15. Semiconservative replication
Each parent strand serves as a template for a
new strand and the two new DNA strands each
have one old and one new strand
Parent strands
New / daughter
strand
16. Meselson and Stahl experiment
[1958] demonstrates
semiconservative replication:
17. Cells broken open
to extract DNA
E. coli grown in the presence
of 15N (a heavy isotope of
Nitrogen) for many generations
E. coli placed in
medium containing
only 14N (a light
isotope of Nitrogen)
• Cells get heavy-labeled DNA
Sampled
at:
0 min
1
2
3
40
min
20
min
Suspended DNA in cesium
chloride (CsCl) solution.
4
15N medium
24. FOUR requirements for DNA to replicate
1. DNA to act as a
template for
complementary
base pairing.
2. The four
deoxyribonucleoside
triphosphates:
dATP, dGTP, dCTP & dTTP.
25. The nucleotides arrive as nucleosides
– DNA bases with P–P–P
• P-P-P = energy for bonding
– DNA bases arrive with their own energy source
for bonding
dATP dGTP dTTP dCTP
3. A source of chemical energy is needed to drive
this highly endergonic reaction.
26. DNA
Polymerase III
4. A DNA polymerase III
enzyme brings
substrates to the
template and catalyses
the reactions.
27. energy
ATPGTPTTPCTP
Energy of Replication
Where does energy for bonding usually come
from?
ADPAMPGMPTMPCMP
modified nucleotide
energy
We come
with our own
energy!
And we
leave behind a
nucleotide!
You
remember
ATP!
Are there
other ways
to get energy
out of it?
Are there
other energy
nucleotides?
You bet!
28. DNA Template & dATP
New strand Template strand
5’ end 3’ end
Sugar A T
Base
C
G
G
C
A
C
OH
P P
3’ end
5’ end 5’ end
A T
C
G
G
C
A
C
T
3’ endPyrophosphate
2 P
OH
Phosphate
5’ end
deoxyribonucleoside
triphosphate
nucleotide
29.
30. DNA replication occurs in two steps:
1. DNA is locally denatured
(unwound)
WHY?
To separate the two
template strands and
make them available
for base pairing.
Unzipping of
DNA
31. DNA replication occurs in two steps:
2. The new
nucleotides are
linked by covalent
bonding to each
growing strand in a
sequence
determined by
complementary
base pairing.
32. REMEMBER:
Nucleotides are always added to the growing
strand at the 3’ end – the end at which the DNA
strand has a free –OH group on the 3’ carbon of
its terminal deoxyribose
33.
34. Three Stages of replication
1) Initiation
– occurs at the origin of replication
2) Elongation
– involves the addition of new nucleotides
based on complementarity of the template
strand
3) Termination
– occurs at a specific termination site
39. Directionality of the DNA strands at a replication fork
Leading strand
Lagging strand
Fork movement
40. Directionality of the DNA strands at a replication fork
Leading strand
Lagging strand
Fork movement
41. Protein Role
DNA helicases Unwinds the double helix
RNA primase Synthesises RNA primers
Single-strand binding
proteins
Keep the two strands separated
DNA polymerase I Erases primer and fills gaps
DNA polymerase II
[not in syllabus]
Proofreading of DNA
DNA polymerase III Synthesises DNA; proofreading
DNA ligase Joins the ends of DNA segments;
DNA repair
42. Replication: 1st step
• Unwind DNA
– helicase enzyme
• unwinds part of DNA helix
• stabilised by single-stranded binding proteins
single-stranded binding proteins replication fork
helicase
43. A primer is :
- required to start
DNA replication—a
short single strand
of RNA.
- synthesised by
primase.
Then DNA
polymerase III begins
adding nucleotides
to the 3′ end of the
primer.
46. • DNA polymerases:
1. can synthesise DNA only in the 5’ to 3’
direction
2. cannot initiate DNA synthesis
Problem at 3’ ends of Eukaryotic Chromosomes
47. Label structures at the Replication Fork
a. Leading strand template
b. Leading strand
c. Lagging strand
d. Lagging strand template
e. RNA primer
f. Okazaki fragment
50. How are Okazaki fragments linked?
Each Okazaki
fragment
requires a
primer.
The final phosphodiester
linkage between
fragments is catalyzed by
DNA ligase.
