Fredrick Griffith
He postulated that information could somehow be transferred between different strains of bacteria. This was long
before the discovery of DNA and was an inspired piece of scientific detective work.
METHODOLOGY
For this study, Griffith used two strains
of Pneumococcus bacteria, type III-S and type II-R.
There is one major difference between these two types; the III-S strain has a smooth polysaccharide coat which
makes it resistant to the immune system of mice, whereas the II-R strain lacks this coat and so will be destroyed by
the immune system of the host.
For the first stage of the transforming principle experiment, Griffith showed that mice injected with III-S died but
when injected with II-R lived and showed few symptoms.
The next stage showed that if the mice were injected with type III-S that had been killed by heat, the mice all lived,
indicating that the bacteria had been rendered ineffective.
The interesting results came with the third part of the experiment, where mice were injected with a mixture of heat
killed III-S and live II-R.
Interestingly enough, the mice all died, indicating that some sort on information had been passed from the dead type
III-S to the live type II-R. Blood sampling showed that the blood of the dead mice contained both live type III-S and
live type II-R bacteria.
Somehow the type III-S had been transformed into the type III-R strain, a process he christened the transforming
principle.
DISCUSSION Follow up experiments performed by Avery, McLeod and McCarty and by Hershey and Chase
established that DNA was the mechanism for this transferal of genetic information between the two bacteria.
In turn, this lead to the discoveries of Crick and Watson, who discovered the exact structure of DNA, and the
mechanisms used for storing and transferring information.
Considering that Griffith did not know the chemical and biological processes behind the transforming principle, it
was inspirational research which built on the theories of scientists such as Mendel. The study opened up avenues of
research into the biochemical principles behind the genetic transference of information.
Genetic engineering, involving the transferring of DNA between organisms, is now more commonplace, but built
upon the research performed by Griffith. Most biology students have heard of Mendel, and Crick and Watson, but
must not forget the work of the other inspiring scientists in between.
Read more: http://explorable.com/transforming-principle.html#ixzz2An7Q0nyO
Oswald Avery
DNA, although now known to be extremely important, was overlooked for quite some time. Until early 1953,
around when the Watson and Crick structure of DNA was published, most major scientists thought that proteins,
rather than DNA, were probably the site of the gene.
In the early 1940s however, experiments performed by Oswald T. Avery and his colleagues at the Rockefeller
Institute for Medical Research made a strong argument for DNA as the source of the genetic material.
Unfortunately, for many years not much attention was paid to Avery’s work.
Streptococcus pneumoniae, also called pneumococcus, was the subject of Avery’s experiment. This bacterium
causes a variety of diseases, including pneumonia and peritonitis. The organism can be found in two forms, smooth
(S) and rough (R) which are designated as such simply because of their appearance when viewed microscopically.
The smooth appearance is a result of the formation of a polysaccharide capsule that encases the bacterial cell. This
capsule protects the cell from immunological defenses, which makes the S form virulent. The R form, on the other
hand, is mutated so that it does not synthesize the enzyme that creates the polysaccharide capsule, and is therefore
not virulent.
The pneucmococcus bacteria can be further characterized into types, which are designated by roman numerals.
Although an S form bacterial cell can be experimentally changed to an R form (and vice versa) provided the cell is
not too far degraded, change of type never suddenly occurs. For example, a type III S cell can be converted to a type
III R cell, but a type II cell will never spontaneously convert to a type III cell.
Although a spontaneous change of type is not possible, a specific experiment had been done showing that a
transformation of type can be induced. This experiment was first performed by injecting a live culture of the Type II
R form into mice along with a dead culture of the Type III S form. Theoretically, none of the mice should have died
because they hadn’t been exposed to a virulent form of the bacteria. However, many of the mice did actually die,
and living Type III S form bacteria was extracted from their blood. Later, the same experiment was accomplished by
growing the bacteria in a glass dish rather than in mice.
Understandably, this transformation from a non-virulent bacteria to a virulent bacteria was troubling to the medical
community. Although some scientists may have been concerned with the mechanism of transformation, Oswald
Avery was more concerned with the identity of the agent performing the transformation. He went to work on
devising an experiment that would allow him to isolate the transforming agent from the rest of the bacterial cell.
