hghxcghdxg

1. HEREDITARY MATERIAL:
DNA STRUCTURE AND FUNCTION 1
Lecture 25 : Proof of DNA’s
hereditary role and structure
Lecture 26 DNA replication
Chapter 11 Life 8th ed
1

2. DNA IS UNIFYING MATERIAL OF INHERITANCE
IN LIVING ORGANISMS
DNA has unique properties as
substance of inheritance in living
organisms:
The capacity to store genetic
information
To copy it and
To pass it from generation to
generation
Some viruses have RNA as genetic
material ( an exception to the rule)
2

3. DNA AND ITS ROLE IN INHERITANCE
What is the evidence that the Gene
is DNA?
What is the structure of DNA?
How is DNA replicated?
How are the Errors in DNA
Repaired
What are some of the applications
of our knowledge of DNA structure
and replication?
3

4. LECTURE 25
 What is the evidence that the gene is
DNA?
 Gene:
 A discrete unit of heredity information consisting of a
specific nucleotide sequence in DNA (or RNA in
some viruses e.g Influenza virus and HIV)
 Most genes of eukaryotes are located in
chromosomal DNA
 Chromosome behaviour during meiosis and
fertilization accounts for inheritance patterns
 A few genes are carried by DNA of Mitochodria and
chloroplast
 In bacteria genes are carried in chromosomal and
plasmid DNA
4

5. WHAT IS THE EVIDENCE THAT THE GENE IS
DNA?
Early in the 20TH century, it was known
that chromosomes consisted of DNA
and protein;
Proteins were thought to be the only
truly complex molecules in cells, and
there fore must be responsible for
heredity
5

6. WHAT IS THE EVIDENCE THAT THE GENE IS DNA?
 1928: Frederic Griffith discovered the
phenomenon of transformation in bacteria
 Used bacteria Streptococcus pneumoniae (
some forms cause pneumonia in man and
mice)
 2 forms of bacteria:
 1, slimy colonies (S strains), form capsule,,
survive attack by macrophages ( defense
cells) in lungs, kills mice
 2 rough colonies (R strains) lacks capsule,
quickly killed by macrophages ,no disease. 6

7. WHAT IS THE EVIDENCE THAT THE GENE IS
DNA?
 Transforming principle: (Griffith)
 Mice were injected with:
 i S: mice died
 ii R: mice lived
 iii heat-killed S: mice lived
 iv R + heat killed S: mice died
 Conclusion: transforming principle from
dead S produced a heritable change in the
R strains 7

8. 8

9. WHAT IS THE EVIDENCE THAT THE GENE IS DNA?
 Identifying the transforming principle;
Oswald Avery
 Fractionated different chemicals in the S-
strains, tested each separately to see what
would cause transformation
 Isolated DNA could transform but no other
isolated fraction could ( RNA, protein, lipids,
polysaccharides)
 Conclusion: DNA is transforming principle
9

10. WHAT IS THE EVIDENCE THAT THE GENE IS DNA?
Hershey & Chase (H&C) proved that
only DNA is responsible for bacterial virus
(phage) infection of bacterial cells
1. Used Phage(virus) infect bacteria
consist of DNA and protein
2.Used different radioactive isotopes to
distinguish DNA from protein
3. Used P-32 ( lots of P in DNA, but none
in protein; for protein, used S-35
(proteins contain S in certain amino acids
but DNA lacks S)
10

11. WHAT IS THE EVIDENCE THAT THE GENE IS
DNA?
 H&C grew phage in hosts with either P or S
radioisotopes
 Then infected different bacteria for short time,
vortexed in blender to separate phage coats from
cells; cetrifugation separated phage (very small)
from cells(larger)
 Results: only P-32 found in cells; which were still
infected and produced new phage
 Conclusion: only DNA not protein ,was responsible
for inheritance
11

12. BACTERIOPHAGE T2 REPRODUCTION CYCLE
12

13. WHAT IS THE EVIDENCE THAT THE GENE IS
DNA?
Transfection in eukaryotic cells:
Genetic transformation of eukaryotic
cells
 a genetic marker is used- a gene that
confers an observable phenotype
Any cell can be transformed, even an
egg cell- results in transgenic organism
( contains recombinant DNA in its
genetic material) 13

