avery experiments

Microbe Notes

DNA Experiments (Griffith & Avery, McCarty, MacLeod & Hershey, Chase)

DNA, deoxyribonucleic acid, is the carrier of all genetic information. It codes genetic information passed on from one generation to another and determines individual attributes like eye color, facial features, etc. Although DNA was first isolated in 1869 by a Swiss scientist, Friedrich Miescher, from nuclei of pus-rich white blood cells (which he called nuclein ), its role in the inheritance of traits wasn’t realized until 1943. Miescher thought that the nuclein, which was slightly acidic and contained a high percentage of phosphorus, lacked the variability to account for its hereditary significance for diversity among organisms. Most of the scientists of his period were convinced by the idea that proteins could be promising candidates for heredity as they were abundant, diverse, and complex molecules, while DNA was supposed to be a boring, repetitive polymer. This notion was put forward as the scientists were aware that genetic information was contained within organic molecules.

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Griffith’s Transformation Experiment

In 1928, a young scientist Frederick Griffith discovered the transforming principle. In 1918, millions of people were killed by the terrible Spanish influenza epidemic, and pneumococcal infections were a common cause of death among influenza-infected patients. This triggered him to study the bacteria Streptococcus pneumoniae and work on designing a vaccine against it . It became evident that bacterial pneumonia was caused by multiple strains of S. pneumoniae, and patients developed antibodies against the particular strain with which they were infected. Hence, serum samples and bacterial isolates used in experiments helped to identify DNA as the hereditary material. 

He used two related strains of S. pneumoniae and mice and conducted a series of experiments using them. 

• When type II R-strain bacteria were grown on a culture plate, they produced rough colonies. They were non-virulent as they lacked an outer polysaccharide coat. Thus, when RII strain bacteria were injected into a mouse, they did not cause any disease and survived.
• When type I S-strain bacteria were grown on a culture plate, they produced smooth, glistening, and white colonies. The smooth appearance was apparent due to a polysaccharide coat around them that provided resistance to the host’s immune system. It was virulent and thus, when injected into a mouse, resulted in pneumonia and death. 
• In 1929, Griffith experimented by injecting mice with heat-killed SI strain (i.e., SI strain bacteria exposed to high temperature ensuing their death). But, this failed to harm the mice, and they survived.
• Surprisingly, when he mixed heat-treated SI cells with live RII cells and injected the mixture into the mice, the mice died because of pneumonia. Additionally, when he collected a blood sample from the dead mouse, he found that sample to contain live S-strain bacteria.

Conclusion of Griffith’s Transformation Experiment

Based on the above results, he inferred that something must have been transferred from the heat-treated S strain into non-virulent R strain bacteria that transformed them into smooth coated and virulent bacteria. Thus, the material was referred to as the transforming principle.

Following this, he continued with his research through the 1930s, although he couldn’t make much progress. In 1941, he was hit by a German bomb, and he died.

Avery, McCarty, and MacLeod Experiment

During World War II, in 1943, Oswald Avery, Maclyn McCarty, and Colin MacLeod working at Rockefeller University in New York, dedicated themselves to continuing the work of Griffith in order to determine the biochemical nature of Griffith’s transforming principle in an in vitro system. They used the phenotype of S. pneumoniae cells expressed on blood agar in order to figure out whether transformation had taken place or not, rather than working with mice. The transforming principle was partially purified from the cell extract (i.e., cell-free extract of heat-killed type III S cells) to determine which macromolecule of S cell transformed type II R-strain into the type III S-strain. They demonstrated DNA to be that particular transforming principle.

• Initially, type III S cells were heat-killed, and lipids and carbohydrates were removed from the solution.
• Secondly, they treated heat-killed S cells with digestive enzymes such as RNases and proteases to degrade RNA and proteins. Subsequently, they also treated it with DNases to digest DNA, each added separately in different tubes.
• Eventually, they introduced living type IIR cells mixed with heat-killed IIIS cells onto the culture medium containing antibodies for IIR cells. Antibodies for IIR cells were used to inactivate some IIR cells such that their number doesn’t exceed the count of IIIS cells. that help to provide the distinct phenotypic differences in culture media that contained transformed S strain bacteria.

