Biology: Genetics: DNA and the Genetic Code

Introduction

On February 28, 1953, two molecular biologists, American James Watson (1928–) and Englishman Francis Crick (1916–2004), announced to the lunchtime regulars at the Eagle Pub in Cambridge that they had discovered life's secret: the chemical structure of DNA, deoxyribonucleic acid. Their realization that DNA was a double helix of two sugar-phosphate backbones on the outside, with paired nucleic acids adenine, guanine, cytosine, and thymine held together by hydrogen bonding on the inside was indeed stupendous. DNA's spiral staircase structure is an icon of science, appearing even as a pattern on the doorknobs of the entrance doors to the Royal Society in London.

Watson and Crick's breakthrough was also the intellectual culmination of many past discoveries in molecular genetics. Johann Friedrich Miescher's (1844–1895) isolation of DNA in the 1870s led to Watson and Crick's discovery for which they and another molecular biologist, New Zealand-born Briton Maurice Wilkins (1916–2004) won the Nobel Prize for physiology or medicine in 1962. The work of Rosalind Franklin (1920–1958) was also invaluable to their discovery. Understanding DNA's structure allowed scientists to comprehend how the genetic code works.

Historical Background and Scientific Foundations

The study of heredity began as a response to Jean-Baptiste Lamarck's (1744–1829) theory of acquired characteristics. This proposed that traits acquired by an individual during its lifetime, whether in response to environmental conditions or caused by its own habits, would be inherited by its offspring. In 1856 an Austrian monk named Gregor Mendel (1822–1884) began to test Lamarck's theories with experiments in hybridizing pea plants.

In contrast to Lamarckian predictions, he found that particular characteristics, such as flower color or seed shape, were passed on to offspring by special cells, which he called “factors,” that determined how those traits were expressed. Through his work, Mendel was able to link an organism's phenotype (its external appearance) to its inherited genotype (its genetic inheritance) and illustrate the effects of dominant and recessive genes. He showed that these factors were inherited in predictable mathematical ratios.

Because Mendel worked on the margins of the scientific community, his findings remained unknown until 1900, when German botanist and geneticist Carl Correns (1864–1933), Dutch botanist Hugo de Vries (1848–1935), and Austrian botanist Erich von Tschermak-Seysenegg (1871–1962) independently discovered Mendel's achievements. While crediting de Vries, Correns connected earlier discoveries about meiosis (the process by which sex cells—egg and sperm—reproduce) and Mendel's laws of inheritance to propose the chromosome theory of heredity, concluding that “each germ cell must receive one or other of the factors that Mendel held responsible for dominant or recessive traits.” Within the cell, genes were strung together on the chromosomes like beads in a necklace, copying themselves during meiosis and separating as the cell divided.

The Discovery of DNA: Johann Friedrich Miescher and Nuclein

Scientists still didn't understand how genetic information in the old chromosome was transferred to the new, however. In 1871, Johann Friedrich Miescher, a Swiss professor of pathology at the University of Basel, identified DNA as one biochemical component of chromosomes. Miescher extracted cell nuclei from white blood cells or lymphocytes that he got from used hospital bandages, since they were filled with pus. While pursuing his research, Miescher realized that the cells' nuclei contained something other than proteins. This strange material did not react with the digestive enzyme pepsin, showing it was not protein in nature. This substance, which he called “nuclein”—renamed nucleic acid by German pathologist Richard Altmann (1852–1900) in 1889—was present in a large variety of cells. He found that it contained phosphorus in addition to carbon, hydrogen, nitrogen, and oxygen. This substance turned out to be DNA. Though Miescher speculated that his “nuclein” could be involved in fertilization in some way, he did not take his hypotheses any further.

Despite the discovery of nuclein, many biologists continued to believe that genetic material was protein in nature. Chromosomes do contain a good deal of protein, and proteins seemed good candidates to be the molecule that contained the genetic code as they were made of up to 20 different amino acids. DNA, on the other hand, contained only four nucleotides, so it was considered too simple to contain the genetic code.

The Work of Oswald Avery, Colin Macleod, and Maclyn McCarty in the 1940s

Miescher's work was largely ignored until the 1940s, with proteins deemed the source of genetic material. As Johns Hopkins biology professor Bentley Glass commented,

“The dismal blindness of scientists to the significance of a chemical substance [nucleic acid] so uniquely limited to the nucleus, and indeed to the very chromosomes themselves, endured until 1944, when the work of Avery, Macleod and McCarty on the transformation phenomenon in Pneumococcus at last reawakened geneticists to the importance of DNA.”

What were their experiments? British microbiologist Frederick Griffith (1881–1941), working with two types of pneumococcus, discovered that the bacteria could be made to change immunological specificity. One was the virulent S strain, which had a smooth cell membrane or “smooth polysaccharide coat.” The other was the considerably milder, rough-coated R strain; its Petri dish colonies actually had ragged edges.