54. Two dimensional view of a replication fork
Direction of synthesis
on leading strand
3’
5’
3’
5’
3’
5’
55. Proofreading procedure
• DNA replication is not perfect due to:
1) the high speed of replication
- (1000 nucleotides per second)
2) spontaneous chemical flip-flops in the bases
• occasionally DNA polymerase incorporates
incorrectly matched bases
56. If bases are paired
incorrectly, the
nucleotide is removed.
Proofreading is done by several DNA
polymerases including DNA
polymerase II
57. Editing & proofreading DNA
• 1000 bases/second =
lots of errors!
• DNA polymerase I
– proofreads & corrects mistakes
– repairs mismatched bases
– removes abnormal bases
• repairs damage
throughout life
– reduces error rate from
1 in 10,000 to
1 in 100 million bases
58. Fast & accurate!
• It takes E. coli <1 hour to copy
5 million base pairs in its single chromosome
– divide to form two identical daughter cells
• Human cell copies its 6 billion bases & divide
into daughter cells in only few hours
– remarkably accurate
– only ~1 error per 100 million bases
– ~30 errors per cell cycle
59. What is the advantage of the one-way
directionality of the DNA structure?
Allows the proofreading enzymes to recognise
the parental strand, running in one direction, as
the ‘right stuff’.
60. Overview
A) CHROMOSOME STRUCTURE
B) SEMICONSERVATIVE REPLICATION
C) THE REPLICATION PROCESS
D) THE DNA BLUEPRINT
E) THE GENETIC CODE
BLUEPRINT: a design plan or
other technical drawing
61. DNA ‘Blueprint’
• every cell in the body has the same "blueprint"
or the same DNA
• blueprint of a house tell the builders how to
construct a house
62. Importance of the DNA ‘Blueprint’
Tells the cell
how to build
the organism.
63. How is it possible for cells to have:
the SAME DNA different structures &
functions?
BUT
64. Proteins are a cell’s “molecular workers”
ANSWER:
Every cell contains a particular set of proteins
Ovum must have
receptors to bind the
sperm head.
Phagocyte must
have receptors to
engulf the microbe.
65. If all body cells have the SAME DNA, explain
why only the pancreas makes insulin?
A cell has the ability to turn off most
genes and only work with the genes
necessary to do a job.
66. DNA ‘Blueprint’
• information by itself, does not do anything – e.g.
a blueprint may describe the structure of a
house in great detail, but unless that
information is translated into action, no house
will ever be built
• likewise, although the base sequence of DNA,
the “molecular blueprint” of every cell contains
an incredible amount of information, DNA
cannot carry out any action on it own
67. Central dogma: flow of information is
from the:
DNA of a
cell’s genes
the proteins that
actually carry out the
cell’s functions
RNADNA
Protein
to
68. What is ‘junk DNA’?
• 98.5% of human DNA does not code for proteins
• Introns (old name: junk DNA) –
- the regions of DNA that do not code for
proteins
• Exons –
- the sections of DNA that code for proteins
71. Evidence for the role of DNA in
inheritance: the
Hershey and Chase experiment (1952)
Martha Chase
Alfred Hershey
72. Hershey and Chase set out to
determine whether the:
protein or DNA enters the bacterial cells.
73. • Bacteriophage - a
particular type of virus
which specifically
attacks bacterial cells
• bacteriophage T2 :
attacks the bacterium
Escherichia coli
consists of a protein
coat and DNA
74. Which elements to follow?
DNA:
in nucleotide
Protein:
BOTH proteins & DNA: C, H, O, N
S
P
in methionine
+ cysteine
75.
76.
77.
78. This experiment confirmed that:
DNA from bacteriophages infected bacteria
Phage
head
Tail
Tail fiber
DNA
Bacterial
cell
100nm
81. What does DNA code for?
DNA specifies only
the production of
protein synthesis
82. DNA nucleotide base sequence:
determines
the amino
acid sequence
of protein
molecules
83. GENETIC CODE is the relationship
between the: bases and amino acids
84. The code
• DNA nucleotide bases:-
adenine, guanine, cytosine and thymine
• RNA has four nucleotide bases:-
adenine, guanine, cytosine and uracil
• this ‘alphabet’ of 4 letters is responsible for
carrying the code that results in the synthesis of
a potentially infinite number of protein
molecules
85. How many bases code for one amino acid? Recall
that there are 20 different amino acids in proteins.
Only 4 amino acids would be possible.
A, T, C, G1?
2?
3?
16 amino acids would be possible: still
not large enough. e.g. AU, CU, or CC.
42 = 16
64 amino acids would be possible: e.g.
