Introduction
The Central Dogma
of Molecular Biology
Cell
DNA
Transcription
mRNA
Translation
Ribosome
Polypeptide
(protein)
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©1998 Timothy G. Sta
The Molecular Nature of the Genetic Material
• Mendel published his work in 1865.
• That work was lost until ca. 1900.
• With the “rediscovery” of Mendel’s conceptual work
the hunt was on for the physical nature of the gene.
• What was it and how did it function?
• These questions were largely answered from 1940’s
through the 1960’s and lead to the biotech revolution
beginning of the 1970’s.
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• DNA, the substance of inheritance
– Is the most celebrated molecule of our time
• Hereditary information
– Is encoded in the chemical language of DNA
and reproduced in all the cells of your body
• It is the DNA program
– That directs the development of many different
types of traits
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The Search for the Genetic Material: Scientific Inquiry
• The role of DNA in heredity
– Was first worked out by studying bacteria and
the viruses that infect them
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Evidence That DNA Can Transform Bacteria
• Frederick Griffith was studying Streptococcus
pneumoniae
– A bacterium that causes pneumonia in
mammals
• He worked with two strains of the bacterium
– A pathogenic strain and a nonpathogenic
strain
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Transformation Of Bacteria
Two Strains Of Streptococcus
Rough Strain
(Harmless)
Capsules
Smooth Strain
(Virulent)
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Transformation Of Bacteria
The Griffith’s 1928 Experiment
OUCH!
+ Control
- Control
- Control
Experimental
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• Griffith found that when he mixed heat-killed
remains of the pathogenic strain
– With living cells of the nonpathogenic strain,
some of these living cells became pathogenic
EXPERIMENT Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they
have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule
and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below:
Living S
(control) cells
Living R
Heat-killed
(control) cells (control) S cells
Mixture of heat-killed S cells
and living R cells
RESULTS
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells
are found in
blood sample.
Figure 16.2
CONCLUSION Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an
unknown, heritable substance from the dead S cells.
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• Griffith called the phenomenon transformation
– Now defined as a change in genotype and
phenotype due to the assimilation of external
DNA by a cell
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Avery, MacLeod and McCarty
• 1944 Avery, MacLeod and McCarty repeated Griffith’s
1928 experiment with modifications designed to discover
the “transforming factor”
• After extraction with organic solvents to eliminate lipids,
remaining extract from heat killed cells was digested with
hydrolytic enzymes specific for different classes of macro
molecules:
Enzyme
Transformation?
Protease
Yes
Saccharase
Yes
Nuclease
No
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Evidence That Viral DNA Can Program Cells
• Additional evidence for DNA as the genetic
material
– Came from studies of a virus that infects
bacteria
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• Viruses that infect bacteria, bacteriophages
– Are widely used as tools by researchers in
molecular genetics
Phage
head
Tail
Tail fiber
Figure 16.3
Bacterial
cell
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100 nm
DNA
• Alfred Hershey and Martha Chase
– Performed experiments showing that DNA is
the genetic material of a phage known as T2
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The HersheyHershey-Chase Experiement
• The Hershey-Chase experiment showed definitively
that DNA is the genetic material
• Hershey and Chase took advantage of the fact that
T2 phage is made of only two classes of
macromolecules: Protein and DNA
OH
H
H2N C C
CH2
CH2
S
CH3
H
O
H2N C C
OH
Methionine
CH2
SH
O
HO P
NH2
O
O
OH
Cysteine
Some amino acids
contain sulfur, thus
proteins contain sulfur,
but not phosphorous.
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OH
H
Nucleotides contain phosphorous,
thus DNA contains phosphorous,
but not sulfur.
Using S35T2 grown in S
35
containing media
incorporate S35
into their proteins
T2 attach to bacteria and
inject genetic material
Bacteria grown in
normal nonradioactive media
When centrifuged,
phage protein coats
remain in the
supernatant while
bacteria form a pellet
The supernatant is
radioactive, but the
pellet is not.
Did protein enter the bacteria?
Blending causes phage
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Is protein the genetic material?
Using P32T2 grown in P
32
containing media
incorporate P32
into their DNA
T2 attach to bacteria and
inject genetic material
Bacteria grown in
normal nonradioactive media
When centrifuged,
phage protein coats
remain in the
supernatant while
bacteria form a pellet
The pellet is
radioactive, but the
supernatant is not.
Did DNA enter the bacteria?
Blending causes phage
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Is DNA the genetic material?
• The Hershey and Chase experiment
EXPERIMENT
In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur
and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells.
1 Mixed radioactively
labeled phages with
bacteria. The phages
infected the bacterial cells.
Phage
2 Agitated in a blender to 3 Centrifuged the mixture
separate phages outside
so that bacteria formed
the bacteria from the
a pellet at the bottom of
bacterial cells.
the test tube.
Radioactive Empty
protein
protein shell
Radioactivity
(phage protein)
in liquid
Bacterial cell
Batch 1: Phages were
grown with radioactive
sulfur (35S), which was
incorporated into phage
protein (pink).
Batch 2: Phages were
grown with radioactive
phosphorus (32P), which
was incorporated into
phage DNA (blue).
4 Measured the
radioactivity in
the pellet and
the liquid
DNA
Phage
DNA
Centrifuge
Radioactive
DNA
Pellet (bacterial
cells and contents)
Centrifuge
Radioactivity
(phage DNA)
Pellet
in pellet
RESULTS
Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells.
When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus.
Figure 16.4
CONCLUSION
Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material.
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