Discussing Molecular Biology and Biologists—Who Were They and What Did They Discover? Oswald Theodore Avery! With Contributions by Manuel Varela and Ann Varela

Sep 16, 2020 by

Reproduced with permission of the Rockefeller Archive Center.http://profiles.nlm.nih.gov/CC/A/A/L/P/_/ccaalp_.jpg

1) Professor Varela, Oswald Theodore Avery’s name is almost synonymous with DNA—so we have to give him a lot of credit, and we will discuss his life, experiments, and discoveries—but to start—where was he born?

Dr. Oswald T. Avery was a scientist extraordinaire who is quite well known for discovering that DNA carries genetic information. On October 21, 1877, Oswald Theodore Avery was born in Halifax, Nova Scotia. Avery and his family came to the United States and set down roots in New York City when he was just a lad of ten. He was the middle child of three boys born to Elizabeth Crowdy and Joseph Francis Avery. Joseph was a Baptist minister who relocated his family to the Lower East Side of New York City to fill the position of pastor at the Mariners’ Temple.

2) What were the early educational experiences like for Avery? Where did he go to school?

Avery graduated from the New York City Male Grammar School in 1893. In 1900, Avery earned his B.A. degree from Colgate University. He took his M.D. degree in 1904 from the College of Physicians and Surgeons of Columbia University.

Avery spent a brief time practicing clinical medicine after graduating from medical school. Avery became Associate Director of the Division of Bacteriology upon his collaboration with Dr. Benjamin White, Director of the Hoagland Laboratory in Brooklyn. This union and field of study was the turning point in Avery’s career and brought him future fame and recognition. While working at the Hoagland Laboratory, Avery developed his analytical thinking skills and developed an interest in the subject of immunity.

In 1913, Dr. Rufus Cole, Director of the Hospital of the Rockefeller Institute, noticed the originality of Avery’s work and requested him to join his staff. It was in Cole’s laboratory that Avery’s interest in bacteriology developed. By 1923, Avery was a member of the Rockefeller Institute faculty. Avery conducted his research at the Rockefeller Hospital until 1943, at which point he retired. Avery continued his research as an emeritus member of the Rockefeller Institute until 1948.

3) You and I are the way we are due to the DNA we received from our parents. How does Avery fit into the picture?

DNA features in our everyday lives in countless ways. It is a famous molecule that has been around since the dawn of life on Earth. It is a molecule that does not die. DNA is well known to dictate the blueprint of ourselves and the lives of all sentient and natural living beings on Earth. DNA harbors the code for determining our body configuration and its biological functioning. DNA is a molecule that is also used daily as a molecular biological tool for progress in biotechnology. Oswald Avery was a key figure in the rise of DNA’s prominence in modern times.

Avery, who worked alongside Maclyn McCarty and Colin MacLeod in the early 1940s, provided groundbreaking evidence that DNA harbored heritable genetic material. DNA is a double-helical molecule that carries genetic information, and Avery and his colleagues provided some of the first compelling experimental evidence that DNA, and not protein, RNA, or sugars, was the molecular carrier of genetic material. Using bacteria and a variety of biochemical experiments, Avery and his colleagues discovered that DNA extracted from pathogenic forms of bacteria carried genetic information that conveyed virulence properties upon new bacteria, transforming them from harmless to harmful versions.

In so doing, Avery’s transformation work with DNA became a focal point for others to expand the novel idea into new molecular territories. One of the primary outcomes of Avery’s studies was that proteins, RNA, and polysaccharide sugars were not carriers of genetic information. The focus, therefore, was on DNA.

For instance, in 1952, Martha Chase and Alfred Hershey used bacteriophage viruses and host bacteria to provide additional evidence that DNA, instead of protein, was the transforming principle. Further clues about the genetic nature of DNA arose out of the pioneering work of Erwin Chargaff, who studied DNA samples extracted from organisms ranging from bacteria to humans. He determined that specific chemical ratios of the nucleotides which constituted DNA were related to certain living species of organisms. Importantly, Chargaff astutely noticed that the amounts of adenosine bases equaled those of thymidine bases. Likewise, he found that cytidine and guanosine base amounts equaled each other.

