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2: All Genetic Information Is Encoded in the Structure of DNA
contain both DNA and protein. Two sets of experiments, one
conducted on bacteria and the other on viruses, provided
pivotal evidence that DNA, rather than protein, was the
The discovery of the transforming principle The first
clue that DNA was the carrier of hereditary information
came with the demonstration that DNA was responsible for
a phenomenon called transformation. The phenomenon was
first observed in 1928 by Fred Griffith, an English physician
whose special interest was the bacterium that causes pneumonia, Streptococcus pneumoniae. Griffith had succeeded in
isolating several different strains of S. pneumoniae (type I, II,
III, and so forth). In the virulent (disease-causing) forms of
a strain, each bacterium is surrounded by a polysaccharide
coat, which makes the bacterial colony appear smooth when
grown on an agar plate; these forms are referred to as S, for
Question: Can an extract from dead bacterial cells
genetically transform living cells?
A mixture of type
IIR bacteria and
heat-killed type IIIS
bacteria are injected
into a mouse.
Conclusion: A substance in the heat-killed virulent
bacteria genetically transformed the type IIR bacteria
into live, virulent type IIIS bacteria.
8.1 Griffith’s experiments demonstrated transformation in
smooth. Griffith found that these virulent forms occasionally mutated to nonvirulent forms, which lack a polysaccharide coat and produce a rough-appearing colony; these
forms are referred to as R, for rough.
Griffith observed that small amounts of living type IIIS
bacteria injected into mice caused the mice to develop pneumonia and die; on autopsy, he found large amounts of type IIIS
bacteria in the blood of the mice (Figure 8.1a). When Griffith
injected type IIR bacteria into mice, the mice lived, and no bacteria were recovered from their blood (Figure 8.1b). Griffith
knew that boiling killed all the bacteria and destroyed their virulence; when he injected large amounts of heat-killed type IIIS
bacteria into mice, the mice lived and no type IIIS bacteria were
recovered from their blood (Figure 8.1c).
The results of these experiments were not unusual.
However, Griffith got a surprise when he infected his mice
with a small amount of living type IIR bacteria along with a
large amount of heat-killed type IIIS bacteria. Because both
the type IIR bacteria and the heat-killed type IIIS bacteria
were nonvirulent, he expected these mice to live.
Surprisingly, 5 days after the injections, the mice became
infected with pneumonia and died (Figure 8.1d). When
Griffith examined blood from the hearts of these mice, he
observed live type IIIS bacteria. Furthermore, these bacteria
retained their type IIIS characteristics through several generations; so the infectivity was heritable.
Griffith finally concluded that the type IIR bacteria had
somehow been transformed, acquiring the genetic virulence
of the dead type IIIS bacteria. This transformation had produced a permanent, genetic change in the bacteria. Although
Griffith didn’t understand the nature of transformation, he
theorized that some substance in the polysaccharide coat of
the dead bacteria might be responsible. He called this substance the transforming principle.
Identification of the transforming principle At the
time of Griffith’s report, Oswald Avery was a microbiologist
at the Rockefeller Institute. At first Avery was skeptical but,
after other microbiologists successfully repeated Griffith’s
experiments with other bacteria, Avery set out to identify the
nature of the transforming substance.
After 10 years of research, Avery, Colin MacLeod, and
Maclyn McCarty succeeded in isolating and purifying the
transforming substance. They showed that it had a chemical
composition closely matching that of DNA and quite different from that of proteins. Enzymes such as trypsin and chymotrypsin, known to break down proteins, had no effect on
the transforming substance. Ribonuclease, an enzyme that
destroys RNA, also had no effect. Enzymes capable of
destroying DNA, however, eliminated the biological activity
of the transforming substance (Figure 8.2). Avery, MacLeod,
and McCarty showed that purified transforming substance
precipitated at about the same rate as purified DNA and that
it absorbed ultraviolet light at the same wavelengths as
DNA. These results, published in 1944, provided compelling
DNA: The Chemical Nature of the Gene
Question: What is the chemical nature of the transforming
The process of transformation indicates that some substance—
the transforming principle—is capable of genetically altering bacteria. Avery, MacLeod, and McCarty demonstrated that the
transforming principle is DNA, providing the first evidence that
DNA is the genetic material.
1 Heat kill virulent
✔ Concept Check 2
2 Treat samples
RNA, or DNA.
a. Protease carries out transformation.