Observation of Avery, McCarty, and MacLeod Experiment

The culture treated with DNase did not yield transformed type III S strain bacteria which indicated that DNA was the hereditary material responsible for transformation. 

Conclusion of Avery, McCarty, and MacLeod Experiment

DNA was found to be the genetic material that was being transferred between cells, not proteins.

Hershey and Chase Experiment

Although Avery and his fellows found that DNA was the hereditary material, the scientists were reluctant to accept the finding. But, not that long afterward, eight years after in 1952, Alfred Hershey and Martha Chase concluded that DNA is the genetic material. Their experimental tool was bacteriophages-viruses that attack bacteria which specifically involved the infection of Escherichia coli with T2 bacteriophage.

T2 virus depends on the host body for its reproduction process. When they find bacteria as a host cell, they adhere to its surface and inject its genetic material into the bacteria. The injected hereditary material hijacks the host’s machinery such that a large number of viral particles are released from them. T2 phage consists of only proteins (on the outer protein coat) and DNA (core) that could be potential genetic material to instruct E. coli to develop its progeny. They experimented to determine whether protein or DNA from the virus entered into the bacteria.

• Bacteriophage was allowed to grow on two of the medium: one containing a radioactive isotope of phosphorus( 32 P) and the other containing a radioactive isotope of sulfur ( 35 S).
• Phages grown on radioactive phosphorus( 32 P) contained radioactive P labeled DNA (not radioactive protein) as DNA contains phosphorus but not sulfur.
• Similarly, the viruses grown in the medium containing radioactive sulfur ( 35 S) contained radioactive 35 S labeled protein (but not radioactive DNA) because sulfur is found in many proteins but is absent from DNA.
• E. coli were introduced to be infected by the radioactive phages.
• After the progression of infection, the blender was used to remove the remains of phage and phage parts from the outside of the bacteria, followed by centrifugation in order to separate the bacteria from the phage debris.
• Centrifugation results in the settling down of heavier particles like bacteria in the form of pellet while those light particles such as medium, phage, and phage parts, etc., float near the top of the tube, called supernatant.

Observation of Hershey and Chase Experiment

On measuring radioactivity in the pellet and supernatant in both media, 32 P was found in large amount in the pellet while 35 S in the supernatant that is pellet contained radioactively P labeled infected bacterial cells and supernatant was enriched with radioactively S labeled phage and phage parts.

Conclusion of Hershey and Chase Experiment

Hershey and Chase deduced that it was DNA, not protein which got injected into host cells, and thus, DNA is the hereditary material that is passed from virus to bacteria.

• Fry, M. (2016). Landmark Experiments in Molecular Biology. Academic Press.
• https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_Introductory_Biology_(CK-12)/04%3A_Molecular_Biology/4.02%3A_DNA_the_Genetic_Material
• https://byjus.com/biology/dna-genetic-material/
• https://bio.libretexts.org/Bookshelves/Genetics/Book%3A_Online_Open_Genetics_(Nickle_and_Barrette-Ng)/01%3A_Overview_DNA_and_Genes/1.02%3A_DNA_is_the_Genetic_Material
• https://www.toppr.com/guides/biology/the-molecular-basis-of-inheritance/the-genetic-material/
• https://www.nature.com/scitable/topicpage/discovery-of-dna-as-the-hereditary-material-340/
• https://www.biologydiscussion.com/genetics/dna-as-a-genetic-material-biology/56216
• https://www.nature.com/scitable/topicpage/discovery-of-the-function-of-dna-resulted-6494318/
• https://www.ndsu.edu/pubweb/~mcclean/plsc411/DNA%20replication%20sequencing%20revision%202017.pdf
• https://www.britannica.com/biography/Frederick-Griffith
• https://ib.bioninja.com.au/higher-level/topic-7-nucleic-acids/71-dna-structure-and-replic/dna-experiments.html
• https://biolearnspot.blogspot.com/2017/11/experiments-of-avery-macleod-and.html
• https://www.khanacademy.org/science/biology/dna-as-the-genetic-material/dna-discovery-and-structure/a/classic-experiments-dna-as-the-genetic-material

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Avery-McCarty-McLeod experiments: The 80th anniversary of identifying DNA as the molecular basis of heredity

