2.5 the nature of dna

DNA discovery, transformation

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3. X-ray diffraction pattern
of DNA
In 1953, James Watsonand Francis Crickpublished
their findings on the discovery of the structure of DNA
and were awarded a Nobel prizefor their work
Watson and Crick interpreted
the X-ray diffraction patterns
produced by Rosalind Franklin
to propose a model for the
molecule of life –Deoxyribose
Nucleic Acid
Using cut-outsof the building blocks
of DNA, Watson and Crick constructed
a helical model of the DNAmolecule
and, in doing so, revolutionised the field
of molecular genetics
Discovering The Structure of DNA

4. Discovering The Structure of DNA
WatsonandCrickused information
from RosalindFranklin’sX-ray
diffraction data and Chargaff’sdata
to build a model of the DNA molecule
Chargaff’s data had shown that the
organicbasesin the molecule
occurred in specificratiosand X-ray
diffraction data revealed a 0.34 nm
repeatin the helical pattern from the
DNA
Watson and Crick built an ingenious
modelwhich demonstrated clearly
that the DNA molecule was a double
helix, with each turn of the helix
occupying a distance of 3.4 nmand
having a width of 2.0 nm

5. DNA or Deoxyribose Nucleic Acid,is the molecule of heredity
It is a long macromolecule or polymerconsisting of twohelical
chains coiled around a common axis
The building blocks or monomersof these chains
are called nucleotides
Each nucleotide consists of a5-carbon sugar,a
phosphate groupand anitrogen containing organic
base
A SINGLE
NUCLEOTIDE
The Structure of DNA
sugar organic
basephosphate
group

6. The Nucleotides
The nucleotides differ by virtue of their organic base
Adenine and Guanine are the two larger bases and are
described as purines
These are the four different nucleotidesor monomers that form
the building blocks of DNA
PURINE –CONTAINING
NUCLEOTIDES
PYRIMIDINE –CONTAINING
NUCLEOTIDES
Thymine and Cytosine are the two smaller bases and are
described as pyrimidines

7. Thymine and Cytosine
are pyrimidine
bases
The Nucleotides
Thymine is a pyrimidine base -
a singlenitrogen -containing ring
is part of its structure
Cytosine is a pyrimidine base-
a singlenitrogen -containing ring
is part of its structure

8. Adenine is a purine base -two
nitrogen -containing rings
are part of its structure
Guanine is a purine base -two
nitrogen -containing rings
are part of its structure
Adenine and Guanine
are
purine bases
The Nucleotides

9. Chargaff’s Data
Erwin Chargaffwas an eminent biochemistwho did
pioneering work in many different fields; he died on
June 20
th
2002 at the age of 96
Chargaff worked for a number of years measuring the
organicbasecompositionof DNA from rats and many other
different organisms
In 1950, he published a paper of his
findings, and from the data he gathered,
Chargaff’s base-pairing rulebecame
established
Sample data from his collection is
shown in the following table
Rat Base Composition
A = 28.6%
G = 21.4%
C = 20.5%
T = 28.4%

10. Chargaff’s Data
Source
Mol % of bases Ratios
Adenine
(A)
Guanine
(G)
Cytosine
(C)
Thymine
(T)
A/T G/C
E. coli24.7 26.0 25.7 23.6
Wheat 27.3 22.7 22.8 27.1
Rat 28.6 21.4 20.5 28.4
Human 29.3 20.7 20.0 30.0
Calculate thebase ratios indicated in the table

11. Chargaff’s Data
Source
Mol % of bases Ratios
Adenine
(A)
Guanine
(G)
Cytosine
(C)
Thymine
(T)
A/T G/C
E. coli24.7 26.0 25.7 23.6 1.051.01
Wheat 27.3 22.7 22.8 27.1 1.001.01
Rat 28.6 21.4 20.5 28.4 1.011.04
Human 29.3 20.7 20.0 30.0 0.981.04
The consistent 1:1 ratiosbetween A and T and between G and C
were to play a key role in understanding the functioningof DNA

12. An important aspect of the DNA double helix is the specific way
in which the bases pair together
The distance between the two backbones does not varyalong the
length of the molecule
This distance accommodates apurine-pyrimidine pair
The Base Pairing Rule
Adenine always
pairs with Thymine
forming twohydrogen
bonds
Adenine
(Purine)
Thymine
(pyrimidine)
Weak hydrogen
Bonds