If Avery, MacLeod, and McCarty had found that samples of heatkilled bacteria treated with RNase and DNase transformed bacteria
but that samples treated with protease did not, what conclusion
would they have made?
b. RNA and DNA are the genetic materials.
c. Protein is the genetic material.
d. RNase and DNase are necessary for transformation.
3 Add the
of type IIR
4 Cultures treated with protease
or RNase contain transformed
type IIIS bacteria,…
5 …but the culture
DNase does not.
Conclusion: Because only DNase destroyed the
transforming substance, the transforming substance is DNA.
8.2 Avery, MacLeod, and McCarty’s experiment revealed the
nature of the transforming principle.
evidence that the transforming principle—and therefore
genetic information—resides in DNA. Many biologists
refused to accept the idea, however, still preferring the
hypothesis that the genetic material is protein.
The Hershey–Chase experiment A second piece of evidence implicating DNA as the genetic material resulted from
a study of the T2 virus conducted by Alfred Hershey and
Martha Chase. The T2 virus is a bacteriophage (phage) that
infects the bacterium Escherichia coli (Figure 8.3a). As stated
in Chapter 6, a phage reproduces by attaching to the outer
wall of a bacterial cell and injecting its DNA into the cell,
where it replicates and directs the cell to synthesize phage
protein. The phage DNA becomes encapsulated within the
proteins, producing progeny phages that lyse (break open)
the cell and escape (Figure 8.3b).
At the time of the Hershey–Chase study (their paper was
published in 1952), biologists did not understand exactly
how phages reproduce. What they did know was that the T2
phage is approximately 50% protein and 50% nucleic acid,
that a phage infects a cell by first attaching to the cell wall,
and that progeny phages are ultimately produced within the
cell. Because the progeny carry the same traits as the infecting phage, genetic material from the infecting phage must be
transmitted to the progeny, but how this takes place was
Hershey and Chase designed a series of experiments to
determine whether the phage protein or the phage DNA is
transmitted in phage reproduction. To follow the fate of protein and DNA, they used radioactive forms, or isotopes, of
phosphorus and sulfur. A radioactive isotope can be used as
a tracer to identify the location of a specific molecule,
because any molecule containing the isotope will be radioactive and therefore easily detected. DNA contains phosphorus
but not sulfur; so Hershey and Chase used 32P to follow
phage DNA during reproduction. Protein contains sulfur but
not phosphorus; so they used 35S to follow the protein.
Hershey and Chase grew one batch of E. coli in a
medium containing 32P and infected the bacteria with T2
phage so that all the new phages would have DNA labeled
with 32P (Figure 8.4). They grew a second batch of E. coli in
Question: Which part of the phage—its DNA or its protein—serves as
the genetic material and is transmitted to phage progeny?
1 Infect E. coli grown in
medium containing 35S.
All other parts of
1 Phage attaches to
E. coli and injects
1 Infect E. coli grown in
medium containing 32P.
is taken up in
phage protein, which
contains S but not P.
3 Phages with 35S
32P is taken up in
phage DNA, which
contains P but not S.
3 Phages with 32P
4 Shear off protein
coats in blender…
2 Bacterial chromosome
breaks down and the
5 …and separate
protein from cells
3 Expression of phage
genes produces phage
4 Progeny phage
6 After centrifugation,
35S is recovered in
the fluid containing
the virus coats.
6 After centrifugation,
form a pellet
containing 32P in the
bottom of the tube.
5 Bacterial wall lyses,
7 No radioactivity is detected,indicating
that protein has not been transmitted
to the progeny phages.
7 The progeny phages are radioactive,
indicating that DNA has been
transmitted to progeny phages.
Conclusion: DNA—not protein—is the genetic material in bacteriophages.
8.3 T2 is a bacteriophage that infects E. coli. (a) T2 phage.
8.4 Hershey and Chase demonstrated that DNA carries the genetic
(b) Its life cycle. [Part a: © Lee D. Simon/Photo Researchers.]
information in bacteriophages.
