Werner Arber

Swiss microbiologist Werner Arber was awarded the Nobel Prize for Medicine or Physiology in 1978, sharing the $165,000 award with Daniel Nathans and Hamilton O. Smith. Observing that when a virus entered bacterium, most of the viral deoxyribonucleic acid (DNA) was destroyed, Arber theorized that the bacterium produced an enzyme that severed the viral DNA into smaller pieces. Nathans and Smith later proved that Arber was correct -- that certain enzymes, called 'restriction enzyme' or 'restriction endonuclease', cleave long strands of DNA into tiny fragments. These fragments, which retain their genetic information, led to the development of gene splicing -- techniques for separating, manipulating, and eventually altering this basic genetic material.

After winning his Nobel honors, Arber became an outspoken participant in the establishment of guidelines to conduct recombinant DNA research safely and ethically. His daughter, Silvia Arber, is a professor of neurobiology at the University of Basel, studying neuronal circuit formation in the developing spinal cord.

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Phillip A. Sharp

Phillip Allen Sharp is an American geneticist and molecular biologist. He was awarded the Nobel Prize in Physiology or Medicine 1993 for his discovery of RNA splicing, the technique of modifying the RNA. He shared the prize with Richard J. Roberts. Sharp discovered that genes in eukaryotes are not contiguous strings but they contain introns. The messenger RNA can be spliced to delete these introns and different proteins can be obtained from the same sequence of DNA.

Phillip Sharp is also an accomplished businessman and is the co-founder of 3 successful companies Biogen, Alnylam Pharmaceuticals and Magen Biosciences. Sharp was awarded the Dickson Prize 1980, Lasker Award 1988, Benjamin Franklin Medal by the American Philosophical Society 1999 and the National Medal of Science 2004.

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Frederick Sanger

Frederick Sanger is an English Biochemist and two time Nobel Laureate in Chemistry. He was awarded the Nobel Prize in Chemistry 1958 for his work on the structure of proteins (especially insulin) and in 1980 he shared the Nobel Prize in Chemistry with Walter Gilbert and Paul Berg. Gilbert and Sanger shared half of the prize for their breakthrough in the determination of nucleic acid base sequence.

Frederick Sanger proved that proteins have a defined chemical composition. He successfully determined the complete amino acid sequence of two polypeptide chains of Bovine Insulin. Sanger developed “dideoxy “chain termination method for sequencing DNA molecule. This method was used to sequence human mitochondrial DNA, bacteriophage DNA and eventually entire human genome.

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Matthew Paul Berg

Molecular biologist who in 1972 created the first recombinant DNA molecules, and, in doing so, created the field of genetic engineering.

Berg, in 1972, combined DNA from the cancer-causing monkey virus SV40 with that of the virus lambda to create the first recombinant DNA molecules. However, upon realizing the dangers of his experiment, terminated it before it could be taken any further. He immediately, in what is now called the "Berg Letter," proposed a one year moratorium on recombinant DNA research, in order for safety concerns to be worked out. Berg made one of the most fundamental technical contributions to the field of genetics in the twentieth century: he developed a technique for splicing together deoxyribonucleic acid (DNA) the substance that carries the genetic information in living cells and viruses from generation to generation--from different types of organisms. His achievement gave scientists a priceless tool for studying the structure of viral chromosomes and the biochemical basis of human genetic diseases. It also let researchers turn simple organisms into chemical factories that churn out valuable medical drugs. In 1980 he was awarded the Nobel Prize in chemistry for pioneering this procedure, now referred to as recombinant DNA technology (RDT).

In 1991, Berg accepted a position as the head of the Scientific Advisory Committee of the Human Genome Project.

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Friedrich Miescher

Friedrich (Fritz) Miescher was born in Basel, Switzerland. The Miescher family was well-respected and part of the intellectual elite in Basel. Friedrich's father was a physician and taught pathological anatomy; Friedrich's uncle, Wilhelm His, was a well-known embryologist.