13. Cytosine always
pairs with Guanine
forming threehydrogen
bonds
Adenine ALWAYS pairs with Thymine A T
Cytosine ALWAYS pairs with GuanineC G
This arrangement
allows for the
maximum number
of hydrogen bonds
between the bases
The bases that pair
together are described
as complementary bases
The Base Pairing Rule
Guanine
(Purine)
Cytosine
(pyrimidine)
Weak hydrogen
Bonds

14. Nucleotides form strong
covalent bonds with each
other when the sugar
groupof one nucleotide
bonds to the phosphate
group of another
nucleotide
The second polynucleotide
chain runs in the opposite
direction to the first.
The bases are aligned in
accordance with the
base-pairing rule
The nucleotides form
strong covalent bonds
between the sugar and
and phosphate groups
Weak hydrogen bonds
between the base pairs
hold the two
polynucleotide
strandstogether
DNA consists of two
polynucleotide strands.
The backbone is a sugar-
phosphate arrangement
and the bases project
toward the inside of the
double helix
The Structure of DNA
The whole molecule is
coiled into a helix
Strong, covalent
sugar -phosphate
bonds hold the
polynucleotide
together

15. Ball and stick model of
DNA showing the
sugar-phosphate
backbone and the
inwardly projecting
pairs of organic bases
A + G = C + T
A = T
G = C
sugar-phosphate
backbone
organic bases
Adenine and thymine are
complementarybases and
form a complementary
base pair; similarly,
cytosine and guanine form
a complementary base pair

16. DNA is the material of heredity.
It stores genetic information.
It is self replicating.
It is able to express its genetic message.
DNA Structure -Summary
DNA is a double-strandedmolecule consisting
of two anti-parallelpolynucleotide strands
The sugar-phosphate backboneis held
together by strong, covalentbonds
The basesproject inwardsand are held
together by weak hydrogen bonds
The bases pair in such a way that
adeninealways pairs with thymineand
guaninealways pairs with cytosine
The ratio of the bases is:
adenine:thymine = 1:1and
guanine:cytosine = 1:1(Chargaff’s rule)

17. DNA Structure -Animation
A = adenine T = thymine C = cytosine G = guanine
Drag the bases to complete the basepairs and the strand of DNA...

19. The Replication of DNA
Watson and Crickproposed that, during replication, the strands of DNA
separate and each strandacts as a templatefor the formation
of a new strand of DNA
This method of DNA duplication became known as
semi-conservative replication and experimental
evidence was provided to support this mechanism
An important property for any molecule that passes genetic information
from one generation to the next is that of self-replication
When Watson and Crickfirst proposed the structure of DNA this was
accompanied by an explanation as to how DNA might replicate

20. The Mechanism of DNA replication
A portion of DNA representing
base pairs held together by
hydrogen bonds
Hydrogen bonds
holding the DNA
strands together are
brokenand the two
strands of the helix
begin to separate
An enzyme called
DNA polymerase
attaches to the DNA
molecule and moves
along its length

21. Free nucleotidesfrom the
nucleoplasm are attracted
to their complementary
bases on each separated
strand of DNA

22. The nucleotides
bond together
Each new DNA molecule consists of one
strandfrom the original DNA double helix
and one newly synthesised strand
(shown here in a different colour)
This mechanism is called
semi-conservative replication
Two identical DNA molecules
are formed
Newly synthesised strands

23. DNA Replication -Summary
A = adenine T = thymine C = cytosine G = guanine

24. Evidence for the Semi-Conservative Mechanism –The
Work of Meselson and Stahl
Meselson and Stahlworked with the DNA from the bacterium E. coli
They created parent DNA moleculesthat were labelled with
15
N,the
heavyisotope of nitrogen
In this way, all the parent DNA moleculescreated were
heavierthen ordinary DNA
The heavy DNA was obtained by growingE. colifor many generations
in a medium that contained
15
NH
4Cl (labelled ammonium chloride)
as the only source of nitrogen
The bacteria containing the heavy DNAwere then transferred to a
growing medium in which the only source of nitrogen was
14
N
(ordinary lighter nitrogen)
The distribution of
15
N and
14
Nin the DNA molecules was then determined
after successive rounds of replication using the technique of
density gradient centrifugation

