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Molecular Basis of Inheritance

The search for genetic material
 Search for Genetic Material

Experience Evidence of DNA as genetic material

Griffith’s and Avery Experiment

Hershey and Chase Experiment

 Search for Genetic Material

The search for the

chemical nature of
1920 genetic material
started around 1920s.
 Search for Genetic Material

1920

1926
Quest at molecular level
 Search for Genetic Material

Griffith’s experiment
1928
1920

1926
 Griffith’s Experiments

He conducted experiments by infecting

lab mice with different strains of
Streptococcus pneumoniae.

Frederick Griffith
 Griffith’s Experiments

❖ In 1928, Frederick Griffith performed transformation experiments.

❖ He performed experiments using two strains of bacterium Streptococcus pneumoniae
(S-III strain and R-II strain).
❖ This bacterium causes pneumonia in mammals, including humans.
 Griffith’s Experiments
Streptococcus
pneumoniae - Two
variants strains

Smooth colonies Rough colonies

S strain R strain
Virulent Non-virulent

No polysaccharide
Polysaccharide
coat giving it a
coat giving it a
rough appearance
smooth
appearance
 Griffith’s Experiments
Streptococcus
pneumoniae - Two
variants strains

S strain R strain

o It is smooth, virulent strain. o Rough, non-virulent strain.

o Protected by polysaccharide o Protective capsule is absent.
capsule. o Forms rough colonies.
o Forms smooth glistening o Does not cause pneumonia
colonies on agar medium.
o It causes pneumonia.
 Griffith’s Experiments
❖ He injected live rough bacteria into mice but the mice did not die

❖ Mice injected with live smooth bacteria died but mice injected with heat killed smooth
bacteria survived.
 Griffith’s Experiments
❖ He injected mice with mixture of live rough bacteria and heat killed smooth bacteria.
❖ This mixture caused pneumonia and killed the mice.
❖ Live smooth bacteria were obtained from blood of dead mice.
Griffith’s Experiments
 Griffith’s Experiments

Step 1

Live S strain Live mice Mice die S strain bacteria

isolated from dead
mice
 Griffith’s Experiments

Step 2

Live R strain Live mice Mice No living bacteria

live isolated from the
mice
 Griffith’s Experiments

Step 3

Heat-killed Live mice Mice No living bacteria

S strain live isolated from the
mice
 Griffith’s Experiments

Step 4

Heat-killed Live mice Mice die S type bacteria

S strain isolated from dead
+ mice

Live R strain
 Griffith’s Experiments
Conclusion

❖ Griffith concluded that some transforming principle (chemical substance) from dead
smooth bacteria had entered living rough bacteria and transformed it into smooth
bacteria.

❖ Transformed smooth bacteria recovered from dead mice produced smooth bacteria
when grown in a culture.
 Griffith’s Experiments

Conclusion

Transforming
+ principle

Heat-killed Live R strain Virulent strain

S strain
 Griffith’s Experiments
Transformation

• It is permanent inheritable change in one strain of bacteria by a substance isolated

from another strain of bacteria.
• Substance which brings about transformation is called transforming agent.
 Griffith’s Experiments

Griffith’s Problems

• Griffith was not able to know:

o Role of mice in change of bacterial strain.
o Component of S-strain bacteria which called virulence.
 What was the
biochemical nature
of the transforming
principle
Carbohydrates RNA Proteins DNA

Four Contestants!
 Search for Genetic Material
Avery, MacLeod,
McCarty experiment
1928 1944
1920

1926

For more than a decade, the

biochemical nature of the genetic
material remained a mystery.
 Avery, MacLeod and McCarty Experiment

Oswald Avery Colin MacLeod Maclyn McCarty

 Avery, MacLeod and McCarty Experiment

○ In 1944, O.T. Avery, C. Macleod, Mc Carty separated extracts of smooth

virulent bacteria into proteins, DNA, RNA and carbohydrate fractions.