14. WHAT IS THE STRUCTURE OF DNA?
 Many lines of evidence used to determine
DNA ‘s Structure
 1.X-ray crystallography
 A purified substance can be made from
crystals; position of atoms is inferred by
patterns of diffraction of X-rays passed
through it
 X-ray pictures of DNA suggested some kind of
helix
14

15. X-RAY CRYSTALLOGRAPHY HELPED REVEAL THE
STRUCTURE OF DNA
15

16. WHAT IS THE STRUCTURE OF DNA?
2.Chemical composition also provided
clues:
DNA is a polymer of nucleotides:
deoxyribose, a phosphate group, and
a nitrogen-containing base.
The bases:
Purines: adenine (A), guanine (G)
Pyrimidines: cytosine (C),
thymine (T)
16

17. 17

18. WHAT IS THE STRUCTURE OF DNA?
 3. .Chargaff published data that % of
A,T,C and G showed certain
regularities
% of bases varies from organism to
organism
% of A= % T, and % C= % G
Or the abundance of of purines = the
abundance of Pyrimidines
this is called the Chargaff’s rule.

19. WHAT IS THE STRUCTURE OF DNA?
 2. .Chargaff published data that % of A,T,C and G
showed certain regularities
 % of bases varies from organism to organism
 % of A= % T, and % C= % G
 Or the abundance of of purines = the abundance of
Pyrimidines
 this is called the Chargaff’s rule.
19

20. WHAT IS THE STRUCTURE OF DNA?
Watson and Crick: (W &C)
Used model building and combined all
the knowledge of DNA to determine its
structure
X-ray crystallography convince them the
molecule was helical
Other evidence suggested there were
two polynucleotide chains that ran in
opposite directions- antiparallel
20

21. WHAT IS THE STRUCTURE OF DNA?
 Watson and Crick
5`- CAGCTAGAGTCATCG-3`
3` - GTCGATCTCAGTAGC-5`
 Two chains of DNA face in opposite direction –
Antiparallel
 W&C suggested that the Chagaff’s rule was due to
base pairing A with T, C with G.
 They also suggested a simple model of replication; if
the DNA uncoiled, each strand could serve as
template for replication of new DNA 21

22. BIO 111 ANNOUNCEMENTS
• Tutorials & Practicals
• Each group is expected to submit ONE writeup: 2 pages
long, 1.5 spacing; font 12. References to be given in text
and list
• The names and IDs of the students in each group must
be included as each student will be allocated the marks
earned by her group: out of 20 marks.
• The students should use this week’s 8 & 9/10
tutorials to work on their group write up. So each group
should meet together in their chosen venue. Therefore
there will be no organized tutorial this week.
The write-ups are due for hand in on Wednesday 14/10/09
during lecture time
No tutorials for 15 & 16/10; due to elections
Groups I , K,U&V Friday 16/10 practical; proposed to be held on
Monday 12/10, 3 to 6 p.m. Need of a feed back from students
involved.

23. LECTURE 26 : DNA REPLICATION
 Key features of DNA
 double –stranded , uniform diameter
 Is antiparallel
 Complementary base paring;
 A pairs with T by 2 hydrogen bonds
 C pairs with G by 3 hydrogen bonds
 Every base pair consists of one purine and one
pyrimidine
 Phosphate groups connect to the 3’ C of one
sugar, and the 5’C of the next sugar
 At one end of the chain – a free 5’ phosphate
group , at the other end a free 3’ hydroxyl
23

24. 24

25. FUNCTIONS OF DNA
 Store genetic material– millions of nucleotides,
base sequence stores and encodes huge
amounts of information
 Susceptible to mutation- change in information
 Genetic material is precisely replicated in cell
division- by complementary base pairing
 Genetic material is expressed as the
phenotype- nucleotide sequence determines
sequence of amino acids in proteins
25

26. HOW IS DNA REPLICATED?
Three possible replication patterns:
Semiconservative replication
Conservative replication
Disperasive replication
Meselson and Stahl showed that
semiconservative replication
was the correct model
26

27. THE MODEL OF DNA REPLICATION
 Thee possible replication patterns:
 Semiconservative replication
 Conservative
 Dispersive replication
 Meselson and Stahl showed that
semiconservative replication was the correct
model
27

28. HOW IS DNA REPLICATED?
Two steps in DNA replication
The double helix is unwound, making
two template strands
New nucleotides are added to the new
strand at the 3’ end; joined by
phosphodiester linkages
Sequence is determined by
complementary base pairing
28