Miescher was an excellent student despite his shyness and a hearing handicap. Miescher initially wanted to be a priest, but his father opposed the idea and Miescher entered medical school. When he graduated in 1868, Miescher ruled out specialties where patient interactions were necessary because of his hearing problem. He decided to base his career on medical research. He went to the University of Tübingen to study under Felix Hoppe-Seyler in the newly established faculty of natural science.

Hoppe-Seyler's laboratory was one of the first in Germany to focus on tissue chemistry. At a time when scientists were still debating the concept of "cell," Hoppe-Seyler and his lab were isolating the molecules that made up cells. Miescher was given the task of researching the composition of lymphoid cells — white blood cells.

These cells were difficult to extract from the lymph glands, but they were found in great quantities in the pus from infections. Miescher collected bandages from a nearby clinic and washed off the pus. He experimented and isolated a new molecule - nuclein - from the cell nucleus. He determined that nuclein was made up of hydrogen, oxygen, nitrogen and phosphorus and there was an unique ratio of phosphorus to nitrogen. He was able to isolate nuclein from other cells and later used salmon sperm (as opposed to pus) as a source.

Although Miescher did most of his work in 1869, his paper on nuclein wasn't published until 1871. Nuclein was such a unique molecule that Hoppe-Seyler was skeptical and wanted to confirm Miescher's results before publication.

Miescher continued to work on nuclein for the rest of his career. He also examined the metabolic changes that occur in salmon when they spawn. In 1872, Miescher was appointed the professor of physiology at the University of Basel, a position previously held by his father and then his uncle. The appointment meant more funds and equipment for research, but it also meant that Miescher had to teach. Although he put in a lot of time and effort, Miescher was not a good teacher. His shyness and preoccupation with his research made it difficult for him to relate to his students. He was a perfectionist and a workaholic, and often worked very long hours to do the nuclein isolations.

It would be years before the role of nucleic acids were recognized. Miescher, himself, believed that proteins were the molecules of heredity. However, Miescher laid the groundwork for the molecular discoveries that followed. Miescher died in 1895 from tuberculosis.

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Isolating Hereditary Material: Frederick Griffith, Oswald Avery, Alfred Hershey, and Martha Chase

In the first half of the twentieth century, Gregor Mendel's principles of genetic inheritance became widely accepted, but the chemical nature of the hereditary material remained unknown. Scientists did know that genes were located on chromosomes and that chromosomes consisted of DNA and proteins. At the time, however, proteins seemed to be a better choice for the genetic material, because chemical analyses had shown that proteins are more varied than DNA in their chemical composition, as well as in their physical properties. Therefore, the eventual identification of DNA as the hereditary material came as a surprise to scientists. This breakthrough resulted from a series of experiments with bacteria and bacteriophages, or viruses that infect bacteria. Together, these experiments demonstrated that DNA was transferred between generations and that this molecule had the ability to transform the properties of a cell.

Frederick Griffith Discovers Bacterial Transformation

In the aftermath of the deadly 1918 flu epidemic, governments across the globe rushed to develop vaccines that could stop the spread of infectious diseases. In England, microbiologist Frederick Griffith was studying two strains of Streptococcus pneumoniae that varied dramatically in both their appearance and their virulence, or their ability to cause disease. Specifically, the highly virulent S strain had a smooth capsule, or outer coat composed of polysaccharides, while the nonvirulent R strain had a rough appearance and lacked a capsule (Figure 1). Mice injected with the S strain died within a few days after injection, while mice injected with the R strain did not die.