Although DNA extraction is now considered a simple process, it was just beginning to emerge during the time when
Avery began his work. The fact that Avery did not know that the transforming agent was in fact DNA complicated
matters even further.
Nevertheless, after years of hard work, Avery and his colleagues were able to develop an experiment that effectively
isolated the transforming agent from the bacterial cells. Type III S form bacteria were grown in large vats of broth
made from beef hearts. The bacteria was then killed, and washed with brine in order to remove the polysaccharide
capsule and whatever protein would come off in the process. The remainder of the bacteria was then precipitated in
pure grain alcohol. After this, the precipitate was washed with chloroform and subjected to a digestive enzyme, both
of which functioned to remove the remaining protein. Finally, after no trace of protein was evident, pure grain
alcohol was once again added, which allowed the transforming agent to be separated. The process was long and
difficult, and in the end only yielded approximately ten to twenty-five milligrams of the agent per seventy-five liters
of culture.
After obtaining enough of the active transforming agent to conduct his tests, Avery and his colleagues set out to
show exactly what the substance was. First, standard qualitative tests for proteins were performed, which came back
negative. Qualitative tests for DNA, however, were strongly positive. Chemical analysis of the substance also
showed that the ratio of nitrogen atoms to phosphorous atoms was approximately 1.67 to 1. This number is very
close to the DNA ratio, and would have been different had there been a significant amount of protein present.
Next, tests with digestive enzymes were performed. The addition of enzymes that digest proteins and RNA left the
agent intact, while enzymes that digest DNA completely destroyed the substance.
Finally, immunological tests involving centrifugation and electrophoresis were performed, which also showed that
proteins and polysaccharides weren’t present, but that DNA was.
Once Avery was satisfied with the results of his tests, he began writing a manuscript that explained the experiment.
In January of 1944 “Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal
Types” by Oswald T. Avery, Colin M. MacLeod, and Maclyn McCarty was published. Although it may seem that
Avery and his colleagues had proven that DNA was the site of the gene, this was not entirely the case. There was
still a possibility that, as Avery puts it in the manuscript, “the biological activity of the substance described is not an
inherent property of the nucleic acid but is due to minute amounts of some other substance adsorbed to it or so
intimately associated with it as to escape detection. . .”
Avery was clearly being very cautious in his conclusions, never stating that he was certain that DNA was the
transforming agent. It is possible that his cautiousness with the matter contributed to his lack of attention received.
However, there were other reasons why Avery wasn’t given serious attention. As James Watson later stated in 1983:
“Both Francis and I had no doubts that DNA was the gene. But most people did. And again, you might say, ‘Why
didn’t Avery get the Nobel Prize?’ Because most people didn’t take him seriously. Because you could always argue
that his observations were limited to bacteria, or that [the transformation of pneumococcus that he described was
caused by] a protein resistant to proteases and that the DNA was just scaffolding.”
Although Avery’s manuscript may not have been received with high praise at the time of its publication, it is now
considered to be a very thorough account of an expertly accomplished experiment. For more information on DNA,
please visit the Race for DNA website. For information on Linus Pauling, a major player in the DNA story, visit the
Linus Pauling Online
Alfred Hershey and Martha Chase
“When asked what his idea of happiness would be, [Hershey] replied, ‘to have an experiment that works, and do it
over and over again.’”
- Jonathan Hodgkin, 2001
In 1944 the Avery-MacLeod-McCarty experiments demonstrated that DNA, rather than proteins, is the carrier of
genetic information. Though the work appeared to be well-supported, and was endorsed by other researchers, the
trio met with resistance from much of the scientific community. For nearly a decade, the Avery group was forced to
repel attacks on the validity of their experiments, defending both their findings and their reputations.
Finally, in 1952, Alfred Hershey, a Carnegie Institution researcher working at Cold Spring Harbor Laboratory, set
out to conclusively settle the issue. Like many of his contemporaries, Hershey believed that proteins, with their
complicated structures, were more likely to be the carriers of genetic information than was the simple DNA
molecule. Hershey, however, was about to make a discovery that would turn his own notions on end.