29. HOW IS DNA REPLICATED?
A large protein complex—the replication
complex—catalyzes the reactions of
replication.
All chromosomes have a base sequence
called origin of replication (ori).
Replication complex binds to ori at start.
DNA replicates in both directions, forming two
replication forks.
Large linear chromosomes have many origins
of replication.
DNA is replicated simultaneously at the
origins.
29

30. TWO VIEWS OF DNA REPLICATION
30

31. HOW IS DNA REPLICATED?
DNA helicase uses energy from ATP hydrolysis to
unwind the DNA.
Single-strand binding proteins keep the strands
from getting back together
Small, circular chromosomes have a single origin
of replication.
As DNA moves through the replication complex, two
interlocking circular chromosomes are formed.
DNA topoisomerase separates the two
chromosomes.
31

32. HOW IS DNA REPLICATED?
DNA polymerases are much larger than their
substrates.
Shape is like a hand; the “finger” regions have
precise shapes that recognize the shapes of the
nucleotide bases.
A primer is required to start DNA replication—a short
single strand of RNA.
Primer is synthesized by primase.
Then DNA polymerase begins adding nucleotides to
the 3′ end of the primer
32

33. DNA POLYMERASE BINDS TO TEMPLATE STRAND
33

34. NO DNA FORMS WITHOUT A PRIMER
34

35. HOW IS DNA REPLICATED?
At the replication fork:
The leading strand is pointing in the “right” direction
for replication.
The lagging strand is in the “wrong” direction.
Synthesis of the lagging strand occurs in small,
discontinuous stretches—Okazaki fragments.
Each Okazaki fragment requires a primer.
The final phosphodiester linkage between fragments
is catalyzed by DNA ligase.
35

36. THE NEW STRANDS FORM IN DIFFERENT WAYS
36

37. HOW IS DNA REPLICATED?
DNA polymerases work very fast:
They are processive: catalyze many polymerizations
each time they bind to DNA
Newly replicated strand is stabilized by a sliding
DNA clamp (a protein)
The new chromosome has a bit of single stranded
DNA at each end (on the lagging strand)—this
region is cut off.
Eukaryote chromosomes have repetitive sequences
at the ends called telomeres.
37

38. HOW ARE ERRORS IN DNA REPAIRED?
DNA polymerases make mistakes in
replication, and DNA can be damaged
in living cells.
Repair mechanisms:
Proofreading
Mismatch repair
Excision repair
38

39. HOW ARE ERRORS IN DNA REPAIRED ?
 As DNA polymerase adds a nucleotide to a growing
strand, it has a proofreading function—if bases are
paired incorrectly, the nucleotide is removed.
The newly replicated DNA is scanned for mistakes by
other proteins.
Mismatch repair mechanism detects mismatched
bases—the new strand has not yet been modified
(e.g., methylated in prokaryotes) so it can be
recognized.
If mismatch repair fails, the DNA is altered.
39

40. HOW ARE ERRORS IN DNA REPAIRED ?
DNA can be damaged by radiation, toxic
chemicals, and random spontaneous
chemical reactions.
Excision repair: enzymes constantly
scan DNA for mispaired bases,
chemically modified bases, and extra
bases—unpaired loops.
40

41. WHAT ARE SOME APPLICATIONS OF KNOWLEDGE OF DNA
STRUCTURE AND REPLICATION?
Copies of DNA sequences can be made by the
polymerase chain reaction (PCR) technique.
PCR is a cyclical process: DNA Replication in a ‘test
tube’
 DNA fragments are denatured by heating.
 A primer, plus nucleosides and DNA polymerase are
added.
 New DNA strands are synthesized.
PCR results in many copies of the DNA fragment—
referred to as amplifying the sequence.
Primers are 15–20 bases, made in the laboratory.
PCR has many applications e.g. detection of disease
agents such as HIV
41

42. WHAT ARE SOME APPLICATIONS OF KNOWLEDGE
OF DNA STRUCTURE AND REPLICATION?
42
DNA sequencing determines the base
sequence of DNA molecules.
Relies on altered nucleosides with fluorescent
tags that emit different colors of light.
DNA fragments are then denatured and
separated by electrophoresis.
Procedure has many uses such detection of
mutations in organisms resistant to drugs
etc..