AUU, GCG, or UGC. This vocabulary
provides more than enough words to
describe the amino acids.
43 = 64
87. Codon:
a set of three adjacent
nucleotides, also called
triplet, in DNA or mRNA
that designates a specific
amino acid to be
incorporated into a
polypeptide
88. Six features of the
genetic code
1. Triplet code
2. Specificity
3. Degeneracy
4. Universality
5. Non-overlapping
6. Punctuated
89. 1) The code is a triplet code
• the DNA code for a protein is first copied into
messenger RNA (mRNA) before a protein is
made
• mRNA is complementary to the DNA
DNA
mRNA
91. One mRNA molecule may contain
hundreds or even thousands of bases
the cell recognises where the code for a
protein starts and stops as the mRNA has:
START
CODON
STOP
CODON
start
and
stop codons
92. 64 codons in all
61 for amino acids
3 ‘stop codons’
(UAA, UAG, UGA)
1 ‘start codon’
(AUG – codes for methionine)
94. Methionine is specified by the codon AUG -
known as the start codon
Note: it may be removed after the protein is
synthesised
All proteins originally begin with the amino
acid methionine. Why?
95. When the ribosome encounters a
stop codon, it releases the :
1. newly synthesised
protein
2. mRNA
96. 2) The code is specific (non ambiguous)
• each triplet code
specifies only one
amino acid
• e.g. UUU =
phenylalanine
97. 3) The code is degenerate
Valine
GUU
GUC
GUA
GUG
a given amino acid may be coded for by more
than one codon
64 codons and only 20
amino acids:
so some amino acids
are coded for by
several codons –
exceptions [next
slide]:
Tyrosine
UAU
UAC
Lysine
AAA
AAG
99. First TWO bases determine the amino acid
• Third Base is usually less specific than the
first two.
• This is also known as the "Wobble Hypothesis"
because often the:
Valine
GUU
GUC
GUA
GUG
third base can change
BUT
the amino acid remains the
same.
Wobble position of a codon refers
to the 3rd nucleotide in a codon
100. What is the advantage of a degenerate code?
This allows for possible
mutations to be less damaging.
102. Polypeptide structure is changed
• deletion or addition of one or two bases,
leads to a change in reading frame (reading
sequence)
THE FAT CAT ATE THE BIG RAT
Delete C: THE FAT ATA TET HEB IGR AT
Insert A: THE FAT ATA ATE THE BIG RAT
103. Six features of the
genetic code
1. Triplet code
2. Specificity
3. Degeneracy
4. Universality
5. Non-overlapping
6. Punctuated
104. 4) The code is nearly universal
• the genetic code is the same in all organisms,
except in:
e.g. AGA = arginine in:
all organisms whose genetic code has
been studied
mitochondria protozoan nuclear DNAand
105. The universality of the genetic code is among the
strongest evidence that all living things share a
common evolutionary heritage
106. What is the importance of the
universality of the code?
GENETIC ENGINEERING IS POSSIBLE
107. Aim:
to map out the entire genetic code of a human
-2.1 million base pairs
-(30,000 – 40,000 protein coding genes)
The Human Genome Project (1990 – 2003)
111. What is the size of a gene?
• average gene in humans: 3000 bases
• but sizes vary greatly
• the largest known human gene:
- 2.4 million bases
112. Six features of the
genetic code
1. Triplet code
2. Specificity
3. Degeneracy
4. Universality
5. Non-overlapping
6. Punctuated
113. 5) The code is non-overlapping
non-overlapping:
- no base of a given triplet contributes to
part of the code of the adjacent triplet
non-overlappingoverlapping
114. • the genetic code is read in groups (or
“words”) of three nucleotides
• after reading one triplet, the “reading frame”
shifts over the next three letters, not just one
or two
115. Six features of the
genetic code
1. Triplet code
2. Specificity
3. Degeneracy
4. Universality
5. Non-overlapping
6. Punctuated
116. 6. The code is punctuated:
REMEMBER: Excluding the start & stop codons, the
actual code determining the sequence of amino acids
is UNPUNCTUATED
NOTE: according to the syllabus, the code is
punctuated due to start and stop codons
however
the majority of text books consider the code
as being unpunctuated i.e. comma less
118. A mutation is a change in the
• amount, arrangement or structure of the
DNA of an organism
119. A mutation produces a change in the genotype & is
passed on when a cell nucleus divides by:
mitosis or
meiosis from the mutant cell
Mutant daughter cells
Mutant daughter cells
Mutant cell
Mutant cell
120. Which type of mutation can be inherited by the
offspring?
germinal
somatic Occur in somatic cells:
are NOT passed on the offspring
Occur in gamete cells:
are passed on to the offspring
121. A mutation may result in the change
in appearance of a characteristic of a
population
e.g. red eyes in Drosophila appeared in 1909
122. e.g. dark-coloured moth appeared in 1848
The "typica" form of
the moth.