The simple but bold 68 th Street entrance to the Rockefeller campus was erected in honor of the man who in many ways embodies the scientific and social spirit of the institute. The inscription on one of the piers guarding the entrance reads,

and is a homage to the seminal work done over many decades by Avery and his colleagues within these gates. “The Professor ” or simply “ Fess ” Avery, as his friends and colleagues fondly call him, had a lot in common with the institute he called home for most of his professional life. Quoting from Rene J. Dubos’ fascinating book The Professor, The Institute, and DNA   (which I have used as the primary reference for this article),

“Avery and the Institute were respectively the human and institutional expressions of the same scientific attitudes. They both emerged and developed in the atmosphere of expectancy generated by a few triumphs of scientific medicine at the end of the nineteenth century; both followed an intellectual course that led them from the study of specific diseases to large problems of theoretical biology; both became part of a culture in which laboratory scientists were regarded as members of a kind of priesthood, willing to accept social constraints for the sake of intellectual privileges.”

Therefore, learning about Avery’s story allows us to delve into the fascinating history of the Rockefeller Institute and the people who shaped it. Furthermore, the 80 th anniversary of Avery’s groundbreaking 1944 paper that identified DNA as the molecular basis of heredity is the perfect opportunity to recall the amazing discoveries that originated at Rockefeller and went on to shape the course of modern biomedical research.

Bridging medical and laboratory sciences

Over centuries, medical science has undergone many paradigm shifts. One such noteworthy transformation occurred during the latter half of the 19th century. Due to limitations in technology, early medical science was largely empirical–the observations regarding transmission and pathologies of diseases were recorded, but the underlying mechanism remained poorly understood. As a result, the medical sciences, which dealt with patient care and treatment, were considered largely disparate from the laboratory sciences, which dealt with the chemical properties of biomolecules. However, by the late 19th century, when infectious diseases were the leading cause of death in humans, scientists like Louis Pasteur, Robert Koch, and others were beginning to demonstrate that bacteria and other microorganisms are the underlying causes of these infectious diseases. This had immediate practical consequences in the prevention and control of these ailments, and, for perhaps the first time, it was evident that progress in medicine could be achieved by laboratory investigations that did not directly involve patient care. This realization began to bridge the divide between the laboratory and medical sciences.

Medical research comes to the U.S.

Across Europe, institutions dedicated to the advancement of medicine through the study of fundamental mechanisms of pathology began emerging, such as the Pasteur Institute in Paris and the Koch Institute in Berlin. Although the prospects for medical research in the United States looked bleak initially, it soon began to change around the turn of the 20 th century. During the late 19 th century, it was becoming increasingly common for young American physicians to spend a few months or years in Europe, familiarizing themselves with the new kind of medical science flourishing across its medical centers. Upon returning home, they brought with them the culture of research-driven medicine that they were eager to emulate. Around the same time, wealthy philanthropists were beginning to shift the emphasis from traditional individualized charities to donating towards programs for social improvement. Together, these two factors catalyzed the creation of institutions where the new model of research-based medical science could be implemented. In addition to places like the Johns Hopkins Institute, one of the main beneficiaries of this new social phenomenon was the Rockefeller Institute, funded by the immense fortune of the oil baron John D. Rockefeller, and created with the ambitious and rather broad vision of promoting any scientific investigation with bearing on health and disease.

Oswald Avery enters the scene

The story of Oswald Avery reflected this larger trend in society. He completed his medical degree at The College of Physicians and Surgeons at Columbia University in 1904. By 1907, he had transitioned from a clinical to a more laboratory-focused role, which was fitting within the increasing research consciousness of medical New York. His first research position was as the assistant director at the Hoagland Laboratory in Brooklyn, which was amongst the first wave of privately endowed medical research laboratories in the United States. Avery spent 6 years at the Hoagland Laboratory studying and researching bacteriology, where his director Benjamin White, a Yale-educated physiological chemist, indoctrinated him with the chemical mode of thinking about biological problems—an approach that greatly inspired Avery’s future research.

