History of DNA
LEARNING OBJECTIVE
• Summarize the evidence that accumulated during the 1940s and early 1950s
demonstrating that DNA is the genetic material.
• State the questions that these classic experiments addressed: Griffith’s
transformation experiment, Avery’s contribution to Griffith’s work, and the Hershey–
Chase experiments.
Transformation of Bacteria
1. In 1928, the bacteriologist Frederick Griffith conducted an experiment in
Diplococcus pneumonia. He studied two strains of virulent diplococcus causing
pneumonia.
2. The virulent strain synthesized a smooth polysaccharide coat and produces smooth
colonies. This strain was called strain-S.
3. Another strain which lacked the proper protein coat is harmless and produces rough
colonies. This strain is called strain-R.
4. When Griffith injected S-type of cells into the mouse, the mouse died.
5. When R-type cells were injected into the mouse, the mice did not die.
6. When he injected heat killed S-type cells into the mouse, the mouse did not die.
7. When the mixture of heat killed S-type cells and R-type cells were injected into the
mouse, the mouse was dead.
8. The living rough strain of diplococcus has been transformed into S-type cells. That is
the hereditary material of heat killed S-type cells had transformed R-type cells into
virulent smooth strains.
9. Thus the phenomenon of changing the character of one strain by transferring the
DNA another strain into the former is called transformation.
Results and Conclusion: although neither the rough strain nor the heat-killed
smooth strain could kill a mouse, a combination of the two did. Autopsy of the dead
mouse showed the presence of living S-strain pneumococci. These results indicated
that some substance in the heat-killed S cells had transformed the living R cells into
a virulent form.
DNA: The Transforming Substance
1. In 1944, Oswald T. Avery and his colleagues Colin M. Mac Leod and Maclyn McCarty
chemically identified Griffith’s transforming factor as DNA.
2. They did this through a series of careful experiments in which they lysed (split open)
heat-killed S cells and separated the cell contents into several fractions: lipids and
polysaccharides.
3. Now proteins and nucleic acids (DNA and RNA) remain.
4. Subject the solution to treatments of enzyme to destroy either proteins (proteinase),
RNA (ribonuclease) or DNA, (DNase).
5. They tested each fraction to see if it could transform living R cells into S cells.
6. Now they add a small portion of each sample to a culture containing R cells. Observe
whether transformation has occurred by testing for the presence of virulent S cell
7. The experiments using destroyed proteins and RNA did not cause transformation.
8. The experiments using the destroyed DNA, however, no transformation occurred –
no smooth colonies were seen on culture plates. Avery and his colleagues published
their discovery that the transforming principle was DNA.
Transformation cannot occur unless DNA is present. Therefore, DNA must be the hereditary
material.
Reproduction of Viruses
In 1952, geneticists Alfred Hershey and Martha Chase performed a series of elegant
experiments on the reproduction of viruses that infect bacteria, known as bacteriophages
or phages. When they planned their experiments, they knew that phages reproduce inside a
bacterial cell, eventually causing the cell to break open and release large numbers of new
viruses. Because electron microscopic studies had shown that only part of an infecting
phage actually enters the cell, they reasoned that the genetic material should be included in
that portion.
a. The purpose of their experiments was to see which of the bacteriophage
components -- the protein coat or the DNA -- entered bacteria cells and
directed reproduction of the virus.
b. Hershey and Chase thought up a clever procedure that made use of the
chemical elements found in protein and DNA. Protein contains sulphur but
very little phosphorus, while DNA contains phosphorous but no sulphur. The
researchers grew phages in cultures that contained radioactive isotopes of
sulphur or phosphorus. Hershey and Chase then used these radioactively
tagged phages in two experiments. In two separate experiments, they
labeled the protein coat with 35S and the DNA with 32P.
The phages in each sample attached to bacteria, and the researchers shook the
phages off by agitating the sample in a kitchen blender. Then they centrifuged the
samples. Centrifuged the mixture so that the bacteria would form a pellet at the
bottom of the test tube, measured the radioactivity in the pellet and in the liquid.
In the sample in which they had labelled the proteins with 35S, they subsequently found
radioactivity in the supernatant, indicating that the protein had not entered the cells.
In the sample in which they had labelled the DNA with 32P, they found radioactivity
associated with the bacterial cells (in the pellet): DNA had actually entered the cells.
Hershey and Chase concluded that phages inject their DNA into bacterial cells, leaving most
of their protein on the outside. This finding emphasized the significance of DNA in viral
reproduction, and many scientists saw it as an important demonstration of DNA’S role as
the hereditary material.
From their results, Hershey and Chase concluded that the phages DNA had entered the
bacteria, but the protein had not. Their findings finally convinced scientists that the genetic
material is DNA and not protein.
Cells agitated in blender and centrifuged. Bacteria, which are heavier, settle into pellet.
Phages and phage parts, which are lighter, remain in supernatant. Pellets and supernatants
tested for radioactivity.
RESULTS AND CONCLUSION: The researchers could separate phage protein coats labelled
with the radioactive isotope 35S from infected bacterial cells without affecting viral
reproduction. However, they could not separate viral DNA labelled with the radioactive
isotope 32P from infected bacterial cells. This demonstrated that viral DNA enters the
bacterial cells and is required for the synthesis of new viral particles. Thus, DNA is the
genetic material in phages.
Nucleotide Data
1. In 1940's, Erwin Chargaff analyzed base content of DNA using new chemical
techniques.
2. It was known DNA contained four different nucleotide:
a. Two with purine bases, adenine (A) and guanine (G); a purine is a type of
nitrogen-containing base having a double-ring structure.
b. Two with pyrimidine bases, thymine (T) and cystosine (C); a pyrimidine is a
type of nitrogen-containing base having a single-ring structure.
3. This constancy is given in Chargaff's rules:
a. The amount of A, T, G, and C in DNA vary from species to species.
b. In each species, the amount of A-T and the amount of G-C.
4. The tetra nucleotide hypothesis proposing DNA has repeating units of one of these
four bases was disapproved.
Discovered a 1:1 ratio of adenine to thymine and guanine to cytosine in DNA samples
from a variety of organisms. The amount of pyrimidines equals the amount of purine.
Diffraction Data
1. Rosalind Franklin, a student at King's College, produced X-ray diffraction
photographs.
2. Franklin's work provided evidence that DNA has the following features:
a. DNA is a helix.
b. One part of the helix is repeated.
The x-ray diffraction pattern of DNA shows that DNA is a double helix. The helical shape is
indicated by the crossed (X) pattern in the center of the photograph. The dark portions at
the top and the bottom of the photograph indicate that some portion of the helix is
repeated. She worked with another scientist Maurice Wilkins (1952).
Linus Pauling (1954) proposed a triple helix structure of DNA.
The Watson and Crick Model
1. American James Watson joined with Francis H.C. Crick in England to work on
structure of DNA.
2. Watson and Crick received the Nobel Prize in 1962 for their model of DNA.
3. Using information generated by Chargaff and Franklin, Watson and Crick built a
model of DNA as double helix; sugar-phosphate molecules on outside, paired bases
on inside.
4. Their model was consistent with both Chargaff's rules and dimensions of DNA
polymer provided by Franklin's photograph of X-ray diffraction of DNA.
5. Complementary base pairing is the paired relationship between purines and
pyrimidines in DNA, such that A is hydrogen-bonded to T and G is hydrogen-bonded
to C.
6. The two DNA strands of the double helix are antiparallel, meaning that the sugarphosphate groups of each strand are oriented in opposite directions. This means that
the 5’ end of one stand is paired to the 3’ end of the other strand, and vice versa