The "carbonaria" form.
123. occur in: any gene at any time
be:
Mutations can
Spontaneous
Induced
124. Spontaneous Mutations:
are permanent changes in the genome that
occur without any outside influence
occur because the machinery of the cell is
imperfect
Both chromatids are
sent to one daughter
cell, the other gets
none.
One chromatid goes
to each daughter
cell.
125. Induced Mutations:
occur when some outside agent causes a
permanent change in DNA
mutagens:
anything that causes a mutation
examples:
• Asbestos
• Tar from tobacco
• Ionising radiation e.g. UV
• Pesticides
• Caffeine
126. Mutation rates vary between
organisms
In general, the mutation rate in:
unicellular eukaryotes
bacteria
Chernobyl disaster was a
catastrophic nuclear accident that
occurred on 26 April 1986
is roughly 0.003 mutations
per genome per generation.
Chernobyl: mutant dog
127. Ionising radiation is radiation that:
carries enough energy to liberate electrons from
atoms or molecules, thereby ionizing them.
130. Mutations
can be:
Chromosomal
[covered in 2nd year]
Gene mutations or point mutations:
INSERTION
INVERSION
DELETION
SUBSTITUTION
describe a change in the structure of DNA
at a single locus
1
2
131. Fig. 12 Gene or point mutation
1) INSERTION: the addition of an extra nucleotide
A GT G C A T A TT G A C A G
2) DELETION: involves the loss of a nucleotide
A GT G C A T A TT C A G
132. Fig. 12 Gene or point mutation
4) SUBSTITUTION:
a particular base is substituted by another (e.g.
sickle-cell anaemia)
A GT G C A T A TT G T A G
3) INVERSION: two nucleotides become arranged in the
wrong order
A GT G C A T T TA G C A G
133. Sickle Cell Anaemia in humans is an
example of base substitution
• a base in one of the genes involved in
producing haemoglobin is substituted
• at position 14 in
the DNA:
thymine is
replaced by
adenine
134. Sickle Cell Anaemia:
at low oxygen tensions, haemoglobin S
crystallises in the red cells distorting them into a
sickle shape
135. Point mutations
No mutation
DNA level TTC TTT ATC TCC
mRNA
level
AAG AAA UAG AGG
Protein
level
Lys Lys STOP Arg
Silent Nonsense Missense
Missense
mutation
Nonsense
mutation
is a point mutation in a sequence of DNA that results
in a premature stop codon
is a point mutation that results in the substitution of
one amino acid in protein for another
136. Frameshift mutations
The addition or
deletion
of a single base
has much more profound
consequences than does the
substitution of one base for another
THE CAT SAW THE DOG
138. Changing the reading frame early in a gene, and
thus in its mRNA transcript, means that the
majority of the protein will be altered.
Amino acid
Deletion of a single nucleotide
DNA
bases
Original DNA code for an amino acid sequence.
Incorrect amino acid sequence, which
may produce a malfunctioning protein.
139. End-Of-Year SEP 2013
Use your knowledge of the genetic code to explain
statements (a) and (b) below. Use your knowledge
of genetic mutations to answer statements (c), (d)
and (e). [5 marks each]
i) Distinguish between a base substitution and an
inversion.
i) Distinguish between a deletion and an insertion.
ii) Explain how deletions and insertions lead to
frameshift mutations
140. Use your knowledge of biology to explain the following.
The structure of the DNA molecule permits vast amounts
of information to be stored. (5 marks)
Question: [SEP, 2007]
1. Information on the DNA molecule is in the form of a
sequence of bases, where three consecutive bases
specify an amino acid. Thus a small number of bases are
needed to code for an amino acid. Considering that DNA
within a eukaryotic cell is 2m long, it allows for a large
amount of information to be stored.
2. In many eukaryotic cells, split genes occur. These
contain regions which code for the protein called exons
and introns which do not code. The way in which the
exons are linked together determines the type of
polypeptide to be formed. Thus one gene can form a
number of closely related polypeptides.