DNA: The Chemical Nature of the Gene
a medium containing 35S and infected these bacteria with T2
phage so that all these new phages would have protein
labeled with 35S. Hershey and Chase then infected separate
batches of unlabeled E. coli with the 35S- and 32P-labeled
phages. After allowing time for the phages to infect the cells,
they placed the E. coli cells in a blender and sheared off the
then-empty protein coats (ghosts) from the cell walls. They
separated out the protein coats and cultured the infected
When phages labeled with 35S infected the bacteria,
most of the radioactivity was detected in the protein ghosts
and little was detected in the cells. Furthermore, when new
phages emerged from the cell, they contained almost no 35S
(see Figure 8.4). This result indicated that, although the
protein component of a phage is necessary for infection, it
does not enter the cell and is not transmitted to progeny
In contrast, when Hershey and Chase infected bacteria
with 32P-labeled phages and removed the protein ghosts, the
bacteria were still radioactive. Most significantly, after the
cells lysed and new progeny phages emerged, many of these
phages emitted radioactivity from 32P, demonstrating that
DNA from the infecting phages had been passed on to the
progeny (see Figure 8.4). These results confirmed that DNA,
not protein, is the genetic material of phages.
Using radioactive isotopes, Hershey and Chase traced the movement of DNA and protein during phage infection. They demonstrated that DNA, not protein, enters the bacterial cell during phage
reproduction and that only DNA is passed on to progeny phages.
✔ Concept Check 3
Could Hershey and Chase have used a radioactive isotope of carbon
instead of 32P? Why or why not?
1 Crystals of a substance are
bombarded with X-rays, which are
diffracted (bounce off).
Watson and Crick’s Discovery of the
Three-Dimensional Structure of DNA
The experiments on the nature of the genetic material set the
stage for one of the most important advances in the history
of biology—the discovery of the three-dimensional structure of DNA by James Watson and Francis Crick in 1953.
Before Watson and Crick’s breakthrough, much of the basic
chemistry of DNA had already been determined by
Miescher, Kossel, Levene, Chargaff, and others, who had
established that DNA consists of nucleotides and that each
nucleotide contains a sugar, a base, and a phosphate group.
However, how the nucleotides fit together in the threedimensional structure of the molecule was not at all clear.
In 1947, William Ashbury began studying the threedimensional structure of DNA by using a technique called
X-ray diffraction (Figure 8.5), in which X-rays beamed at
a molecule are reflected in specific patterns that reveal
aspects of the structure of the molecule. But his diffraction
pictures did not provide enough resolution to reveal the
structure. A research group at King’s College in London, led
by Maurice Wilkins and Rosalind Franklin, also used X-ray
diffraction to study DNA and obtained strikingly better
pictures of the molecule. Wilkins and Franklin, however,
were unable to develop a complete structure of the molecule; their progress was impeded by the personal discord
that existed between them.
Watson and Crick investigated the structure of DNA,
not by collecting new data but by using all available information about the chemistry of DNA to construct molecular
models (Figure 8.6). By applying the laws of structural
chemistry, they were able to limit the number of possible
structures that DNA could assume. They tested various
structures by building models made of wire and metal plates.
With their models, they were able to see whether a structure
was compatible with chemical principles and with the X-ray
2 The spacing of the atoms within the crystal
determines the diffraction pattern, which
appears as spots on a photographic film.
8.5 X-ray diffraction provides information about the structures of molecules. [Photograph
from M. H. F. Wilkins, Department of Biophysics, King’s College, University of London.]
3 The diffraction pattern provides
information about the structure
of the molecule.
8.3 DNA Consists of Two
Strands That Form a
DNA, though relatively simple in structure, has an elegance
and beauty unsurpassed by other large molecules. It is useful
to consider the structure of DNA at three levels of increasing
complexity, known as the primary, secondary, and tertiary
structures of DNA. The primary structure of DNA refers to
its nucleotide structure and how the nucleotides are joined
together. The secondary structure refers to DNA’s stable
three-dimensional configuration, the helical structure
worked out by Watson and Crick. Later, we will consider
DNA’s tertiary structures, which are the complex packing
arrangements of double-stranded DNA in chromosomes.
The Primary Structure of DNA
8.6 James Watson (left) and Francis Crick (right) provided a
three-dimensional model of the structure of DNA. [A.
Barrington Brown/Science Photo Library/Photo Researchers.]
The primary structure of DNA consists of a string of
nucleotides joined together by phosphodiester linkages.