Through a series of experiments, Griffith established that the virulence of the S strain was destroyed by heating the bacteria. Thus, he was surprised to find that mice died when they were injected with a mixture of heat-killed Sbacteria and living R bacteria, neither of which caused mice to die when they were injected alone. Griffith was able to isolate live bacteria from the hearts of the dead animals that had been injected with the mixed strains, and he observed that these bacteria had the smooth capsules characteristic of the S strain. Based on these observations, Griffith hypothesized that a chemical component from the virulent S cells had somehow transformed the R cells into the more virulent S form (Griffith, 1928). Unfortunately, Griffith was not able to identify the chemical nature of this "transforming principle" beyond the fact that it was able to survive heat treatment.

DNA Is Identified as the “Transforming Principle”

The actual identification of DNA as the "transforming principle" was an unexpected outcome of a series of clinical investigations of pneumococcal infections performed over many years (Steinman & Moberg, 1994). At the same time that Griffith was conducting his experiments, researcher Oswald Avery and his colleagues at the Rockefeller University in New York were performing detailed analyses of the pneumococcal cell capsule and the role of this capsule in infections. Modern antibiotics had not yet been discovered, and Avery was convinced that a detailed understanding of the pneumococcal cell was essential to the effective treatment of bacterial pneumonia. Over the years, Avery's group had accumulated considerable biochemical expertise as they established that strains of pneumococci could be distinguished by the polysaccharides in their capsules and that the integrity of the capsule was essential for virulence. Thus, when Griffith's results were published, Avery and his colleagues recognized the importance of these findings, and they decided to use their expertise to identify the specific molecules that could transform a nonencapsulated bacterium into an encapsulated form. In a significant departure from Griffith's procedure, however, Avery's team employed a method for transforming bacteria in cultures rather than in living mice, which gave them better control of their experiments.

 Avery and his colleagues, including researchers Colin MacLeod and Maclyn McCarty, used a process of elimination to identify the transforming principle (Avery et al., 1944). In their experiments, identical extracts from heat-treated S cells were first treated with hydrolytic enzymes that specifically destroyed protein, RNA, or DNA. After the enzyme treatments, the treated extracts were then mixed with live R cells. Encapsulated S cells appeared in all of the cultures, except those in which the S strain extract had been treated with DNAse, an enzyme that destroys DNA. These results suggested that DNA was the molecule responsible for transformation.

Avery and his colleagues provided further confirmation for this hypothesis by chemically isolating DNA from the cell extract and showing that it possessed the same transforming ability as the heat-treated extract. We now consider these experiments, which were published in 1944, as providing definitive proof that DNA is the hereditary material. However, the team's results were not well received at the time, most likely because popular opinion still favored protein as the hereditary material.

Hershey and Chase Prove Protein Is Not the Hereditary Material

Protein was finally excluded as the hereditary material following a series of experiments published by Alfred Hershey and Martha Chase in 1952. These experiments involved the T2 bacteriophage, a virus that infects the E. coli bacterium. At the time, bacteriophages were widely used as experimental models for studying genetic transmission because they reproduce rapidly and can be easily harvested. In fact, during just one infection cycle, bacteriophages multiply so rapidly within their host bacterial cells that they ultimately cause the cells to burst, thus releasing large numbers of new infectious bacteriophages. The T2 bacteriophage used by Hershey and Chase was known to consist of both protein and DNA, but the role that each substance played in the growth of the bacteriophage was unclear. Electron micrographs had shown that T2 bacteriophages consist of an icosahedral head, a cylindrical sheath, and a base plate that mediates attachment to the bacterium, shown schematically in Figure 5. After infection, phage particles remain attached to the bacterium, but the heads appear empty, forming "ghosts."

