DNA as the genetic material

1. HERSHEY–CHASE EXPERIMENT
Dr. P. Samuel
Assistant Professor,
Department of Biotechnology,
ANJA College, Sivakasi

2. Introduction
• The Hershey–Chase experiments were a series of experiments conducted
in 1952 by Alfred Hershey and Martha Chase that helped to confirm
that DNA is genetic material.
• While DNA had been known to biologists since 1869, many scientists still
assumed at the time that proteins carried the information for inheritance
because DNA appeared simpler than proteins.
• In their experiments, Hershey and Chase showed that when bacteriophages,
which are composed of DNA and protein, infect bacteria, their DNA enters
the host bacterial cell, but most of their protein does not.
• Although the results were not conclusive, and Hershey and Chase were
cautious in their interpretation, previous, contemporaneous, and subsequent
discoveries all served to prove that DNA is the hereditary material.
• Hershey shared the 1969 Nobel Prize in Physiology or Medicine with Max
Delbrück and Salvador Luria for their “discoveries concerning the genetic
structure of viruses.”

3. Overview of experiment and observations

4. Historical background
• In the early twentieth century, biologists thought that proteins
carried genetic information. This was based on the belief that
proteins were more complex than DNA.
• Phoebus Levene's influential "tetranucleotide hypothesis", which
incorrectly proposed that DNA was a repeating set of
identical nucleotides, supported this conclusion.
• The results of the Avery–MacLeod–McCarty experiment, published
in 1944, suggested that DNA was the genetic material, but there was
still some hesitation within the general scientific community to
accept this, which set the stage for the Hershey–Chase experiment.
• Hershey and Chase, along with others who had done related
experiments, confirmed that DNA was the biomolecule that carried
genetic information.
• Before that, Oswald Avery, Colin MacLeod, and Maclyn
McCarty had shown that DNA led to the transformation of one
strain of Streptococcus pneumoniae to another that was
more virulent. The results of these experiments provided evidence
that DNA was the biomolecule that carried genetic information.

5. Methods and results
• Hershey and Chase needed to be able to examine
different parts of the phages they were studying
separately, so they needed to isolate the phage
subsections.
• Viruses were known to be composed of a protein shell
and DNA, so they chose to uniquely label each with a
different elemental isotope.
• This allowed each to be observed and analyzed
separately. Since phosphorus is contained in DNA but
not amino acids, radioactive phosphorus-32 was used to
label the DNA contained in the T2 phage.
• Radioactive sulfur-35 was used to label the protein
sections of the T2 phage, because sulfur is contained in
amino acids but not DNA.

6. Structure of a T2 Phage

7. • Hershey and Chase inserted the radioactive elements into the
bacteriophages by adding the isotopes to separate media within
which bacteria were allowed to grow for 4 hours before
bacteriophage introduction.
• When the bacteriophages infected the bacteria, the progeny
contained the radioactive isotopes in their structures.
• This procedure was performed once for the sulfur-labeled phages
and once for phosphorus-labeled phages.
• The labeled progeny were then allowed to infect unlabeled bacteria.
The phage coats remained on the outside of the bacteria, while
genetic material entered.
• Disruption of phage from the bacteria by agitation in a blender
followed by centrifugation allowed for the separation of the phage
coats from the bacteria.
• These bacteria were lysed to release phage progeny. The progeny of
the phages that were originally labeled with 32P remained labeled,
while the progeny of the phages originally labeled with 35S were
unlabeled. Thus, the Hershey–Chase experiment helped confirm that
DNA, not protein, is the genetic material.

8. • Hershey and Chase showed that the introduction
of deoxyribonuclease (referred to as DNase), an enzyme that breaks
down DNA, into a solution containing the labeled bacteriophages
did not introduce any 32P into the solution.
• This demonstrated that the phage is resistant to the enzyme while
intact.
• Additionally, they were able to plasmolyze the bacteriophages so
that they went into osmotic shock, which effectively created a
solution containing most of the 32P and a heavier solution containing
structures called “ghosts” that contained the 35S and the protein coat
of the virus.
• It was found that these “ghosts” could adsorb to bacteria that were
susceptible to T2, although they contained no DNA and were simply
the remains of the original bacterial capsule.
• They concluded that the protein protected the DNA from DNAse,
but that once the two were separated and the phage was inactivated,
the DNAse could hydrolyze the phage DNA. However, it
subsequently became clear that in some viruses, RNA is the genetic
material.

9. Experiment and conclusions
• Hershey and Chase were also able to prove that the DNA from the phage is inserted into the
bacteria shortly after the virus attaches to its host. Using a high speed blender they were able
to force the bacteriophages from the bacterial cells after adsorption.
• The lack of 32P labeled DNA remaining in the solution after the bacteriophages had been
allowed to adsorb to the bacteria showed that the phage DNA was transferred into the
bacterial cell. The presence of almost all the radioactive 35S in the solution showed that the
protein coat that protects the DNA before adsorption stayed outside the cell.
• Hershey and Chase concluded that DNA, not protein, was the genetic material. They
determined that a protective protein coat was formed around the bacteriophage, but that the
internal DNA is what conferred its ability to produce progeny inside a bacterium. They
showed that, in growth, protein has no function, while DNA has some function. They
determined this from the amount of radioactive material remaining outside of the cell. Only
20% of the 32P remained outside the cell, demonstrating that it was incorporated with DNA in
the cell's genetic material. All of the 35S in the protein coats remained outside the cell,
showing it was not incorporated into the cell, and that protein is not the genetic material.
• Hershey and Chase's experiment concluded that little sulfur containing material entered the
bacterial cell. However no specific conclusions can be made regarding whether material that
is sulfur-free enters the bacterial cell after phage adsorption. Further research was necessary
to conclude that it was solely bacteriophages' DNA that entered the cell and not a combination
of protein and DNA where the protein did not contain any sulfur.