In the early 1950s, Rosalind Franklin and Maurice Wilkins used a powerful technique called X-ray diffraction to examine the crystallized structure of DNA. Franklin produced a remarkable set of DNA-based X-ray diffraction pictures. One, in particular, was called photo 51. See Figure 1.

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Figure 1. Rosalind Franklin X-ray diffraction Photo 51.

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The pioneering studies of Avery, Chase, Hershey, Chargaff, and especially of Franklin were utilized by Francis Crick and James Watson to deduce a novel molecular model for the structure of DNA. Subsequently, Wilkins provided supporting evidence for the validity of the proposed Crick-Watson model of the DNA double helix.

4) Much of his work focused on the bacterium that was thought to be responsible for pneumonia, Streptococcus pneumoniae, which I remember scantily studying years ago. What insights did he provide, and why is his work important?

One of Avery’s most important studies of the Streptococcus pneumoniae bacteria resulted in his major discovery of a potent virulence factor. This new factor is a biological substance produced by the bacteria that reside in the urine and blood of patients suffering from lobar pneumonia, a severe form of respiratory disease in humans.

The name of these bacteria, Streptococcus pneumoniae, is a relatively modern term, starting in 1974. In Avery’s time, however, shortly after 1910, the bacterium was known more popularly as Pneumococcus, and this was the term he used in his publications. In 1920, the name was changed to Diplococcus pneumoniae to reflect better the morphological nature of its appearance under the microscope. The genus Streptococcus was then used instead because the bacterium was biologically related to the streptococcal-based species.

Avery’s interest in the infectious disease field began in 1909 when he acquired new skills as a medical bacteriologist. His first known publication in the area involved his interest in Treponema pallidum, the syphilis-causing bacterium. Avery found that these bacteria could be visualized under the microscope when lesions of syphilis patients were sampled. Working with B. White, he published the work in a medical journal. Avery examined other bacteria that were associated with infectious diseases, such as the tuberculosis-causing bacterium Mycobacterium tuberculosis. He also had an interest in the bacterium called Hemophilus influenzae, which was widely, but inaccurately, thought at the time to cause the flu, but was nevertheless a serious causative agent of diseases such as ear and throat infections.

Avery studied other microorganisms, such as the non-pathogenic yogurt-based lactic acid bacteria, presenting the work in abstract form at a scientific conference in 1910. His main focus, however, was on the pathogenic Pneumococcus.

One of his first studies of Streptococcus pneumoniae involved examining the various types of these bacteria that caused lobar pneumonia. He participated with a research team devoted to grouping the various forms of these bacteria into distinctive types. Avery discovered a few new bacterial types of Streptococcus pneumoniae, based on their serological differences. Publishing the work, in 1918, in the Journal of the American Medical Association, known today as J.A.M.A. Avery also studied the relationship between pH and bacterial growth. In 1918, working with K.G. Dernby and G.E. Cullen, Avery cleverly realized that the pH optimum for growth of Streptococcus pneumoniae bacteria seemed to correspond to the pH optimum for enzymes. The new work permitted the development of a rapid test for differentiating between streptococci bacteria that infected human versus bovine hosts.

In the 1920s, Avery partook in a series of groundbreaking studies with Streptococcus pneumoniae. Working with G.E. Cullen, Avery studied internal sugar- and protein-metabolizing enzymes that were produced by Streptococcus pneumoniae. These studies were published, beginning in 1919 and ending in 1923.

Additionally, Avery and J.M. Neill found that an oxidation process could affect the activities of specific enzymes involved with blood. They studied the bacteria in the laboratory under two conditions, with and without heat. First, they grew the bacteria to saturation amounts. Then, they made cell extracts of the bacteria without their cell walls. Next, they washed the bacteria by centrifugation, replacing the old buffer with fresh buffer, and he then applied heat.

Neill and Avery found two exciting results. One outcome was the presence of a heat-sensitive substance that stayed with the bacterial extracts after buffer washing with the centrifuge. The second outcome was another substance, which was heat-resistant but was lost upon washing with the buffer and the centrifuge.