To determine the roles that the T2 bacteriophage's DNA and protein play in infection, Hershey and Chase decided to use radioisotopes to trace the fate of the phage's protein and DNA by taking advantage of their chemical differences. Proteins contain sulfur, but DNA does not. Conversely, DNA contains phosphate, but proteins do not. Thus, when infected bacteria are grown in the presence of radioactive forms of phosphate (32P) or sulfur (35S), radioactivity can be selectively incorporated into either DNA or protein. Hershey and Chase employed this method to prepare both 32P-labeled and 35S-labeled bacteriophages, which they then used to infect bacteria. To determine which of the labeled molecules entered the infected bacteria, they detached the phage ghosts from the infected cells by mechanically shearing them off in an ordinary kitchen blender. The ghosts and bacterial cells were then physically separated using a centrifuge. The larger bacterial cells moved rapidly to the bottom of the centrifuge tube, where they formed a pellet. The smaller, lighter phage ghosts remained in the supernatant, where they could be collected and analyzed. During analysis, Hershey and Chase discovered that almost all of the radioactive sulfur remained with the ghosts, while about one-third of the radioactive phosphate entered the bacterial cells and could later be recovered in the next generation of bacteriophages.

From these experiments, Hershey and Chase determined that protein formed a protective coat around the bacteriophage that functioned in both phage attachment to the bacterium and in the injection of phage DNA into the cell. Interestingly, they did not conclude that DNA was the hereditary material, pointing out that further experiments were required to establish the role that DNA played in phage replication. In fact, Hershey and Chase circumspectly ended their paper with the following statement: "This protein probably has no function in the growth of intracellular phage. The DNA has some function. Further chemical inferences should not be drawn from the experiments presented" (Hershey & Chase, 1952). However, a mere one year later, the structure of DNA was determined, and this allowed investigators to put together the pieces in the question of DNA structure and function

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Barbara McClintock and the Discovery of Jumping Genes (Transposons)

Some of the most profound genetic discoveries have been made with the help of various model organisms that are favored by scientists for their widespread availability and ease of maintenance and proliferation. One such model is Zea mays (maize), particularly those plants that produce variably colored kernels. Because each kernel is an embryo produced from an individual fertilization, hundreds of offspring can be scored on a single ear, making maize an ideal organism for genetic analysis. Indeed, maize proved to be the perfect organism for the study of transposable elements (TEs), also known as "jumping genes" which were discovered during the middle part of the twentieth century by American scientist Barbara McClintock. McClintock's work was revolutionary in that it suggested that an organism's genome is not a stationary entity, but rather it is subject to alteration and rearrangement—a concept that was met with criticism from the scientific community of the time. Eventually, however, the significance of McClintock's work became widely appreciated, and she was awarded the Nobel Prize in 1983.

McClintock and the Origins of Cytogenetics

Barbara McClintock began her scientific career at Cornell University, where she pioneered the study of cytogenetics—a new field in the 1930s—using maize as a model. Indeed, the marriage of cytology and genetics became official in 1931, when McClintock and graduate student Harriet Creighton provided the first experimental proof that genes were physically positioned on chromosomes by describing the crossing-over phenomenon and geneticrecombination. Although Thomas Hunt Morgan was the first person to suggest the link between genetic traits and the exchange of genetic material by chromosomes, 20 years elapsed before his ideas were scientifically proven, largely due to limitations in cytological and experimental techniques (Coe & Kass, 2005). McClintock's own innovative cytogenetic techniques were what allowed her to confirm Morgan's ideas, and these techniques are thus among her greatest contributions to science.

Discovering TEs through Experimentation with Maize

As previously mentioned, McClintock is best known not for her innovations in cytogenetic techniques, but rather for her discovery of transposable elements through experimentation with maize. In order to understand McClintock's observations (and logic) that led to her discovery of TEs, however, it's first necessary to be aware that the phenotypic system that McClintock studied—the variegated color pattern of maize kernels—involved three alleles rather than the usual two. Think of every maize kernel as essentially a single individual, originating as an ovule that undergoes (or has undergone) double fertilization. During double fertilization, one sperm fuses with the egg cell's nucleus, producing a diploid zygote that will develop into the next generation. Meanwhile, the other sperm fuses with the two polar nuclei to form a triploid endosperm. As a result, the colored (or colorless, as the case may be) tissue that makes up the aleurone (or outer) layer of the endosperm is triploid, not diploid.