10. Discussion
 Confirmation
• Hershey and Chase concluded that protein was not likely to be the
hereditary genetic material. However, they did not make any
conclusions regarding the specific function of DNA as hereditary
material, and only said that it must have some undefined role.
• Confirmation and clarity came a year later in 1953, when James D.
Watson and Francis Crick correctly hypothesized, in their journal
article "Molecular Structure of Nucleic Acids: A Structure for
Deoxyribose Nucleic Acid", the double helix structure of DNA, and
suggested the copying mechanism by which DNA functions as
hereditary material.
• Furthermore, Watson and Crick suggested that DNA, the genetic
material, is responsible for the synthesis of the thousands of proteins
found in cells. They had made this proposal based on the structural
similarity that exists between the two macromolecules, that is, both
protein and DNA are linear sequences of amino acids and
nucleotides respectively.

12. Genetic Code: Meaning, Types and
Properties
• Meaning of Genetic Code
The genetic code may be defined as the exact
sequence of DNA nucleotides read as three
letter words or codons, that determines the
sequence of amino acids in protein synthesis.
In other words, the genetic code is the set of
rules by which information encoded in genetic
material (DNA or RNA sequences) is
translated into proteins (amino acid sequences)
by living cells.

13. The main points related to genetic code
• The genetic code is ‘read’ in triplets of bases
called codons. In other words, a set of three
nucleotide bases constitutes a codon.
• In a triplet code, three RNA bases code for one
amino acid.
• There are 64 codons which correspond to 20
amino acids and to signals for the initiation
and termination of transcription.
• The code uses codons to make the amino acids
that, in turn, constitute proteins.

14. • Each triplet [codon] specifies one amino acid
in a protein structure or a start signal or stop
signal in protein synthesis.
• The code establishes the relationship between
the sequence of bases in nucleic acids (DNA
and the complementary RNA) and the
sequence of amino acids in proteins.
• The code explains the mechanism by which
genetic information is stored in living
organisms.

15. Types of Genetic Code
• The genetic code is of two types. The genetic code can
be expressed as either RNA codons or DNA codons.
• RNA codons occur in messenger RNA (mRNA) and are
the codons that are actually “read” during the synthesis
of polypeptides (the process called translation).
• But each mRNA molecule acquires its sequence of
nucleotides by transcription from the corresponding
gene [DNA], Because DNA sequencing has become so
rapid and because most genes are now being discovered
at the level of DNA before they are discovered as
mRNA or as a protein product, it is extremely useful to
have a table of codons expressed as DNA. Both tables
are given here.

16. DNA Codons
• These are the codons as they are read on the
sense (5′ to 3′) strand of DNA. Except that the
nucleotide thymine (T) is found in place of
uracil (U), they read the same as RNA codons.
However, mRNA is actually synthesized using
the antisense strand of DNA (3′ to 5′) as the
template.

18. Types of Codon
• The genetic code consists of 64 triplets of
nucleotides. These triplets are called codons. With
three exceptions, each codon encodes for one of
the 20 amino acids used in the synthesis of
proteins. This produces some redundancy in the
code.
• Most of the amino acids are encoded by more than
one codon. One codon that is AUG serves two related
functions. It signals the start of translation and codes
for the incorporation of the amino acid methionine
(Met) into the growing polypeptide chain.

19. Properties of Genetic Code
• The genetic code is:
(i) Triplet,
(ii) Universal,
(iii) Comma-less,
(iv) Non-overlapping,
(v) Non-ambiguous,
(vi) Redundant, and
(vii) Has polarity.

20. • The Code is Triplet
 The genetic code is triplet. The triplet code has 64
codons which are sufficient to code for 20 amino
acids and also for start and stop signals in the
synthesis of polypeptide chain. In a triplet code three
RNA bases code for one amino acid.
• The Code is Universal
 The genetic code is almost universal. The same
codons are assigned to the same amino acids and to
the same START and STOP signals in the vast
majority of genes in animals, plants, and
microorganisms.

21. • The Code is Commaless
It is believed that the genetic code is
commaless. In other words, the codons are
continuous and there are no demarcation lines
between codons. Deletion of a single base in a
commaless code alters the entire sequence of
amino acids after the point of deletion as given
below.

22. • The Code is Non-Overlapping
Three nucleotides or bases code for one amino
acid. In a non-overlapping code, six bases will
code for two amino acids.
In a non-overlapping code, one letter is read
only once. In overlapping code, six nucleotides
or bases will code for 4 amino acids, because
each base is read three times.

23. • The Code is Non-ambiguous
The genetic code has 64 codons. Out of these,
61 codons code for 20 different amino acids.
However, none of the codons codes for more
than one amino acid. In other words, each
codon codes only for one amino acid.
This clearly indicates that the genetic code is
non-ambiguous. In case of ambiguous code,
one codon should code for more than one
amino acid. In the genetic code there is no
ambiguity.

24. • The Code is Redundant
In most of the cases several codons code for
the same amino acid. Only two amino acids,
viz. tryptophan and methionine are coded by
one codon each. Nine amino acids are coded
by two codons each, one amino acid
[Isoleucine] by three codons, five amino acids
by 4 codons each, and three amino acids by 6
codons.

25. • The Code Has Polarity
 The code has a definite direction for reading of message,
which is referred to as polarity. Reading of codon in opposite
direction will specify for another amino acid due to alteration
in the base sequences in the code.
 In the following codons, reading of message from left to right
and right to left will specify for different amino acids. Because
the codon in the following case will be read as UUG from left
to right and as GUU from right to left which codes for another
amino acid.