DNA:

THE CHEMICAL NATURE OF

THE GENES
(STRUCTURE AND ANALYSIS)
 Characteristics of a Genetic Material

• replication
• storage of information
• expression of information
• variation by mutation
 Until 1944, Observations Favored
Protein as the Genetic Material

• Beginning in the late nineteenth century,

research into the structure of biomolecules
progressed considerably, setting the stage for
describing the genetic material in chemical
terms. Although proteins and nucleic acid
were both considered major candidates for
the role of genetic material, until the 1940s
many geneticists favored proteins
• DNA was first studied in 1868 by a Swiss
chemist, Friedrich Miescher. He isolated cell
nuclei and derived an acidic substance, now
known to contain DNA, that he called nuclein.

• Phoebus A. Levene’s observations in 1910 that

DNA contained approximately equal amounts
of four similar molecular building blocks called
nucleotides
Levene’s tetranucleotide hypothesis
for DNA structure
 DNA Molecules Have Distinctive
Base Compositions
• A most important clue to the structure of DNA
came from the work of Erwin Chargaff and his
colleagues in the late 1940s. They found that
the four nucleotide bases of DNA occur in
different ratios in the DNAs of different
organisms and that the amounts of certain
bases are closely related.
Chargaff’s rule disproved the tetranucleotide theory
Base composition of DNA from different sources and ratios of bases
 These data, collected from DNAs of a great many different
species, led Chargaff to the following conclusions:

• The base composition of DNA generally varies from one species to

another.
• DNA specimens isolated from different tissues of the same species
have the same base composition.
• The base composition of DNA in a given species does not change
with an organism’s age, nutritional state, or changing
environment.
• In all cellular DNAs, regardless of the species, the number of
adenosine residues is equal to the number of thymidine residues
(that is, A + T), and the number of guanosine residues is equal to
the number of cytidine residues (G + C). From these relationships
it follows that the sum of the purine residues equals the sum of
the pyrimidine residues; that is, A+ G = T+C.
While chemists were working out
the structure of DNA, biologists
were attempting to identify the
source of genetic information.
The discovery of the transforming principle

• The phenomenon was

first observed in 1928
by Fred Griffith, an
English physician
whose special interest
was the bacterium that
causes pneumonia,
Griffiths experiments demonstrated transformation in bacteria
• 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.
 The critical question, of
course, was what molecule
serves as the transforming
principle?
Summary of Avery, MacLeod, and McCarty’s
experiment demonstrating that DNA is the
transforming principle
 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
The Hershey–Chase experiment: A second piece of
evidence implicating DNA as the genetic material
The Hershey–Chase experiment: A second piece of
evidence implicating DNA as the genetic material
 Indirect and Direct Evidence
Supports the Concept that DNA
Is the Genetic Material in
Eukaryotes
 Indirect Evidence: Distribution of
DNA

The amount of DNA and the number of sets of chromosomes is closely correlated. No such
consistent correlation can be observed between gametes and diploid cells for proteins.
 Indirect Evidence: Mutagenesis
• Ultraviolet (UV) light is one of a
number of agents capable of
inducing mutations in the genetic
material.
• UV light is most mutagenic at the
wavelength (l) of 260 nanometers
(nm), and both DNA and RNA
absorb UV light most strongly at
260 nm. On the other hand,
protein absorbs most strongly at
280 nm, yet no significant
mutagenic effects are observed at
that wavelength.
Direct Evidence: Recombinant
DNA Studies
RNA Serves as the Genetic Material
in Some Viruses
 Knowledge of Nucleic Acid
Chemistry Is Essential to the
Understanding of DNA Structure
Nucleotides: Building Blocks of
Nucleic Acids
pentose sugars found in nucleic acids
Nucleosides and nucleotides are named according to the specific
nitrogenous base (A, T, G, C, or U) that is part of the molecule
Polynucleotides: the phosphodiester bond
The Structure of DNA Holds the Key
to Understanding Its Function
• From 1940 to 1953, many scientists were interested in solving
the structure of DNA. Among others, Erwin Chargaff, Maurice
Wilkins, Rosalind Franklin, Linus Pauling, Francis Crick, and
James Watson sought information that might answer what
many consider to be the most significant and intriguing
question in the history of biology:
• How does DNA serve as the genetic basis for life?
• The answer was believed to depend strongly on the chemical
structure and organization of the DNA molecule, given the
complex but orderly functions ascribed to it.
In 1953, James Watson and Francis Crick proposed that
the structure of DNA is in the form of a double helix.