In order to show that proteins carry genetic information, Hershey and his lab technician, Martha Chase, decided to
track the transfer of proteins and DNA between a virus and its host. For their experiment, they chose to use the T2
bacteriophage as the vehicle for delivering genetic material. Like all bacterial viruses, the T2 is comprised of only a
protein-based outer wall and a DNA core, its simple structure making it the perfect research candidate. The phage
reproduces by injecting its genetic material into a bacterium, leaving its protein shell attached to the host. Then,
through a microscopic takeover, the virus seizes control of the bacterium’s reproductive mechanisms and uses them
to duplicate itself, destroying the host in the process.
Though it was known that the protein shell remained outside the bacterium, researchers thought it possible that
certain proteins were transferred from the virus to the bacterium upon attachment. If genetic material was in fact
carried by proteins, this would explain how a phage is able to reproduce within a bacterium without the entirety of
the protein shell penetrating the bacterium’s membrane. In order to prove that proteins are the carriers of genetic
information, Hershey and Chase needed to demonstrate that at least a portion of the phage’s protein mass was
transferred to the interior of the bacterium.
In their first experiment, Hershey and Chase tagged the T2 phage DNA with Phosphorous-32, a radioactive form of
the element. Because phosphorous can be found in large quantities in DNA, but in only trace amounts in protein,
the researchers could track the location of DNA and protein according to the radiation concentrations. They then
allowed the tagged phages to begin infecting samples of E. coli. After introducing to the phage culture to the
bacterial sample, they used a Waring blender to violently disturb the infected bacteria, causing the protein shells to
detach from their hosts. Then, using a centrifuge, they separated the bacterium from the phages and protein.
Once the separation was complete, they measured the radiation concentrations in the E. coli cells and the protein
shells. The phosphorous tracer appeared in large quantities only in the bacterial sample, demonstrating that DNA
was transferred from the bacteriophage to the host organism. Further, despite the protein shells being detached
while reproduction of the phage should have been taking place, the virus was still copied in each of the host cells.
This, in turn, suggested that the proteins shell itself was not necessary to the replication process following the initial
insertion of genetic material.
Shocked by their findings, Hershey and Chase decided to perform the test once again, this time using a different
tracer molecule. They chose sulfur for the second test, because it appears in the amino acids that make up proteins,
but is not present in DNA. This allowed them to track the same process as in the first experiment, but in
reverse. After tagging the proteins, infecting the E. coli cells, and separating the shells from the host, the researchers
tested for the presence of sulfur. In accordance with their previous results, the sulfur could only be found in the
protein shells and not in the bacteria. And again, the phage’s genetic material was replicated despite the protein shell
being disconnected from the bacteria via the blending process.
The Hershey-Chase Blender Experiment. Diagram by Eric Arnold.
Sufficiently impressed by the significance of his findings, Hershey returned to the phosphorous-tagged batch to
engage in some follow-up research. Upon examining the offspring of the phages, the researchers found that the
young bacteriophages also possessed phosphorous-tagged DNA, but their protein lacked any trace of
radioactivity. The implications of their first experiments were reinforced.
At first, the pair was inclined to believe that the experimental or data-collection procedures were flawed. They
rechecked the experiment design, the equipment, and the bacterial cultures. It was all in vain, though. Hershey was
a notoriously cautious researcher and his experiments were always well-planned and precisely executed. The results
were no mistake and the import of their work was clear: Hershey and Chase had elucidated direct, irrefutable
evidence that DNA, not protein, is the source of genetic material.
Later that year, the pair reported their findings in a short paper in The Journal of General
Physiology titled “Independent Functions of Viral Protein and Nucleic Acid in Growth of
Bacteriophage.” This publication catalyzed a storm of activity in the scientific community,
with researchers all over the world clamoring for details on the experiments. Alfred
Hershey’s lectures on the subject were attended by the greatest scientific minds in Europe
and North America; Pauling was one of hundreds to hear him speak. In the years following
the discovery, DNA became a major focus for researchers all over the world, resulting in
Pauling’s own attempts to deduce its structure and the eventual success of Watson and
Crick. Even today, our genetic research traces its roots from the work of Alfred Hershey
and Martha Chase.