○ Each fraction was separately added to a culture medium containing live

rough bacteria.
 Avery, MacLeod and McCarty Experiment

They purified carbohydrates,

proteins, DNA and RNA, from
the heat-killed S cells

Sugars RNA Proteins DNA

 Avery, MacLeod and McCarty Experiment

To the purified macromolecule

solution, they added the live R
strain bacteria

Sugars RNA Proteins DNA

+ + + +
R strain
bacteria
 Avery, MacLeod and McCarty Experiment

To the macromolecule + R
strain solution, heat killed S
strain bacteria were added,
incubated and then each
injected into 4 different lab
mice.

Sugars RNA Proteins DNA

+ + + +
R strain
bacteria

S strain
No Transformation bacteria
 Griffith’s Experiments

DNA
+

Heat-killed Live mice Mice die S type bacteria isolated

S strain from dead mice

Live R strain
 Avery, MacLeod and McCarty Experiment

Sugars RNA Proteins DNA

+ + + +
Carbohydrase Carbohydrase Carbohydrase Carbohydrase
+ + + +
R strain
bacteria

S strain
No Transformation bacteria
 Avery, MacLeod and McCarty Experiment

Sugars RNA Proteins DNA

+ + + +
RNase RNase RNase RNase
+ + + +
R strain
bacteria

S strain
No Transformation bacteria
 Avery, MacLeod and McCarty Experiment

Sugars RNA Proteins DNA

+ + + +
Proteinase Proteinase Proteinase Proteinase
+ + + +
R strain
bacteria

S strain
No Transformation bacteria
 Avery, MacLeod and McCarty Experiment

Sugars RNA Proteins DNA

+ + + +
DNase DNase DNase DNase
+ + + +
R strain
bacteria

No Transformation
Carbohydrates RNA Proteins DNA

Four Contestants!
 DNA

One Winner !
 Transforming Principle = DNA

They concluded that DNA is the hereditary

material, but not all biologists were convinced!! At
the time, the belief was that DNA was just a chain
of four repeating nucleotides, and thus was not
complex enough to be genetic material.
 NO! NO! NO!
Some scientists believed that the active
component of the transforming substance was
a protein associated with nucleic acids in the
chromosomes. Avery and his colleagues were
not able to purify the DNA and thus it was
suggested that the DNA could have been
contaminated by traces of protein.

Nucleic Proteins
acid
Proteins DNA

Two Contestants!
 Search for Genetic Material

1928 1944
1920

1926 1952
Hershey and Chase
experiment
 Hershey and Chase’s Experiment

Martha Chase Alfred Hershey

 Hershey and Chase’s Experiment
● Alfred D. Hershey and Martha Chase performed experiments in 1952 using
viruses that infect bacteria.

● Such viruses are called bacteriophages.

● Bacteriophage gets attached over the surface of bacterium and inserts its
genetic material into bacterial cell.
 Recall: Bacteriophage

Bacteriophages are the viruses which infect bacteria.

Bacteriophage: Attaches to the surface of the
bacteria
Bacteriophage: Penetration into the bacteria
Life Cycle of Bacteriophage

Transduction
The process by which DNA
is introduced into a cell
by a virus
Bacteriophage: Insertion of the genetic
material
Bacteriophage: Viral genome integration
into bacterial genome
Life Cycle of Bacteriophage
Life Cycle of Bacteriophage
 Hershey and Chase’s Experiment
● Experiment was performed using Escherichia coli and bacteriophage T2.
● T2 bacteriophage has DNA core and a protein coat.
● T2 phage injects its DNA core into bacterium but the protein coat remains outside.
 Hershey and Chase’s Experiment

Experimental Procedure

Labelling

Infection

Blending

Centrifugation
 Hershey and Chase’s Experiment

Labelling

❖ They grew some viruses on a medium that contained radioactive phosphorus and
some others on medium that contained radioactive sulfur.
❖ Viruses grown in the presence of radioactive phosphorus contained radioactive DNA
but not radioactive protein because DNA contains phosphorus but protein does not.
❖ Viruses grown on radioactive sulfur contained radioactive protein but not radioactive
DNA because DNA does not contain sulfur.
 Hershey and Chase’s Experiment