During his time at the Hoagland laboratory, Avery published nine papers related to tuberculosis, vaccinations, and secondary infections, catching the attention of Dr. Rufus Cole, the then-director of the Rockefeller Institute Hospital. In 1913, Cole recruited Avery to the pneumonia research program at the hospital, where in a few years, Avery was quickly promoted to the highest rank of a full Member. During his early years at Rockefeller, Avery’s research style also changed markedly from the more methodical but perhaps unimaginative experiments he did during his time at the Hoagland Laboratories to a more creative but still equally thorough approach that would come to characterize much of his later work. This shift in approach was likely due to the carefully cultivated intellectual culture at the Rockefeller Institute that encouraged bold and imaginative scientific pursuits largely unencumbered with funding or logistical concerns, which fit well with Avery’s innate scientific temperament. Thus began Avery’s decades-long scientific journey toward fundamental problems in biological chemistry that eventually led to the landmark 1944 paper for which he is best remembered. 

The DNA revolution

In the first half of the 20 th century, driven by the revolutions in genetics and molecular biology, life sciences underwent a radical transformation from a largely descriptive science to an information science. The emerging interpretation was one where the information is stored in the genetic blueprint carried by each organism, and life processes are an outcome of “reading out” this blueprint (see Figure 1). This revolution in biology was heralded by Darwin’s theory of evolution and Mendel’s genetics experiments, which laid the framework for thinking about heredity and information transfer. However, neither of their theories talked about the physicochemical mechanisms underlying their observations. The first inklings of the molecular substrate of heredity can be traced back to Walther Flemming who observed structures in the nucleus of cells that were stained by various dyes and thus named them “colored bodies” or chromosomes. Later Boveri and Sutton as well as T.H. Morgan observed how these chromosomes are transmitted during cell divisions and between generations, and noted that they followed all the rules outlined for the heredity “factors” proposed by Mendel’s theory. In parallel, other scientists pioneered techniques to isolate different chemical components of cells based on differences in their chemical properties, leading to the isolation and characterization of several important biomolecules such as RNA, DNA, lipids, and proteins. Due to the rich biochemical diversity in the composition and properties of proteins, the scientific community was quick to nominate them as the best molecular candidates for encoding genetic information. Ultimately, this protein-based heredity dogma in the field of genetics was challenged from unlikely quarters–the seemingly disconnected field of bacterial immunology.

The key experiments that established DNA’s role in heredity

During the period of 1920s-30s that came to be known as “The Golden Era of Immunology at The Rockefeller Institute,” Avery and his contemporaries made key discoveries regarding bacterial metabolism, the chemical basis of virulence and immunity, and the heritable variability in these properties between different subclasses of pneumococci. While this was happening in New York, across the Atlantic an English scientist named Fred Griffith performed his now iconic experiment (see Figure 2) where he observed that when a mixture of avirulent R strain and heat-killed virulent S strain pneumococcal bacteria is injected in mice, there is a transfer of these heritable virulence-conferring chemical properties from the latter to the former. These experiments sent shockwaves across the international immunology community and were also widely discussed in Avery’s department at the Rockefeller Institute. Avery’s group meticulously replicated these results and even extended them to demonstrate that this transformation between different bacterial cells can occur in vitro . They further showed that this in vitro transformation could be brought about not just by whole heat-killed S cells but also with a soluble fraction produced by dissolving the S cells in sodium deoxycholate (an ionic detergent) and filtering the cellular debris; the active material could be precipitated from the filtrate with alcohol and was described as “a thick syrupy precipitate” that was “fairly stable.”

Characteristic of his thorough and disciplined approach towards science, Avery spent many subsequent years trying to establish the chemical identity of this viscous precipitate that they had named the “transforming principle,” perhaps because he had sensed its broader significance as the potential molecular candidate for heredity that the field of genetics was desperately hunting. In this endeavor, Avery collaborated with many of the newer members in the department amongst which two key figures were Colin MacLeod, who helped optimize the technique for extracting highly pure samples of this transformation substance, and later Maclyn McCarty who performed many chemical tests to help establish the identity of this purified transformation substance. Some of the key chemical tests performed showed that the transforming principle was stable to the action of a myriad of proteases and ribonucleases whilst only responding to enzymes previously shown to attack deoxyribonucleic acids, showing that its elemental phosphorus-nitrogen ratios closely resembled that of DNA, and roughly estimating the molecular weight of the substance being consistent with it being a long polymer. Eventually, these results were compiled in the now classic paper by Avery, MacLeod, and McCarty submitted to the Journal of Experimental Medicine in November 1943 and published in 1944. The somewhat unflashy name of the article “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III” contrasts the extraordinary findings detailed within, which for the first time implicated that DNA could be the molecular identity of the information blueprint prevalent in all life forms. While Avery himself was conservative about making such broad claims, the scientific community understood his discovery as pivotal.