When Griffith injected mice with the S-type bacteria, they died quickly; injecting another group with the R-strain did not affect their health. He then took the virulent S-bacteria and subjected it to intense heat, which seemed to destroy its ability to infect mice. But when he mixed the heat-treated S-type with the milder rough-coated strain and injected this mixture into mice, the mice died in a few days. Blood samples from the dead mice showed high levels of virulent pneumococcus, leading him to conclude that something caused the mild strain to adopt a new smooth polysaccharide coat and become lethal. The source of this genetic transformation was unknown, however.

In 1943, though he was near retirement, Canadian-born American bacteriologist Oswald Avery (1877–1955), a physician and researcher at the Rockefeller Institute, decided to investigate which agent caused this bacterial transformation. Using centrifugation, he and his coworkers Maclyn McCarty (1911–) and Colin MacLeod (1909–1972) used centrifugation to remove the large cellular structures from S-type bacteria, which they treated with proteases, enzymes that removed the proteins from the cells, then mixed the S and R strains together.

The R-bacteria were transformed into a virulent strain, so proteins could not carry the genes. The rest of the S bacteria were treated with an enzyme that removed the DNA; when this was mixed with the R bacteria there was no change. This meant that DNA contained cellular hereditary information. Avery, McCarty, and MacLeod thus demonstrated that DNA, not proteins, contained a cell's genes. DNA's physical structure, however, was still a mystery.

Determining the Structure of DNA

Before World War II (1939–1945), two chromatographers, Irish crystallographer John Desmond Bernal (1901–1971) at Cambridge and English physicist William Astbury (1898–1961) at Leeds, used x-ray diffraction to determine crystals' molecular structure. The crystal's atomic planes caused entering x-ray beams to interfere with one another as they left the crystal. The interference patterns revealed the molecule's structure.

Using hundreds of x-ray diffraction pictures, Astbury built a DNA model to see how its sugar and phosphate bases fit together. The results convinced him that the molecule's bases were stacked on each other like a pile of pennies, spaced 3.4 Angstroms apart (an Angstrom [Å], equals one ten-billionth of a meter).

The remaining question of DNA's structure would be partially answered by Erwin Chargaff (1905–2002), an Austrian biochemist and war refugee working at Colombia University. Having read Avery's paper about bacterial transformation in 1944, he turned his attention to the study of DNA and its four chemical bases, or nucleotides: adenine (A), cytosine (C), guanine (G), and thymine (T). Using chromatography to analyze plant and animal tissue, Chargaff realized that the proportions of A and T were almost the same as those of C and G.

The total amount of the pyrimidines molecules (T and C) were also equal to the total amount of the purines (A and G). Although Chargaff sensed that this 1:1 ratio was important, he did not understand how it related to DNA's overall structure.

The Question of Rosalind Franklin

Rosalind Franklin (1920–1958) was an x-ray crystallographer who until 1951 was known primarily for her groundbreaking work with the crystalline structure of coal and other carbons. Educated at Cambridge, she received her doctorate in 1945, and worked at the Laboratoire Central des Services Chimique de L'Etat in Paris. In 1951 Franklin left coal research to go to King's College, London, to study the structure of DNA with Maurice Wilkins (1916–2004).

Franklin discovered that there were two different forms of DNA, A and B, which had to be separated to get clear x-ray diffraction images of each. Franklin even developed a special camera to improve the DNA-fiber orientation in the x-ray beam to photograph the B form. One picture from this apparatus, photograph 51, verified Astbury's work, showing that each helical curve in B-form DNA was 34Å long and contained 10 base pairs.

From her data, Franklin proposed a double-helix structure for the DNA molecule, with sugar phosphates composing its external backbone, and hydrophobic (water-hating) base pairs of purines and pyrimidines on the inside. In 1951, James Watson attended a talk she gave on her work. The following year, he was also given a copy of a report of Franklin's work in this area by one of Franklin' co-workers. This proved instrumental to his and Crick's later discovery of DNA's structure.

James Watson and Francis Crick

Watson and Crick, based at Cambridge University, were also working on the DNA problem. Crick, trained as a physicist, had designed circuits for acoustic and magnetic mines for the British Admiralty during World War II. Watson, 12 years younger, had studied zoology and microbiology at Indiana University. After meeting Maurice Wilkins at a symposium and seeing the x-ray diffraction pattern of crystalline DNA, Watson changed research directions. In October 1951 he took a fellowship to work on DNA with Crick at the Cavendish laboratory at Cambridge.