Next, Avery and Neill studied the oxidation-reduction relationships in the Streptococcus pneumoniae extracts. He discovered that the presence of oxygen could activate the heat-sensitive bacterial-based substance! It was a remarkable discovery. The bacterial substance, when oxygen was about, used up the oxygen, and the oxidized products elicited the formation of peroxide. The new bacterial products proceeded to oxidize hemoglobin and destroy it.

Neill and Avery then learned that the heat-resistant substances reacted slowly with oxygen to form their oxidizing end-products. Without oxygen, they found that methemoglobin, a form of hemoglobin, was reduced by the gain of electrons. Conversely, in the presence of the heat-sensitive substance, Avery and Neill learned that the oxidation-reduction occurred at a rapid pace, but only when oxygen was not around, suggesting that an enzyme was responsible. These research studies formed the basis for a series of papers published in 1924 and 1925.

Today, we know specific species of Streptococcus produce hemolytic substances that destroy red blood cells. For instance, the Streptococcus pneumoniae and Streptococcus viridans make hydrogen peroxide. The biochemical converts hemoglobin into methemoglobin, turning the red blood on an agar plate into a dark green color. The blood cell destroying oxidative process is known as alpha (α)-hemolysis. The α-hemolytic nature of these bacteria permits them to cause a pathological condition called sub-acute endocarditis in which valves of the heart are damaged, potentially causing heart failure.

Avery was also a key figure in the process known as a beta (β)-hemolysis, which destroys red blood cells. We know today that the oxygen-sensitive virulence factor called streptolysin O (SLO) makes a pore by which the cellular contents of red blood cells leaks out, thus, killing the cells. Another β-hemolytic substance, called streptolysin S (SLS), is oxygen stable. The SLS is an enzyme called phospholipase S, which degrades the membrane lipids of red blood cells, killing them. Group A, B, and C Streptococcus bacteria can readily produce SLO and SLS, making these bacteria quite pathogenic.

5) He worked with something called “polysaccharide,” (and I know about mucopolysaccharides, that are linked to Hunter’s Syndrome).  But first, why are polysaccharides important? And I know there are special kinds of them.

The polysaccharides are long stretches of sugar monomers that form long chains and branches of attached sugars. One type of these carbohydrates serves to store individual glucose sugars in plants in the form of starch, a more complex molecule than simple sugars. The starch within animals is referred to as glycogen. Some of these polysaccharides provide storage for energy generation. Another polysaccharide type, called cellulose, provides supportive roles for cell structures in eukaryotes and prokaryotes. The second type of structural polysaccharide is called chitin, which is found in insects and some fungi. Polysaccharides also play essential roles in conferring virulence to specific bacterial pathogens.

In modern times, the term mucopolysaccharides represent a type of polysaccharide known as the proteoglycans. These proteoglycans are gigantic biochemicals that consist of large segments of carbohydrates bound to a much smaller protein component. The molecular structure of a typical proteoglycan appears like a test-tube brush. The central core is composed of proteins, and the bristles consist of specialized carbohydrates called glycosaminoglycans, which are another type of proteoglycan.

In general, proteoglycans function in the human body by forming gelatinous filler substances in the eye and umbilical cord. The proteoglycans also lubricate moving skeletal bones. Proteoglycans can further constitute specific cartilage material, such as those found in the nose or the ear. In the body, proteoglycans can reside on the outer shells of living cells to form so-called extracellular matrix substances. As such, they function to hold cells and tissues together, sort of like a cellular glue. See Figure 2.

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Figure 2. Proteoglycan structure.

Hunter syndrome is known as mucopolysaccharidosis type II (MPS II). Here, the enzyme called iduronate-2-sulfatase (I2S) that is responsible for the glycosaminoglycan breakdown is missing or defective. Thus, the proteoglycan part of the extracellular matrix fails to properly break down glycosaminoglycan into heparan- and dermatan-sulfates. The result is the buildup of an overabundance of the enzyme’s substrates, the glycosaminoglycans. The abnormal amounts of glycosaminoglycans interfere with cellular and organ function, leading to disease.

6) Molecular genetics—what was his contribution to this field?