Since this discovery overturned the long-held protein-based heredity dogma, there was great resistance from the scientific community both within and outside the institute to accept these findings. Amongst the many criticisms leveled against these findings was that despite the meticulous efforts at purification, Avery’s DNA sample was somehow contaminated by small amounts of some protein which was the true “transforming principle”, or that there was some nucleoprotein so tightly associated with the DNA that it became chemically inseparable. Avery himself had anticipated this backlash and therefore had sent the manuscript of his paper for critical review to many of his friends and associates before submitting it for publication. Even after its publication, Avery was not one to broadcast his findings as a turning point in science, and characteristic of his personality, his response to the criticisms was to begin planning further experiments that could vindicate his results. Subsequently, many experiments done at Rockefeller and elsewhere provided additional evidence for DNA’s role in the transmission of hereditary characteristics. Avery’s introverted nature meant that his reactions to the happenings were rarely expressed in public, but his mood of excitement tempered with caution was evident in a letter he wrote to his brother Roy in 1943, where he recognized the broad implications of his findings, but advised him not to “shout it around” because “It’s hazardous to go off half-cocked – and embarrassing to retract it later…”.

It is hard to estimate when this tide of opinion slowly shifted, but some 8 years later, when the Hershey-Chase experiment at the Cold Spring Harbor laboratory beautifully corroborated Avery-MacLeod-McCarty’s findings, the last remaining skeptics were converted. DNA was thus incorporated into the standard genetic theory, and, in 1953, the identification of the structure of DNA by Watson and Crick with data from Rosalind Franklin ushered in an age of biology united under a few fundamental principles. This led to the coming of age of molecular biology, where the information transfer in biology was established in concrete molecular terms, compiled in what is now known as the central dogma . In turn, this paved the way for the Genomics Era, setting the stage for a time where DNA/RNA sequencing has now become a routine part of many biological experiments. 

The staggering implications of these discoveries have touched every area of biology, and the ability to use a molecular language to describe essential life processes has far-reaching consequences for all of medical science. In a sense, all modern chemical genetics owes its roots to the studies in bacterial heritability done by Avery and colleagues here at the Rockefeller Institute, and it surely gives us immense pride to be a part of this great scientific legacy.

Oswald Avery and the Avery-McLeod-McCarthy Experiment

Oswald Avery and his colleagues showed that DNA was the key component of Griffith’s experiment , in which mice are injected with dead bacteria of one strain and live bacteria of another, and develop an infection of the dead strain’s type

On February 1 , 1944 , physician and medical researcher Oswald Avery together with his colleagues Colin MacLeod and Maclyn McCarty announced that DNA is the hereditary agent in a virus that would transform a virus from a harmless to a pathogenic version. This study was a key work in modern bacteriology .

Prelude – The Griffith Experiment

The achievement by the scientists Avery, MacLeod, and McCarty were based on Frederick Griffith’s studies on bacteria, believing that bacteria types were not changeable from one to another generation. His also famous attempt is called the Griffith experiment, and was published in 1928. In it, the medical officer Griffith identified a principle in pneumococcal bacteria, in which they could transform from one to another type. After several years of research on the disease pneumonia , he found out that types changed into another rather than multiple types being present at the same time. His later research proved, that the transformation occurred, when dead bacteria of a virulent and live bacteria of a non-virulent type were injected in mice, they would suffer an infection and die shortly after. The other case proved, that the injected virulent bacteria was to be isolated from an infected mouse, depicted in the picture above. The German bacteriologist Fred Neufeld was the first to prove Griffith’s findings right and soon, renowned institutes, like the Koch Institute or the Rockefeller Institute took over the case, doing further research on Griffith’s great accomplishments.