Erwin Chargaff
“We have created a mechanism that makes it practically impossible for a real genius to appear. In my own field the
biochemist Fritz Lipmann or the much-maligned Linus Pauling were very talented people. But generally, geniuses
everywhere seem to have died out by 1914. Today, most are mediocrities blown up by the winds of the time.”
-Erwin Chargaff, 1985.
Erwin Chargaff, (1905-2002) a biochemist born in Austria, became interested in DNA earlier than most. In the
1930s, while he was working with the bacteria Rickettsi, he became aware of nucleic acids, and decided to educate
himself about them.
In 1944, after Oswald Avery published his paper detailing the transforming principle of the Pneumococcus bacteria,
Chargaff decided to devote his laboratory almost entirely to the chemistry of nucleic acids. Experimenting with
these delicate substances was not an easy task, but eventually a chromatographic technique was developed that
would allow for the separation and analysis of the base rings in DNA. This work would later lead to the
development of Chargaff’s Rules, the topic of today’s post.
The guanine-cytosine base pair.
DNA has two main structural components – a backbone made up of sugar and phosphate groups, and a series of
bases found in the middle of the molecule. There are four different bases found in DNA: Adenine (A), Cytosine (C),
Guanine (G), and Thymine (T). These four bases can be divided into two categories, pyrimidines and purines. The
pyrimidine bases, Cytosine and Thymine, contain only one ring, while the purine bases, Guanine and Adenine,
contain two rings. In the DNA structure, the bases pair complementarily, meaning that a purine base will bind with a
pyrimidine base. More specifically, Adenine binds with Thymine and Cytosine binds with Guanine.
The adenine-thymine base pair.
Although this information is now considered fundamental biology, it wasn’t fully understood until after Watson and
Crick discovered the structure of DNA in 1953. However, Chargaff’s research in the late 1940s had suggested that
the four bases paired in the manner described above.
When Chargaff first decided to devote his laboratory to nucleic acids, he allowed a postdoctoral student named Ernst
Vischer to choose his research program from a list of suggested topics. Vischer decided to analyze the purines and
pyrimidines in nucleic acids, and went to work developing the chromatographic technique so crucial to isolating the
bases. Although his technique was rather crude, it did the trick and Vischer achieved great success. The results of
the base analysis showed that the amounts of Adenine and Thymine were about equal, and also that the amounts of
Guanine and Cytosine were about equal. Eventually, Chargaff came to the conclusion that in a single molecule of
DNA, Guanine/Cytosine = Adenine/Thymine = 1. This concept would later become known as Chargaff’s Rules.
Chargaff’s Rules were officially announced in a lecture delivered in June of 1949 and were first published in May of
1950. However, Linus Pauling had heard about the ratios much earlier – straight from Chargaff in late 1947, while
traveling to England for his six-month stay as a professor at Oxford University. Pauling, who considered the trip by
ship across the Atlantic Ocean with his family to be a vacation, did not pay attention to what Chargaff told him.
Crellin Pauling, the youngest child of Linus and Ava Helen Pauling, mentioned the remarkable background to the
incident in a speech given during a symposium to celebrate Pauling’s life that was held here at Oregon State
University in 1995.
Rosalind Franklin
During their so-called race to discover the structure of DNA, Linus Pauling and the unlikely pair of James Watson
and Francis Crick utilized remarkably similar approaches in attempting to solve the riddle of the genetic material. In
fact, one of the main tactics used by Watson and Crick was to approach the problem in the same manner that they
assumed Pauling would. Although Pauling and Watson and Crick did, at one point, come up with nearly identical,
yet incorrect, structures, it was Watson and Crick who would eventually solve DNA. Why then, if the pair were
thinking like Pauling, were they able to beat him to the structure?