• The data available to Watson and Crick, crucial

to the development of their proposal, came
primarily from two sources:
– (1) base composition analysis of hydrolyzed
samples of DNA
– (2) X-ray diffraction studies of DNA.
Base-Composition Studies
Base-Composition Studies
 Chargaff’s data were critical to the creation of the successful
model of DNA put forward by Watson and Crick. On the basis of
these data, the following conclusions may be drawn:

• the amount of adenine residues is proportional to

the amount of thymine residues in DNA Also, the
amount of guanine residues is proportional to the
amount of cytosine residues.
• Based on this proportionality, the sum of the purines
(A+G) equals the sum of the pyrimidines (C + T)
• The percentage of (G+C) does not necessarily equal
the percentage of (A+T). As you can see, this ratio
varies greatly among organisms
 X-Ray Diffraction Analysis

The technique had been attempted on DNA as early as 1938 by William Ast- bury. By 1947, he
had detected a periodicity of 3.4 angstroms (3.4-Å) repetitions within the structure of the
molecule, which suggested to him that the bases were stacked like coins on top of one another.
The Watson–Crick Model
 The Watson–Crick Model
• Two long polynucleotide chains are coiled around a central axis, forming a
right-handed double helix.
• The two chains are antiparallel; that is, their C-5¿-to-C-3¿ ori- entations
run in opposite directions.
• The bases of both chains are flat structures lying perpendicular to the axis;
they are “stacked” on one another, 3.4 Å (0.34 nm) apart, on the inside of
the double helix.
• The nitrogenous bases of opposite chains are paired as the result of the
formation of hydrogen bonds; in DNA, only A “ T and G ‚ C pairs occur.
• Each complete turn of the helix is 34 Å (3.4 nm) long; thus, each turn of
the helix is the length of a series of 10 base pairs.
• A larger major groove alternating with a smaller minor groove winds along
the length of the molecule.
• The double helix has a diameter of 20 Å (2.0 nm)
The Structure of RNA Is Chemically
Similar to DNA, but Single Stranded
Many Analytical Techniques Have
Been Useful during the
Investigation of DNA and RNA
 Sedimentation Behavior
• Nucleic acid mixtures can be separated into
different components by several possible
centrifugation procedures
• successive fractions are eluted from the tube
and measured spectrophotometrically for
absorption at 260 nm. In this way, the position
of a nucleic acid fraction along the gradient
can be determined and the fraction isolated
and studied further.
Two major types of gradient centrifugation techniques
are employed in the analysis of nucleic acids:
sedimentation equilibrium and sedimentation velocity.

• Sedimentation equilibrium
– centrifugation (sometimes called density gradient centrifugation),
a density gradient is created that overlaps the densities of the
individual components of a mixture of molecules.
– During centrifugation, the molecules migrate until they reach a
point of neutral buoyant density.
– At this point, the centrifugal force on them is equal and opposite
to the upward diffusion force, and no further migration occurs.
– If DNAs of different densities are present, they will separate as the
molecules of each density reach equilibrium with the corre-
sponding density of CsCl.
 Sedimentation equilibrium centrifugation
studies can also be used to generate data on the
base composition of double-stranded DNA.
Two major types of gradient centrifugation techniques
are employed in the analysis of nucleic acids:
sedimentation equilibrium and sedimentation velocity.

• sedimentation velocity
– employs an analytical centrifuge that uses ultraviolet
absorption optics to monitor the migration of the molecules
during centrifugation and determine the “velocity of
sedimentation” (Sevedverg unit)
– In this technique, the molecules are loaded on top of the
gradient, and the gravitational forces created by
centrifugation drive them toward the bottom of the tube.
– Two forces work against this down- ward movement: (1) the
viscosity of the solution creates a frictional resistance, and
(2) part of the force of diffusion is directed upward.
 Molecular Hybridization
• This technique derives its name from the fact
that single strands need not originate from
the same nucleic acid source in order to
combine to form duplex structures.
• For example, if DNA strands are isolated from
two distinct organisms and a reasonable
degree of base complementarity exists
between them, under the proper temperature
conditions, double-stranded molecular
hybrids will form during renaturation.
Molecular Hybridization
 Fluorescent in situ Hybridization
(FISH)
• A refinement in the molecular
hybridization technique has
led to the use of DNA present
in cytological preparations as
the “target” for hybrid
formation.
• When this approach is
combined with the use of
fluorescent probes to monitor
hybridization, the technique is
called fluorescent in situ
hybridization, or simply by the
acronym FISH
Fluorescent in situ Hybridization
(FISH)
 Electrophoresis of Nucleic Acids
• Electrophoresis, also useful
in protein studies, can be
applied to the separation of
different-sized fragments of
DNA and RNA chains and is
invaluable in current
research investigations in
molecular genetics.
• Smaller molecules migrate
at a faster rate through the
gel than larger molecules