Oswald Avery (1877-1955)

The Avery-MacLeod-McCarty Experiment

Avery and his collaborators Colin MacLeod and Maclyn McCarty at Rockefeller University (then Rockefeller Institute) in New York wanted to elucidate the chemical nature of the transforming substance. They refined the purification process until the result was a cell extract whose amounts of carbon, hydrogen, nitrogen and phosphorus corresponded to those of DNA.[ 4 ] To ensure that the transformation was not induced by residues of RNA or proteins, they treated the cell extract with different enzymes prior to the transformation. One of these enzymes had a deoxyribonucleode polymerase activity described by Greenstein in 1940. Only this neutralized the transformation activity of the extract, while trypsin, chymotrypsin (two protein cleaving enzymes), ribonuclease, protein phosphatases and esterase had no effect on transformation activity. They were also able to show that all offspring inherited the S-properties and that the repetition of the experiment with extracts from these offspring led to the same results.

This experiment shows that the genetic information must lie on the DNA, since the R-cells needed information from the S-cells to form a mucus capsule, i. e. to become S-cells. And only the DNA made it possible to transform R cells into S cells. In the counterexample with an enzyme, it became even clearer that the genetic information must lie in the DNA, since only R cells develop when a DNAse is added, because the DNA was broken down by the enzyme.

The achievements of the Avery-MacLeod-McCarty experiment quickly spread out into the scientific community and it was proven right just as fast. However, only very few scientists would accept the thought that genetics were to be applied to bacteria, but as Joshua Lederberg , himself an American molecular biologist suggested, the three scientists paved the early way for molecular genetics. The experiment opened up new possibilities and research fields for following biologists. Avery was awarded the Copley Medal for his bacterial transformations, but neglected by many scientists and organizations for his work.

References and Further Reading:

• [1] Lederberg J (February 1994). “The transformation of genetics by DNA: an anniversary celebration of Avery, MacLeod and McCarty (1944)” . Genetics 136 (2): 423–6
• [2] History of DNA Researc h Timeline
• [3] Isolating Hereditary Material: Frederick Griffith, Oswald Avery, Alfred Hershey, and Martha Chase at Nature
• [4] Crick and Watson Decipher the DNA , SciHi Blog, February 28, 2013
• [4] Louis Pasteur – The Father of Medical Microbiology , SciHi Blog, December 27, 2012
• [5] National Academy of Sciences Biographical Memoir
• [6]  Oswald T. Avery Collection (1912-2005)  – National Library of Medicine finding aid
• [7]  DNA: The Search for the Genetic Material  Avery, MacLeod and McCarty’s Experiment for the Advanced Science Hobbyist.
• [8] Oswald T. Avery at Wikidata
• [9]  How 3 Scientists Found that DNA is the Molecule of Heredity – Avery, MacLeod, and McCarty , 2020, YourekaScience @ youtube
• [10] Timeline of famous Biology Experiments , via DBpedia and Wikidata

Harald Sack

Related posts, norman borlaug and the green revolution, alec jeffreys and the genetic fingerprint, maurice wilkins and the riddle of the dna structure, seymour benzer and his experiments in behavioural genetics, leave a reply cancel reply.

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1944: DNA is \"Transforming Principle\"

1944: dna is "transforming principle".

Avery, MacLeod and McCarty identified DNA as the "transforming principle" while studying Streptococcus pneumoniae , bacteria that can cause pneumonia. The bacteriologists were interested in the difference between two strains of Streptococci that Frederick Griffith had identified in 1923: one, the S (smooth) strain, has a polysaccharide coat and produces smooth, shiny colonies on a lab plate; the other, the R (rough) strain, lacks the coat and produces colonies that look rough and irregular. The relatively harmless R strain lacks an enzyme needed to make the capsule found in the virulent S strain.

Griffith had discovered that he could convert the R strain into the virulent S strain. After he injected mice with R strain cells and, simultaneously, with heat-killed cells of the S strain, the mice developed pneumonia and died. In their blood, Griffith found live bacteria of the deadly S type. The S strain extract somehow had "transformed" the R strain bacteria to S form. Avery and members of his lab studied transformation in fits and starts over the next 15 years. In the early 1940s, they began a concerted effort to purify the "transforming principle" and understand its chemical nature.