McClintock worked with what is known as the Ac/Ds system in maize, which she discovered by conducting standard genetic breeding experiments using plants with an unusual phenotype. Through these experiments, McClintock recognized that breakage occurred at specific sites on maize chromosomes. Indeed, the first transposable element she discovered was a site of chromosome breakage, aptly named "dissociation" (Ds). Although McClintock eventually found that some TEs can "jump" autonomously, she noted that the movements of Ds are regulated by an autonomous element called "activator" (Ac), which can also promote its own transposition.

Of course, these discoveries were preceded by extensive breeding experimentation. It was known at the time from previous work by Rollins A. Emerson, another American maize geneticist, that maize had genes encoding variegated, or multicolored, kernels; these kernels were described as colorless (although they were actually white or yellow), except for spots or streaks of purple or brown. Emerson had proposed that the variegated streaking was due to an "unstable mutation," or a mutation for the colorless phenotype that would sometimes revert back to its wild-type variant and result in an area of color. However, he couldn't explain why or how this occurred. As McClintock discovered, the unstable mutation Emerson puzzled over was actually a four-gene system.

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The Watson and Crick Structure of DNA

Today, our series on models of DNA is concluded with a discussion of the correct structure determined by James Watson and Francis Crick. Although they made an unlikely pair, the two men succeeded where one of the era’s leading scientists – Linus Pauling – failed, and in the process they unraveled the secrets of what may be the most important molecule in human history.

In the fall of 1951, James Watson was studying microbial metabolism and nucleic acid biochemistry as a postdoctoral fellow in Europe. It didn’t take long for him to tire of these subjects and to begin looking for more inspiring research. He became interested in DNA upon seeing some x-ray photos developed by Maurice Wilkins. He then tried to talk his way into Wilkins’ lab at King’s College, but was denied and ended up studying protein x-ray diffraction in the Cavendish Laboratory at Cambridge University. Here he was assigned space in an office to be shared with an older graduate student named Francis Crick, a crystallographer. At the time, Crick was studying under Max Perutz, and was also becoming bored with his research. Watson and Crick hit it off immediately and before long, Watson’s interest in DNA had worn off on Crick. Although neither of them were experts in structural chemistry, they decided to attempt to solve the structure of DNA. As Watson put it, their planned method of attack would be to “imitate Linus Pauling and beat him at his own game.”

The pair’s first attempt at the structure in the fall of 1951 was very quick, and also unsuccessful. Interestingly, however, it was quite similar to Linus Pauling and Robert Corey‘s own attempt about a year later. Watson and Crick came up with a three stranded helix, with the base rings located on the outside of the molecule and the phosphate groups found on the inside. This left them with the problem of fitting so many negatively charged phosphates into the core without the molecule blowing itself apart. In order to solve this problem, they turned to Pauling’s own The Nature of the Chemical Bond. They were looking for positive ions that would fit into the core of DNA, therefore canceling the negative charge. They found magnesium and calcium to be possibilities, but there was no significant evidence that these ions were in DNA. However, there was no evidence against it either, so they ran with the idea.

Watson and Crick assumed – as would Pauling in his later attempt – that the finer details would fall into place. Overjoyed at solving DNA so quickly, they invited Wilkins and his assistant, Rosalind Franklin, to have a look at their structure. Expecting praise, they were undoubtedly surprised when Franklin verbally destroyed their work. She told them that any positive ions found in the core would be surrounded by water, which would render them neutral and unable to cancel out the negative phosphate charges. She also noted that DNA soaks up a large amount of water, which indicates that the phosphate groups are on the outside of the molecule. All in all, Franklin had no positive feedback for Watson and Crick.  And she was, at it turned out, correct. After the visit, Watson and Crick attempted to persuade Wilkins and Franklin to collaborate with them on another attempt at the structure of DNA, but their offer was declined.

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