When, in 1944, Oswald Avery discovered that the transformation principle was DNA, it helped to launch the field of molecular genetics. Avery’s discovery that the transforming substance of these virulent Streptococcus pneumoniae bacteria was nucleic acid in nature represents his most significant contribution to molecular biology. The field deals with the study of genes, but at the level of the molecule. Molecular geneticists are primarily concerned with the genetics of living beings, including the molecular nature of the hereditary material and how it is regulated. Avery’s work paved the way for molecular biologists to examine more closely these gene structural and regulatory mechanisms.

Avery’s finding on the critical nature of DNA as genetic material pointed to this famous molecule as the main focus of research. Investigators such as Rosalind Franklin, Maurice Wilkins, James Watson, and Francis Crick were led by Avery’s discovery to propose a structural molecular model for DNA. The contribution of Avery initiated their focus on DNA. Similarly, investigators focused on isolating these genes to produce large quantities in the lab, a process we call gene cloning. The new cloning methodologies then provided unique opportunities to alter the nucleotide sequences of genes to examine the gene products and their activities more closely. New advances in DNA sequencing technologies became possible with the dawn of molecular genetics.

The birth of molecular genetics also led to the novel development of genomics, the study of the entire genomes of living organisms. The DNA sequences of intact genomes could be closely compared to those genomes of distinctive organisms to generate phylogenetic relationships, i.e., evolutionary information about the degrees of relatedness between different groups of organisms.

Lastly, advances in molecular genetics led to the inventions of many new biotechnological methods for producing needed biological products for the use or development of diagnostic tests for medical diseases and forensic science pathologies.

7) The chemistry of immunological processes—what were his contributions to this realm?

Avery studied specific polysaccharide molecules that belong to the protective outer capsules of infectious disease-causing bacteria. In this case, the polysaccharide capsules confer virulence capabilities to the Streptococcus pneumoniae bacteria. Avery discovered that the bacteria secreted polysaccharides, which ended up in the blood and urine of patients with lobar pneumonia. He studied the polysaccharide composition of Streptococcus pneumoniae variants to classify them.

Avery also discovered that the polysaccharide component of the bacterial capsules induced the immune response to produce antibodies that were specific to these carbohydrate parts of the bacterial cell wall. The fact that a sugar molecule could stimulate the immune response, a process called immunogenicity, was a novel finding, and it was Avery who was the first investigator to demonstrate that a substance other than a protein could serve as an antigen. The findings by Avery that sugars of bacteria influence pathogenesis, virulence, and immunological specificity, were significant contributions to immunohistochemistry.

8) Who did Avery work with, and what was this “transforming principle” that we study in science?

The “transforming principle” is DNA. We know that when non-native DNA is introduced to the internal cellular milieu of a new cell, this new host cell can acquire new traits that are encoded in the foreign DNA. The host cell, thus, becomes transformed into a new version, with different properties. The famous experiment indicating that DNA was the genetic material was performed by Avery and colleagues Maclyn McCarty and Colin MacLeod in the early 1940s.

The transforming principle story begins in 1928 with the pioneering work of Frederick Griffith. He studied two types of Streptococcus pneumoniae. The first was the harmless R (for rough) type (called Type II at the time), which formed rough-looking colonies on Petri plates, and the second was the harmful S (for smooth) type (called Type III at the time), which formed smooth-looking colonies on the culture plates. See Figure 3.

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Figure 3. Griffith’s early transformation experiment of 1928.

Griffith had found that when living but non-lethal Streptococcus pneumoniae R bacteria were mixed with its virulent S, but heat-killed, dead version of the bacteria and injected the combination into laboratory mice, they quickly succumbed and died. The outcome was shocking because separately, neither treatment (live S nor dead R bacteria) killed the mice. The non-lethal living R bacteria did not kill the mice when injected, and likewise, the injection of the heat-killed dead virulent S bacteria did not kill the mice. Separately, these treatments were non-lethal to the mice.

