMed: 11609896]
9. Morgan T.H. (1922) Croonian Lecture:—On the mechanism of heredity. Proceedings of the Royal Society B: Biological Sciences 94: 162–197.
10. Nature conference: Thirty years of DNA. Nature 302: 651–654 (1983). https://doi​.org/10.1038/302651a0. [PubMed: 6835401]
11. Nilsson E.E., Maamar M.B. and Skinner M.K. (2020) Environmentally induced epigenetic transgenerational inheritance and the Weismann Barrier: The dawn of Neo-Lamarckian theory. Journal of Developmental Biology 8: 28. [PMC free article: PMC7768451] [PubMed: 33291540]
12. Olby R. (1994) The Path to the Double Helix: The Discovery of DNA (Dover Publications, New York).
13. Satzinger H. (2008) Theodor and Marcella Boveri: Chromosomes and cytoplasm in heredity and development. Nature Reviews Genetics 9: 231–238. [PubMed: 18268510]
14. Stent G.S. (1972) Prematurity and uniqueness in scientific discovery. Scientific American 227: 84–93. [PubMed: 4564019]
15. Strauss B.S. (2016) Biochemical genetics and molecular biology: The contributions of George Beadle and Edward Tatum. Genetics 203: 13–20. [PMC free article: PMC4858768] [PubMed: 27183563]
16. Teich M. and Needham D. (1992) A Documentary History of Biochemistry 1770–1940 (Leicester University Press, New York).
17. Williams G. (2019) Unravelling the Double Helix: The Lost Heroes of DNA (Weidenfeld & Nicolson, New York).