Nucleotides DNA is typically a very long molecule and is
The key to solving the structure came when Watson recognized that an adenine base could bond with a thymine base
and that a guanine base could bond with a cytosine base; these
pairings accounted for the base ratios that Chargaff had discovered earlier. The model developed by Watson and Crick
showed that DNA consists of two strands of nucleotides
wound around each other to form a right-handed helix, with
the sugars and phosphates on the outside and the bases in the
interior. They published an electrifying description of their
model in Nature in 1953. At the same time, Wilkins and
Franklin published their X-ray diffraction data, which demonstrated experimentally the theory that DNA was helical in
Many have called the solving of DNA’s structure the most
important biological discovery of the twentieth century. For
their discovery, Watson and Crick, along with Maurice
Wilkins, were awarded a Nobel Prize in 1962. Rosalind
Franklin had died of cancer in 1957 and thus could not be
considered a candidate for the shared prize.
therefore termed a macromolecule. For example, within each
human chromosome is a single DNA molecule that, if
stretched out straight, would be several centimeters in
length. In spite of its large size, DNA has a quite simple structure: it is a polymer—that is, a chain made up of many
repeating units linked together. The repeating units of DNA
are nucleotides, each comprising three parts: (1) a sugar, (2)
a phosphate, and (3) a nitrogen-containing base.
The sugars of nucleic acids—called pentose sugars—
have five carbon atoms, numbered 1Ј, 2Ј, 3Ј, and so forth
(Figure 8.7) The sugars of DNA and RNA are slightly different in structure. RNA’s sugar, called ribose, has a hydroxyl
group (–OH) attached to the 2Ј-carbon atom, whereas DNA’s
sugar, or deoxyribose, has a hydrogen atom (–H) at this position and therefore contains one oxygen atom fewer overall.
By collecting existing information about the chemistry of DNA
and building molecular models,Watson and Crick were able to discover the three-dimensional structure of the DNA molecule.
8.7 A nucleotide contains either a ribose sugar (in RNA) or
a deoxyribose sugar (in DNA). The carbon atoms are assigned
DNA: The Chemical Nature of the Gene
(present in DNA)
(present in RNA)
8.8 A nucleotide contains either a purine or a pyrimidine base. The atoms of the rings in the
bases are assigned unprimed numbers.
This difference gives rise to the names ribonucleic acid (RNA)
and deoxyribonucleic acid (DNA). This minor chemical difference is recognized by all the cellular enzymes that interact
with DNA or RNA, thus yielding specific functions for each
nucleic acid. Furthermore, the additional oxygen atom in the
RNA nucleotide makes it more reactive and less chemically
stable than DNA. For this reason, DNA is better suited to
serve as the long-term repository of genetic information.
The second component of a nucleotide is its nitrogenous base, which may be of two types—a purine or a
pyrimidine (Figure 8.8). Each purine consists of a six-sided
ring attached to a five-sided ring, whereas each pyrimidine
consists of a six-sided ring only. Both DNA and RNA contain
two purines, adenine and guanine (A and G), which differ
in the positions of their double bonds and in the groups
attached to the six-sided ring. Three pyrimidines are common in nucleic acids: cytosine (C), thymine (T), and uracil
(U). Cytosine is present in both DNA and RNA; however,
thymine is restricted to DNA, and uracil is found only in
RNA. The three pyrimidines differ in the groups or atoms
attached to the carbon atoms of the ring and in the number
of double bonds in the ring. In a nucleotide, the nitrogenous
base always forms a covalent bond with the 1Ј-carbon atom
of the sugar (see Figure 8.7). A deoxyribose or a ribose sugar
and a base together are referred to as a nucleoside.
The third component of a nucleotide is the phosphate
group, which consists of a phosphorus atom bonded to four
oxygen atoms (Figure 8.9). Phosphate groups are found in
every nucleotide and frequently carry a negative charge,
which makes DNA acidic. The phosphate group is always
bonded to the 5Ј-carbon atom of the sugar (see Figure 8.7)
in a nucleotide.
The DNA nucleotides are properly known as deoxyribonucleotides or deoxyribonucleoside 5Ј-monophosphates.
Because there are four types of bases, there are four different
kinds of DNA nucleotides (Figure 8.10). The equivalent
RNA nucleotides are termed ribonucleotides or ribonucleoside 5Ј-monophosphates. RNA molecules sometimes contain additional rare bases, which are modified forms of the
four common bases. These modified bases will be discussed
in more detail when we examine the function of RNA molecules in Chapter 10.
ϪO 9 P " O
8.9 A nucleotide contains a phosphate group.
The primary structure of DNA consists of a string of nucleotides.
Each nucleotide consists of a five-carbon sugar, a phosphate, and
a base. There are two types of DNA bases: purines (adenine and
guanine) and pyrimidines (thymine and cytosine).