25. Bacterial cells grown in
15
N mediumfor several
generations –DNA extracted from a sample
and subjected to density gradient centrifugation
First generationof bacterial cells after transfer
to
14
N medium–DNA extracted from a sample
and subjected to density gradient centrifugation
Second generationof bacterial cells after transfer
to
14
N medium–DNA extracted from a sample
and subjected to density gradient centrifugation
Third generationof bacterial cells after transfer
to
14
N medium–DNA extracted from a sample
and subjected to density gradient centrifugation

26. Density Gradient Centrifugation
Solutions of decreasing densityof
caesium chlorideare placed into
centrifuge tubes
The most densecaesium chloride
is at the bottomof the tube
The extracted DNAmolecules are
pipetted onto the top of the most
dilute caesium chloride solution

27. The tubes are spun in a centrifuge
location of
heavy DNA
14
N
15
N hybrid
DNA
light
DNA
The DNA molecules in the
gradient are observed under
ultraviolet light
As the tubes are centrifuged, the DNA molecules move to positions where
their densitycorresponds with that of the caesium chloride solution
Density Gradient Centrifugation

28. Heavy, light and intermediate DNA molecules separate into
positions where their densities correspond to that of the caesium
chloride gradient within the centrifuge tubes

29. Meselson and Stahl’s Results
heavy
DNA
before transfer
to
14
N
hybrid
14
N
15
N
DNA
one cell generation
after transfer
to
14
N
light
DNA
hybrid
14
N
15
N
DNA
two cell generations
after transfer
to
14
N
¾ light
DNA
¼ hybrid
DNA
three cell generations
after transfer
to
14
N
These results support the hypothesis that DNA replication is
semi-conservative as follows:

30. first generation
after transfer to
14
N medium
all intermediate
(hybrid) DNA
second generation
after transfer to
14
N medium
50% light –50%
hybrid
third generation after
transfer
to
14
N medium
75% light –25%
hybrid
bacterial DNA following
growth of cells in
15
N medium
all heavy DNA
14
N
light DNA
15
N
heavy DNA
KEY

31. Summary
bacterial DNA following
growth of cells in
15
N
medium;
all heavy DNA
first generation
after transfer to
14
N medium;
all intermediate
(hybrid) DNA
third generation after
transfer to
14
N
medium; 75% light
–25% hybrid
second generation after
transfer to
14
N
medium; 50% light
–50% hybrid

32. Semi-Conservative Replication
During replication, the DNA double helix unzipsunder the
influence of DNA polymerase
Each of the separated strands serves as a templatefor the
assembly of a new complementarystrand
Freenucleotidesfrom the nucleoplasm are attracted to their
exposed complementary bases on the separated strands of DNA
Following replication, each new DNA molecule consists of one
strand from the original DNA double helix and onenewly
synthesisedstrand
This mechanism, which was first predicted by Watson and Crick
in their first publication of the structure of DNA (1953), is called
Semi-Conservative Replication

34. Every human cell contains:
46 chromosomes
2 metres of DNA
Around 3 billion nucleotides
Approximately 30 000 genes
that code for the proteins that
determine the nature and
development of organisms

35. Extension Work

37. During the 1920sit was generally believed that proteinrather
than DNA was the genetic material of cells
Many years of experimental research was needed before the scientific
community finally accepted that DNA is indeed the molecule of heredity
This revolution in thinking began with Griffith’s Transformation
Experiments in 1928and finally concluded with the work of
Hershey and Chase involving the use of bacteriophages in 1952
THE WORK OF GRIFFITH -1928
Griffith, a bacteriologist, conducted research on techniques for
distinguishing between different strains of pneumococcus, the bacterium
that causes pneumonia
Griffith had identified two strains of Pneumococcus:one strain
produced a capsule enabling the bacterium to evade the host immune
system and thus cause pneumonia, while the other non-encapsulated
strainfailed to cause disease
Griffith injected micewithboth of the Pneumococcus strains
and obtained the following results:
Evidence that DNA is The Molecule of Heredity

38. Live, non-encapsulated
R strainbacteria
injected into mice
Live, encapsulated
S strainbacteria
injected into mice
Heat killed R strain
orS strain bacteria
injected into mice
Heat killed S strain
andlive R strain
injected as a mixture
into mice
Mice live
Mice die
Mice live
Mice die!!