Step 1

Bacterial medium with Bacterial medium with

radioactive sulfur (35S) radioactive
phosphorus (32P)
But why did they do that?
 Hershey and Chase’s Experiment

But why ? Sulphur is present

in proteins but not
C- in nucleic acids
peptide

-COOH
-NH2
S
S
S
S
S A- chain
S

B- chain
Protein
 Hershey and Chase’s Experiment
Phosphorus is
But why ? present in
nucleic acids
G C Bases Phosphate
A
C
T
G
but not in
G C
proteins
Guanine A
G C
Y
Cytosine
C G
Thymine Y A Adenine
Sugar- G
A T
C

Phosphate C
A T
G

backbone Nucleotide

Nucleotide P
S
C
 Hershey and Chase’s Experiment

Labelling

❖ Radioactive sulphur was incorporated into sulphur containing amino acids (cysteine
and methionine), therefore it became a part of bacterial proteins.

❖ Radioactive phosphorus gets incorporated into nucleotides which form nucleic acids,
mostly DNA.
 Hershey and Chase’s Experiment

Infection

❖ Both types of labelled phages were allowed to infect normally cultured bacteria in
separate experiments.

❖ DNA was introduced and protein coat was left out.

 Hershey and Chase’s Experiment
One set of viruses were made
Step 1 to infect bacteria containing
radioactive phosphorus.
One set of viruses were were
made to infect bacteria
containing radioactive sulphur.
 Hershey and Chase’s Experiment
The viral progenies were
Step 1 isolated from both the groups.
 Hershey and Chase’s Experiment
The viral progenies were
Step 2 allowed to infect E.coli
without any radioactive
elements.
 Hershey and Chase’s Experiment

Blending

❖ As the infection proceeded the bacteria were agitated in a blender to break the
contact between virus and bacteria.
 Hershey and Chase’s Experiment
The viral progenies attach to E. coli
Step 2 bacteria and penetrate them. After some
time, the two sets were blended to
separate the viral coat from the bacteria.
 Hershey and Chase’s Experiment

Step 3
 Hershey and Chase’s Experiment

Step 3
 Hershey and Chase’s Experiment
Blended mixture was
Step 4 transferred to centrifuge
tubes.
 Hershey and Chase’s Experiment

Centrifugation

❖ The virus particles were separated from the bacteria by spinning them in a
centrifuge.
❖ Heavier bacteria settled down in the form of pellet.
❖ Supernatant contains lighter viral coats which do not enter bacterial cells.
 Hershey and Chase’s Experiment

Centrifugation

❖ Pellet as well as supernatant was analysed.

❖ Phage with labelled protein did not make bacteria labelled.
❖ Radioactivity was restricted to supernatant which contained only empty phage capsids.
 Hershey and Chase’s Experiment
Centrifugation
Step 4 separated the bacterial
cells and the coats as
bacterial cells are
heavier than virus coats
 Hershey and Chase’s Experiment
Bacteria which was infected
Results
by the viral progenies that
had radioactive phosphorus,
showed radioactivity inside
the cell.
 Hershey and Chase’s Experiment
Bacteria that were infected with
Results viruses that had radioactive
sulfur were not radioactive. This
showed that proteins did not
enter the bacteria from the
viruses.
 Hershey and Chase’s Experiment

Analysis

❖ The progeny of the two types of bacteriophages was again tested for radioactivity.
❖ Radioactive was absent in the viruses derived from parents having labelled protein.
❖ The viruses derived from parents having labelled DNA possessed radioactivity.
❖ This shows that the genetic material is DNA and not the protein.
 DNA

One Winner !
 Hershey and Chase’s Experiment

Step 1

Genetic Material of all

living organisms = DNA

Phage grown Phage grown

With With radioactive
radioactive phosphorus (35P)
sulfur (35S)
What would be the ideal
genetic material ?
 Ideal Genetic Material

❖ It should be able to replicate

❖ It should be chemically and structurally

stable
❖ It should provide scope for slow changes
(mutation) required for evolution

❖ It should be able to express itself in the

form of 'Mendelian Characters’
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