Their first attempts in November 1951 were not successful, based as they were on the ideas of American biochemist Linus Pauling (1901–1994), who was also close to solving the structure. Pauling had discovered in 1948 that a large number of proteins were shaped like a spring coil, termed the alpha-helix. Watson and Crick, taking Pauling's idea of a helix but misinterpreting Franklin's data, initially proposed a model in which the nucleotide bases were on the outside of the helix. So unpromising was their initial model that laboratory chiefs told Watson and Crick to abandon their efforts. Pauling incorrectly proposed a triple-helical structure in January 1952, and Watson persuaded his bosses to let the model building resume at Cambridge before Pauling saw his error and corrected it.

The data from Franklin's 1951 lecture had convinced Watson and Crick that the nucleotide bases belonged on the inside of the DNA molecule. Chargaff, who consulted with the duo, later recalled in his work Heraclitean Fire: Sketches from a Life before Nature.

So far as I could make out, they wanted, unencumbered by any knowledge of the chemistry involved, to fit DNA into a helix. The main reason seemed to be Pauling's alpha-helix model of a protein. I told them all I knew. If they had heard before about the pairing rules, they concealed it. But as they did not seem to know much about anything, I was not unduly surprised. I mentioned our early attempts to explain the complementarity relationships by the assumption that, in the nucleic acid chain, adenylic was always next to thymidylic acid and cytidylic next to guanylic acid. I believe that the double-stranded model of DNA came about as a consequence of our conversation; but such things are only susceptible of a later judgment.

In 1953, Watson visited King's College, London, where Franklin and Wilkins worked. Without her knowledge, Wilkins showed Franklin's photograph 51 to Watson. Franklin's colleague and member of a research oversight committee, Austrian-born chemist Max Perutz (1914–2002), also gave Watson a copy of Franklin's 1952 MRC Report. Watson later admitted, “Rosy [Franklin], of course, did not directly give us her data. For that matter, no one at King's realized they were in our hands.”

After seeing photograph 51 and the MRC report and consulting with Chargaff, Watson and Crick had all the pieces of the DNA puzzle. Crick realized, as Franklin did not, that while the molecule consisted of two spiral chains in a helix, the strands ran parallel to each other—in opposite directions. Watson, now knowing

the base pairs were inside the molecule, made cardboard cutouts of the four DNA bases, and put them together in various ways. When he noticed that an A—T pair matched the shape of a G—C pair, he realized that in the DNA double helix, a base on one side is matched by its opposite on the other. When separated, each half of the chain becomes the template for a new, identical sequence.

Watson, Crick, and Wilkins shared the 1962 Nobel Prize for medicine or physiology for their discovery. Like Marie Curie before her, however, Rosalind Franklin's work had exposed her to high doses of radiation before its dangers were known; she died of ovarian cancer in 1958. Despite her enormous contributions to the research, she was not mentioned by the prize committee, as the Nobel is not awarded posthumously. Cambridge University later named a building in Newnham College in her honor.

After DNA: Deciphering the Genetic Code

Once the structure of DNA had been determined, the challenge in the late 1950s and early 1960s was to determine how its information expressed genetic traits. How do the four nucleic acids code for specific proteins that comprise the cell's working parts? As Crick noted in his Nobel lecture,

It now seems certain that the amino acid sequence of any protein is determined by the sequence of bases in some region of a particular nucleic acid molecule. Twenty different kinds of amino acid are commonly found in protein, and four main kinds of base occur in nucleic acid. The genetic code describes the way in which a sequence of twenty or more things is determined by a sequence of four things of a different type.

The main issue in cracking the genetic code was 1) to determine how many base pairs would code for the 20 amino acids that composed proteins, and 2) discover what series of bases specified which amino acids. Simple mathematics indicated that because there were 20 possible amino acids, and there were four bases (A, T, C, G), there had to be at least three bases, a codon, to code for each protein. As Crick again noted,

This can hardly be done by a pair of bases, as from four different things we can only form 4 × 4 = 16 different pairs, whereas we need at least twenty and probably one or two more to act as spaces or for other purposes. However, triplets of bases would give us 64 possibilities. It is convenient to have a word for a set of bases which codes one amino acid and I shall use the word “codon” for this.

But could this theoretical supposition be proven in the laboratory? In a 1961 experiment, Crick and his colleague, South African-born biologist Sydney Brenner (1927–), created genetic mutations in the DNA of a bacteriophage, a virus that infects bacteria. Crick and Brenner induced the mutations using a chemical called proflavine to change individual bases in the DNA, destroying the function of a particular and crucial phage gene. They found that if there were two or four mutations together, the gene was still inactive, but if three mutations were put together in the same gene, the gene started working. In other experiments, Crick and Brenner used proflavine to delete bases one by one in the DNA, demonstrating that only after three bases next to each other were eliminated did the DNA transcribing system come back to its correct phase, and the gene would begin to work again.