Bacteriologists suspected the transforming factor was some kind of protein. The transforming principle could be precipitated with alcohol, which showed that it was not a carbohydrate like the polysaccharide coat itself. But Avery and McCarty observed that proteases - enzymes that degrade proteins - did not destroy the transforming principle. Neither did lipases - enzymes that digest lipids. They found that the transforming substance was rich in nucleic acids, but ribonuclease, which digests RNA, did not inactivate the substance. They also found that the transforming principle had a high molecular weight. They had isolated DNA. This was the agent that could produce an enduring, heritable change in an organism.

Until then, biochemists had assumed that deoxyribonucleic acid was a relatively unimportant, structural chemical in chromosomes and that proteins, with their greater chemical complexity, transmitted genetic traits.

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As taught in, learning resource types, fundamentals of biology, dna structure, classic experiments.

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• DNA Structure and Classic experiments, Excerpt 1

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Although the fractions were not completely pure, the fractions that had transforming abilities were greatly enriched for nucleic acid, specifically DNA.

What functional group is found on the 3’ end of a nucleotide? carboxyl close hydroxyl check nitrogenous base close phosphate close Check

Which of the following is found in DNA but not in protein? Carbon close Nitrogen close Oxygen close Phosphorus check Sulphur close Check

• DNA Structure and Classic experiments, Excerpt 2

In density gradient centrifugation, which of the following DNA molecules will travel the farthest down the tube? a 14N-14N duplex close a 14N-15N duplex close a 15N-15N duplex check Check

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• Classic experiments by Avery, Griffith, and Hershey/Chase
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Isolating Hereditary Material: Frederick Griffith, Oswald Avery, Alfred Hershey, and Martha Chase

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 S bacteria and living R bacteria (Figure 2), 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 (Figure 3), 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

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.

References and Recommended Reading

Avery, O. T., et al . Studies on the chemical nature of the substance inducing transformation of pneumococcal types. Journal of Experimental Medicine 79 , 137–157 (1944)

Griffith, F. The significance of pneumococcal types . Journal of Hygiene 27 , 113–159 (1928)

Hershey, A. D., & Chase, M. Independent functions of viral protein and nucleic acid in growth of bacteriophage. Journal of General Physiology 36 , 39–56 (1952)

Steinman, R. M., & Moberg, C. L. A triple tribute to the experiment that transformed biology . Journal of Experimental Medicine 179 , 379–384 (1994)

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A Pioneer of Genetics: Oswald Avery's landmark experiment

Dec 06, 2023 · 2 mins read

@ForwardThinking

Oswald Avery was one of the first molecular biologists and a pioneer in immunochemistry, but he's best known for one experiment that changed life as we know it. In 1944, he spearheaded the groundbreaking Avery-McLeod-McCarthy Experiment: a milestone in genetics research.

Working alongside Colin MacLeod and Maclyn McCarty, Avery investigated the transformation of non-virulent bacteria into virulent strains, targeting the responsible element by examining the pneumococcus bacterium.

The experiment aimed to identify whether DNA, RNA, or proteins carried the hereditary information necessary for transformation.

The purification procedure Avery undertook consisted of isolating and purifying the bacterial components, treating them with enzymes to degrade proteins and RNA, leaving behind DNA.

Astonishingly, when DNA remained, the non-virulent bacteria transformed into virulent forms, establishing DNA as the carrier of genetic information.

Avery's meticulous approach and rigorous experimental design solidified the understanding of DNA as the hereditary material, reshaping the course of genetics.

Despite initial scepticism, the findings of the Avery–MacLeod–McCarty experiment were quickly confirmed and profoundly influenced subsequent genetic research, paving the way for the DNA-focused era in biology.

Avery's findings, initially overlooked, gained recognition posthumously, earning him acclaim as a pivotal figure in the discovery of DNA's role in heredity.

His research laid the groundwork for Watson and Crick's elucidation of DNA's structure in 1953, further validating Avery's pivotal contributions.

Oswald Avery's relentless pursuit of scientific truth revolutionized genetics, unraveling the fundamental role of DNA in passing on genetic information, marking a watershed moment in biological understanding.

“Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types: Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III” (1944) by Oswald Avery, Colin MacLeod and Maclyn McCarty