The combination of the two treatments, however, was as potently lethal as the live virulent S form of the Streptococcus pneumoniae. When necropsied, the lab animals all had the living lethal virulent S forms of the Streptococcus pneumoniae swimming in their tissues! Griffith had reasoned that a substance in the dead lethal S bacteria had somehow transformed the non-virulent R type into its potently virulent S counterpart. A transformation of a biological nature had taken place in the laboratory.

It is at this point, in the early 1940s, that Avery and his colleagues MacLeod and McCarty arrive in the picture. They had become interested in deducing the nature of Griffith’s transforming principle. They wanted to identify it and study its chemistry. Most investigators at the time, in 1943, including Avery, believed that the responsible transforming principle was protein. They nevertheless purified, as much as was possible, samples of protein, DNA, and RNA, from the S type III virulent Streptococcus pneumoniae.

Next, they tested their protein, DNA, and RNA samples using chemical analytical means to convince themselves that their preparations were as pure possible. They measured the elemental composition of their DNA and RNA preparations. They found that their samples were consistent with what was known about the chemical constitution of these types of nucleic acids at the time. Likewise, they tested the chemical composition of their protein for the presence of amino acids. They convinced themselves that protein extracted from the S type III virulent Streptococcus pneumoniae was pure.

They were suitably convinced that their extracted bacterial substances represented pure DNA, RNA, and protein. Next, they conducted an in vitro transformation experiment, trying to convert the R type (harmless) Streptococcus pneumoniae into an S type (virulent) using their DNA, RNA, and protein purified samples. Avery and his colleagues quickly found that the R type II bacteria had to be in a “reactive phase,” a condition we call, in modern times, “competent.” The reactive phase host would need to be in a state that permitted the host cells to respond to the transforming stimulation by external agents—the putative transformation substance! They added competent R type II bacteria (the harmless one) that had been grown in blood media to new test tubes containing their pure DNA, RNA, or protein samples. Avery and colleagues then incubated these tubes at 37 °C for up to one full day and plated on Petri dishes containing culture media. Next, they assessed whether transformation from the R to the S forms occurred by closely examining the types of colonies formed on the Petri plates.

Avery and colleagues found on the plates that the samples containing DNA grew smooth S-type III bacteria! The pure S type DNA had transformed the type II R host bacteria into type III S Streptococcus pneumoniae. The plates contained the unmistakable smooth colonies, indicative of the dangerous type—the harmless bacteria had been definitively transformed into the harmful type by DNA. On the other hand, neither RNA nor protein seemed to result in profound transformation as had their DNA.

Next, they followed up on their newly discovered bacterial transforming properties that were conferred upon the type II R bacteria by the pure DNA. They destroyed it—on purpose! The reasoning behind this DNA destruction method lies as follows. If DNA truly transforms a new host into a completely different type, then adding an enzyme that destroys DNA should then make it lose its transforming ability—destroy the DNA—destroy its capacity transform. That was the logic behind Avery’s next experiment. It was indeed a brilliant deduction.

As before, Avery and his co-workers added purified DNA from the dead virulenttype II S bacteria to host R type III bacteria. This time, however, the mixture contained an enzyme that they called “depolymerase,” which we call DNase today—the enzyme removes nucleotides from DNA, essentially destroying it and its transformative properties along with it. Thus, adding the DNase to their mixture resulted in its inability to transform. That is, destroying DNA also destroyed its transformation properties. In contrast, Avery and his colleagues noted that neither proteases nor ribonucleases did not destroy the transformative properties that DNA had conferred.

Therefore, Avery, MacLeod, and McCarty had provided definitive evidence that DNA was the transformation principle, and hence, the hereditary material. Their discovery was published in a series of papers between 1943 and 1945.

9) I know he won the COPLEY award—but what would you say are his major contributions to molecular biology?

Dr. Oswald Avery was granted the Copley Medal award in 1945 by the Royal Society of London. The accolade is typically bestowed to individuals who have made outstanding achievements in scientific research, irrespective of the nature of the scientific discipline—investigators of all fields of science, are considered on an annual basis. Avery’s Copley Medal award was based on his discoveries having to do with the immunological basis of infectious disease pathology. Avery’s discovery that polysaccharides from bacterial cell walls mount an immune response by inducing specific antibodies against these sugars was significant. The relationship between the immune system chemistry and bacteria proved to be a significant one. The discovery provided the path to advance the field of medical science in new ways. One could, for instance, exploit the findings to rapidly identify the various types of Streptococcus pneumoniae pathogens, which could then inform the diagnosis of infection and provide for new avenues of chemotherapy.