Their results indicated that the genetic code was one in which three bases coded for an amino acid. As DNA unwinds during replication, it is read by a molecule called RNA polymerase; this, in turn, makes a template for protein synthesis called messenger RNA (mRNA), a process called transcription. An organelle called a ribosome then “reads” each codon in the mRNA and another carrier molecule called transfer RNA (tRNA) brings the appropriate amino acid called for by the

codon. The amino acids are then linked together to make the protein by the ribosome. This process is called translation.

After Crick and Brenner's discovery, a key question remained: Which particular triplet of nucleotides (codon) coded for which particular amino acid? In 1961 American zoologist Marshall Nirenberg (1927–) and German biochemist Heinrich Matthaei, working at the National Institutes of Health in Bethesda, Maryland, finally cracked the genetic code. Nirenberg took Escherichia coli (E. coli) bacteria and ground them up with a mortar and pestle to release their cytoplasm, which contained all the organelles, such as ribosomes, needed for protein synthesis.

Nirenberg then made a synthetic RNA made of just the base uracil and combined it with the E. coli cytoplasm to see which amino acid would be created. This created a protein chain made only of phenyalanine, so Nirenberg realized the codon coded for phenylalaline. Similar experiments revealed which codons coded for the nineteen other amino acids. By 1965, Nirenberg understood the language of DNA.

Modern Cultural Connections

Starting in the 1960s and 1970s, it became possible to both read off the base sequence of DNA from specific cells and to manipulate DNA by adding, altering, or deleting genes in living organisms. These technologies have had profound scientific and cultural impacts.

The ability to decipher (sequence) DNA base by base, which has become rapidly cheaper and quicker in recent years thanks to new laboratory techniques and computer controls, has allowed the rapid discovery of new genetic knowledge. In biology, the evolutionary relationships between species are being rapidly clarified by examining the precise differences between their DNA. Detailed trees of evolutionary relationship are being worked out based on the examination

of individual genes and (by the early 2000s) of entire genomes. Strong confirmatory evidence for evolutionary theory, along with much new detail about the mechanisms of evolution, has been provided by DNA-based methods. These developments are socially important in societies such as the United States, where over 40% of the population still believes some form of Creationism (the belief that living things came into

being at least partly by miraculous processes, not by evolution).

The ability to sequence DNA has also impacted many individual lives by application to criminology. In many cases, criminals leave hair, skin cells, blood, or semen at a crime scene: all these substances contain DNA. Since each person's DNA is unique (except in the case of identical multiple births such as identical twins), comparing DNA from a crime scene with the DNA of an accused or convicted person can prove guilt or innocence. As of early 2008, over 210 persons convicted of rape or murder had been proven innocent by DNA evidence since 1992. On the other hand, there has been concern among citizens and medical ethicists that insurance companies will deny coverage to people based on defects in their DNA, leading to concerns over DNA privacy and discrimination.

Technologies allowing the manipulation of DNA have also given rise to new dilemmas and debates. The genetic alteration of crop plants and livestock is controversial, with corporations hoping for higher profits, proponents claiming benefits for consumers, and critics claiming that such technologies are an irresponsible interference with the basis of life that may get out of control as altered genes make their way into the environment. The manipulation of human DNA is particularly contentious: some persons believe that such technologies will allow humans to control their own evolution, visualizing the enhancement of height, intelligence, strength, or other characteristics. Others see such a path as fundamentally wrong or dangerous, possibly leading toward the creation of a genetically-modified elite in a world already rife with inequality. Botched attempts to engineer the human genome would, on the other hand, cause suffering in children endowed with modified genes. The manipulation of human DNA for the cure or treatment of genetic diseases (gene therapy) is less controversial, but has proven difficult to translate into safe clinical practice. No safe and effective DNA-altering treatment for a genetic disease was yet available as of 2008.

See Also Biology: Cell Biology; Biology: Evolutionary Theory; Biology: Genetics; Biology: Genetics: The Human Genome.

bibliography

Books

Watson, James D. The Double Helix: A Personal Account of the Discovery of the Structure of DNA. London: Weidenfeld & Nicolson, 1968.

Wilson, E.B. The Cell in Development and Heredity, 3rd ed. New York: Macmillan, 1925.

Periodicals

Elkin, Lynne Osman. “Rosalind Franklin and the Double Helix.” Physics Today (February 2003): 63.

Glass, Bentley. “A Century of Biochemical Genetics.” Proceedings of the American Philosophical Society 109, no. 4 (August 18, 1965): 227–236.

Wade, Nicholas. “A Revolution at 50: Watson and Crick, Both Aligned and Apart, Reinvented Biology.” New York Times (February 25, 2003).

Wright, Pearce. “Erwin Chargaff: Disillusioned Biochemist Who Pioneered Our Understanding of DNA.” Guardian (July 2, 2002).

Anna Marie Eleanor Roos