Although there were a variety of reasons behind Watson and Crick’s success, a good portion of it can be attributed
to the relative superiority of resources available to them. Watson and Crick obviously had each other to keep
themselves in check, but they also benefited from other voices of criticism such as Rosalind Franklin, Maurice
Wilkins, and later Jerry Donohue. Linus Pauling also shared his ideas with his colleagues, but none of them were
very familiar with DNA, and therefore couldn’t offer much feedback. (And they were largely ignored even when
they did offer criticisms of Pauling’s structure.)
Another vital resource available to Watson and Crick was an excellent X-ray crystallography pattern, the famous
photo 51, taken by Rosalind Franklin. Although, in all likelihood, Pauling could have also viewed Franklin’s
photographs had he tried, he settled on using blurry patterns published by William T. Astbury several years before
Franklin’s superior images. These X-ray photographs are the main topic of today’s post. In particular, the factors
accounting for the difference in quality between Franklin’s and Astbury’s patterns will be discussed. Before delving
into this subject, however, a brief overview of X-ray crystallography is necessary.
X-ray crystallography, also sometimes known as X-ray diffraction, is used to determine the arrangement of atoms
within a crystalline molecule. It is a rather complicated procedure, and the photos taken in the process can be
interpreted only by a person with significant training. The steps to obtaining these photos are as follows.
First, an adequate crystal must be obtained. This is a very difficult step because the crystal must be large enough to
observe and also sufficiently uniform. If it does not meet these specifications, errors – such as blurriness – will
occur, often rendering the resulting crystallographic patterns useless, at least for purposes of determining atomic
arrangement.
After an adequate crystalline specimen is obtained, a beam of X-rays is shined through it. When the beam strikes the
electron clouds of the atoms in the crystal, it is scattered. These scattered beams can then be observed on a screen
placed behind the crystal. Based on the angles and intensities of the scattered beams, a crystallographer can create a
three dimensional picture of the electron density of the crystal.
Finally, from the electron density information, the mean positions of the atoms within a crystal can be determined,
and the structure of the molecule can be considered “solved.” That said, just one image is not nearly enough to
determine the structure of an entire crystal. Therefore, the crystal must be rotated stepwise through angles up to and
even slightly beyond 180 degrees, depending on the specimen. Patterns are required at each step, and complete data
sets may contain hundreds of photos.
Clearly, because the process of X-ray crystallography is so cumbersome, there are many opportunities for mistakes
that may have led to the poor quality of Astbury’s photographs. However, Astbury’s techniques seem to have been
excellent. He was a very experienced crystallographer, and had achieved great success in his earlier work with X-ray
diffraction on substances such as keratin.
As it turns out, Astbury’s photos were of poor quality because of the DNA sample he was using. In the early 1950s,
Rosalind Franklin had discovered that DNA came in two forms – a dry condensed form and a wet extended form.
Astbury’s DNA sample was well prepared from calf thymus, but it contained a mixture of the two forms. This
turned out to be the major reason why Astbury’s photographs were so blurry
Astbury's images, from "X-Ray Studies of Nucleic Acids," 1947. Plate 1.
Astbury's images, 1947. Plate 2.
It is important to note that, even if Astbury had known he was using a poor crystalline sample of DNA, he probably
still wouldn’t have been able to compete with the quality of Franklin’s photos. In 1950, three years after Astbury’s
images were published, Maurice Wilkins developed a way to obtain much better X-ray patterns of DNA through the
use of a solution of sodium thymonucleate. This solution is highly viscous, and Wilkins found that thin strands could
be drawn out by gently dipping a glass stirring rod into a sample and slowly pulling it out. These thin strands were
pure DNA, and Wilkins was able to get excellent X-ray patterns from them.
Before long, Wilkins had also acquired better equipment and had also hired Rosalind Franklin to run it. Franklin,
essentially working independently, used the same basic technique developed by Wilkins. She did, however, add
several of her own smaller experimental refinements, which made the photographs even better. Eventually, she
developed photo 51, which would later be shown to Watson and Crick. The rest, as they say, is history.
Rosalind Franklin and William Astbury were both excellent crystallographers, but Franklin’s experience with DNA
gave her a clear advantage when working with the molecule. Her brilliant X-ray patterns would later prove to be a
major determining factor in the “race for DNA”. For more information on DNA, please visit the Race for DNA
website.