Arguably the most significant discovery by Avery was the new experimental evidence that DNA was the genetic material that conferred biological traits. He found that DNA was the key molecule that was responsible for holding genetic information and passing it from generation to generation or from harmful to harmless bacteria, as Avery had elegantly demonstrated in the lab. As such, Avery and his associates played a major role in the birth of molecular genetics.

Furthermore, Avery can most certainly be considered by historians of science to be a founding parent of molecular biology. Because his DNA work forced others who were astutely focused on genetics to turn their attention to the then seemingly unlikely DNA molecule as a candidate for harboring genetic information, Avery helped to enhance the progress of the fledgling field of molecular biology.

With DNA as their main point of focus, investigators could now seek to understand how the miracle molecule specified biological traits. Molecular biologists could now learn about the mechanisms for DNA synthesis (replication), RNA synthesis (transcription), and protein synthesis (translation). Protein biochemists could now use molecular biology to clone the genes that encoded them to study their protein behaviors more closely. This proved to be quite necessary for those proteins which strongly resisted purification in the lab.

Investigators could also seek to understand better how DNA made its way to new generations. Avery’s work paved the way to new advancements in medicine, genomics, and bioinformatics. Avery’s contributions permitted the rapid enhancement in recombinant DNA technology and biotechnology, which in turn, permitted advancements in virtually all areas of the biological, biochemical, cell biological, immunological, and ecological sciences. Many scientific divisions in the biological sciences and their smaller fields of study have gone molecular. To “go molecular” means that the genes responsible for transformation to new traits are isolated and studied in close mechanistic detail. Avery played a significant role in making further advancements in each of these disparate sciences happen.

10) Is there anything that I have neglected to ask?

It noteworthy that the DNA transformation discovery by Avery and his associates was met with widespread disbelief. At the time, it was incorrectly accepted that protein should be the transforming principle because of its complexity. Such investigators were under the impression that DNA itself was too simple of a molecule to confer the massive complexity known to exist in living organisms. Alfred Hershey and Martha Chase supported the DNA idea with radioactively-labeled bacteriophages and infection of Escherichia coli. Chase and Hershey observed that primarily labeled DNA entered the bacteria, rather than labeled protein. Even these biologically based experimental data were met with disbelief.

Above you mentioned the Copley Medal, which was award to Avery in 1945 for his contributions to immunological specificity. But he was widely celebrated in his day. For example, Avery received several honorary degrees, including the following: Sc.D., from Colgate University (1921), L.L.D., from McGill University (1935), Sc.D., from New York University (1947), and Sc.D., from Rutgers University (1953). Avery also received the coveted Paul Ehrlich Gold Medal. Avery was elected in 1933 to the National Academy of Sciences, a most prestigious honor. He took the George M. Kober Medal, awarded by the Association of American Physicians, in 1946. Avery was awarded the Albert Lasker Basic Medical Research Award, 1947, a tribute that is often a precursor to the Nobel—although no Nobel was to ever go to Avery—it is a mystery that remains to this day and still confounds historians of science. Avery received the Passano Award by the Passano Foundation in 1949. He was given the Pasteur Gold Medal in 1950, and he became a Foreign Member of the Royal Society of London.

Dr. Avery Became a United States citizen on August 1, 1918. He had studied music in college before he discovered his enthusiasm for the field of medicine. Avery used his talents and scientific knowledge to assist his country’s efforts in World War I and World War II. He served on many committees concerned with restraining several important infectious diseases, which are widespread among troops under environments of combat. After Avery’s retirement, he moved to Tennessee to be near his brother’s family. Avery died of liver cancer on February 20, 1955, in Nashville, Tennessee.

For more information about this pioneering molecular biologist, visit the following link:

Video about Transformation Agents: https://sites.google.com/site/dnatimelinejghs/home/oswald-theodore-avery

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