39. Griffith concluded that the presence of the dead, encapsulated
bacterial cells had transformedthe live non-encapsulated strain into a
pathogenic encapsulated S strain
The transformed bacteria and their offspringcontinued to produce
capsules and hence the change was permanentand heritable
Griffith deduced that some agent was being passedto the
non-encapsulated strain from the killed S strain
The nature of thistransforming agentwas not to be determined until
the work of Avery, MacLeod and McCartyhad been completed
Heat killed S strain
andlive R strain
injected as a mixture
into mice
Mice die!!
A blood sample taken from the mice inoculated
with killed S strain and live R strainshowed the
presence of both Live R and Live Sstrain bacteria
in the systems of the mice

40. Avery, MacLeod and McCarty
Avery and his colleagues carried out extensive chemical analysisof the
different molecules of the heat killed encapsulated Pneumococci
After purifying the different molecules of the heat killed S strain,
they tested the ability of each of these molecules to bring about
transformation of the live R cells
They identified DNAas the transforming substance and published their
findings in the JournalofExperimentalMedicinein 1944
Despite their findings, the work of Avery and his co-workers was largely
overlookeduntil the early 1950s although Avery himself was aware
of the potential of his discovery:
“If we are right, and of course that’s yet not proven, then it means that nucleic
acids are not merely structurally important but functionally active substances
in determining the biochemical and special characteristics of cells…..But today
it takes a lot of well documented evidence to convince anyone that DNA could
possible be endowed with such biologically active and specific properties and
that evidence we are now trying to get.”
Letter from Avery to his brother in 1943

41. The Experiments of Hershey and Chase
DNA and protein are the only two molecules that a bacteriophage
contains and the experiments of Hershey and Chase were designed to
demonstrate which of these molecules entered bacterial cells and
whether or not they were passed on to the new generation of viruses
In 1952 Hershey and Chaseturned their attention to virusesas an
approach to the search for the nature of the genetic material
By 1950 thelife cycle of the bacteriophagewas
well known and it was recognised that viral
genetic material was injected into bacteria to
direct the formation of new viral particles
Hershey and Chase carried out a series of ingenious
experiments to clarify once and for all whether DNA
or proteinwas the genetic material of life
They worked with a type of virus known as the
bacteriophage–a virus that infects and
multiplies within bacterial cells

42. Bacteriophage Structure
Hershey and Chase worked with
the T2 type bacteriophagethat
infects the bacterium E. coli
As the protein coat is composed
of some sulphur-containing amino
acidsand DNAhas the element
phosphorusin its structure, then it
was possible to distinguish between
DNA and Protein
They prepared two batches
of bacteriophagefor infection:
One batch contained
radioactively labelled proteins
(
35
S-proteins) and the other batch
contained radioactively labelled
DNA (
32
P-DNA)
SinceDNA lacks sulphur atoms
and protein lacks phosphorus
atoms,the radioactive atoms
provided a method oflabelling
that allowed for the trackingof
DNA and protein during the
course of their experiments
Head consisting of
a protein coat and
containing
the viral DNA
Collar
Tail Sheath
Base Plate
Tail Pin
Tail Fibres

43. T2 phage was used to infect
E. colicells that had been grown
on agar plates containing a
medium that included either
radioactive phosphorus or
radioactive sulphur
Sulphur-containing protein coat
Phosphate-containing DNA
E.coli grown in a medium
containingradioactive phosphates(
32
P)
E.coli grown in a medium
containingradioactive sulphates (
35
S)
The new viral particles that
emerged from the bacterial cells
formed radioactive DNAfrom
one set of cells and radioactive
protein coatsfrom the other
These radioactively -labelled
phage particleswere then
allowed to infect non –
radioactive E. coli cells

44. The phage particles inject
“material” intonon –
radioactiveE. coli cells
The only radioactively labelled compound that entered the
bacterial cells and directed the formation of new phage particles
was DNA, thus supporting the view that DNAis the molecule
that carries genetic information –The Molecule Of Heredity
New phage particles
are produced in both
populations of bacterial cells
and the ‘ghost’ phagesare
stripped off the E. coli cells
Radioactivityis detected onlyin the population of
bacterial cells infected with phage particles
that contained radioactive DNA

45. Bacteriophage Life Cycle
bacterial cell
mature bacteriophage
particle
bacteriophage
attaches to
bacterial cell
and injects its
DNA
The bacterial chromosome
is degraded and viral
DNA multiplies
Lysis of the bacterial
cell occurs releasing
new viral particles that
infect further bacterial cells
New viral particles
are assembled
within the host cell

46. Bacteriophage Life Cycle
The following animation summarises how a bacteriophage multiplies...

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