Transcript Chapter 11 DNA: The Carrier of Genetic Information

Chapter 11
DNA: The Carrier of Genetic
Information
Experiments in DNA
• ???Protein as the genetic material
• 20 AA – many different combinations = unique
properties
• Genes control protein synthesis
• DNA and RNA – only 4 nucleotides = dull
Experiments in DNA
• Frederick Griffith – 1928
– Bacteria – pneumococcus – 2 strains
– (S) smooth strain – virulent (lethal)
• Mice – pneumonia - death
– (R) rough strain – avirulent
• Mice survive
– Heat killed (S) strain
• Mice survive
– Heat killed (S) + live (R)
• Mice died
• Found living (S) in dead mice
• Griffith continued
–  transformation - type of permanent genetic
change where the properties of 1 strain of dead
cells are conferred on a different strain of living
cells
– “transforming principle” was transferred from
dead to living cells
Fig. 16-2
Mixture of
heat-killed
Living S cells Living R cells Heat-killed
S cells and
(control)
(control)
S cells (control) living R cells
EXPERIMENT
RESULTS
Mouse dies Mouse healthy Mouse healthy Mouse dies
Living S cells
• Avery, MacLeod, McCarty - 1944
– Identified Griffith’s transforming principle as DNA
– Live (R) + purified DNA from (S)  R cells
transformed
– R + (S) DNA  die
– R = (S) protein  live
– DNA responsible for transformation
– Really?
• Hershey and Chase – 1952
– Bacteriophages
– Radioactive labels
• Viral protein – sulfur
• Viral DNA - phosphorus
– infect bacteria, agitate in blender, centrifuge
– Found
• Sulfur sample – all radioactivity in supernatant (not
cells)
• Phosphorus sample – radioactivity in pellet (inside
cells)
– SO – bacteriophages inject DNA into bacteria,
leaving protein on outside
– DNA = hereditary material
Fig. 16-3
Phage
head
Tail
sheath
Tail fiber
Bacterial
cell
100 nm
DNA
Fig. 16-4-3
EXPERIMENT
Phage
Empty
Radioactive protein
shell
protein
Radioactivity
(phage
protein)
in liquid
Bacterial cell
Batch 1:
radioactive
sulfur (35S)
DNA
Phage
DNA
Centrifuge
Pellet (bacterial
cells and contents)
Radioactive
DNA
Batch 2:
radioactive
phosphorus (32P)
Centrifuge
Pellet
Radioactivity
(phage DNA)
in pellet
• Rosalind Franklin (in lab of Wilkins)
– X-ray diffraction on crystals of purified DNA
– (X-ray crystallography)
– Determine distance between atoms of molecules
arranged in a regular, repeating crystalline
structure
• Helix structure
• Nucleotide bases like rungs on ladder
Fig. 16-6
(a) Rosalind Franklin
(b) Franklin’s X-ray diffraction
photograph of DNA
• James Watson and Francis Crick – 1953
– Model for DNA structure = double helix
– DNA now widely accepted as genetic material
– Took all available info on DNA and put together
– Showed –
• DNA can carry info for proteins
• Serve as own template for replication
Structure of DNA
• Nucleotides
– Deoxyribose
– Phosphate
– Nitrogenous base (ATCG)
• Purines – adenine, guanine – 2 rings
• Pyrimidines – thymine, cytosine – 1 ring
– covalent bonds link = sugar-phosphate backbone
• 3’ C of sugar bonded to 5’ phosphate = phophodiester
linkage
• 5’ end – 5’ C attached to phosphate
• 3’ end – 3’ C attached to hydroxyl
Chargaff - 1950
• # purines = # pyrimidines
– #A = #T
– #C = # G
• Each cross rung of ladder
– 1 purine + 1 pyrimidine
Fig. 16-UN1
Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width
consistent with X-ray data
•
•
•
•
Hydrogen bonding between N bases
A-T = 2 H bonds
G-C = 3 H bonds
Complementary base pairs
• # possible sequences virtually unlimited
•  many genes, much info
Fig. 16-8
Adenine (A)
Thymine (T)
Guanine (G)
Cytosine (C)
Fig. 16-5
Sugar–phosphate
backbone
5 end
Nitrogenous
bases
Thymine (T)
Adenine (A)
Cytosine (C)
DNA nucleotide
Phosphate
Sugar (deoxyribose)
3 end
Guanine (G)
Fig. 16-7a
5 end
Hydrogen bond
3 end
1 nm
3.4 nm
3 end
0.34 nm
(a) Key features of DNA structure (b) Partial chemical structure
5 end
DNA Replication
• Semiconservative – each strand of DNA is
template to make opposite new strand
• Meselson and Stahl
– E. coli and isotopes of N
– 15N – heavy/dense; 14N “normal”
– Bacteria with 15N in DNA replicated with medium
having 14N
– Centrifuge 
– Supports semiconservative model
• Explains how mutagens can be passed on
Fig. 16-11a
EXPERIMENT
1 Bacteria
cultured in
medium
containing
15N
2 Bacteria
transferred to
medium
containing 14N
RESULTS
3 DNA sample
centrifuged
after 20 min
(after first
application)
4 DNA sample
centrifuged
after 20 min
(after second
replication)
Less
dense
More
dense
Fig. 16-9-3
A
T
A
T
A
T
A
T
C
G
C
G
C
G
C
G
T
A
T
A
T
A
T
A
A
T
A
T
A
T
A
T
G
C
G
C
G
C
G
C
(a) Parent molecule
(b) Separation of
strands
(c) “Daughter” DNA molecules,
each consisting of one
parental strand and one
new strand
Fig. 16-10
Parent cell
(a) Conservative
model
(b) Semiconservative model
(c) Dispersive
model
First
replication
Second
replication
Steps of DNA Replication
•
•
•
•
•
•
1. DNA helicase –
2. Helix-destabilizing proteins –
3. Topoisomerases –
4. RNA primer –
5. DNA polymerase –
6. Origin of replication –
– Leading strand
– Lagging strand
• 7. DNA Ligase
Leading Strand
Fig. 16-12b
Origin of replication Double-stranded DNA molecule
Parental (template) strand
Daughter (new) strand
0.25 µm
Bubble
Replication fork
Two daughter DNA molecules
(b) Origins of replication in eukaryotes
Fig. 16-14
New strand
5 end
Sugar
5 end
3 end
T
A
T
C
G
C
G
G
C
G
C
T
A
A
Base
Phosphate
Template strand
3 end
3 end
DNA polymerase
A
Pyrophosphate 3 end
C
Nucleoside
triphosphate
5 end
C
5 end
Fig. 16-15a
Overview
Origin of replication
Leading strand
Lagging strand
Primer
Leading strand
Lagging strand
Overall directions
of replication
Fig. 16-17
Overview
Origin of
Leading
strand Lagging
replication
strand
Singlestrand
Helicase
binding
protein
5
3
3
Parental DNA
DNA pol III
PrimerPrimase
5 3
5
Leading
Lagging strand
Overall strand
directions
of
Leading strand
replication
DNA pol III Lagging strand
DNA pol I
4
35
3
2
DNA
ligase1
3
5
Telomeres
• Telomeres – caps end of chromosome; short
non-coding sequences repeated many times
• Cell can divide many times before losing
crucial info
• Lagging strand is discontinuous, so DNA
polymerase unable to complete replication ,
leaving small part unreplicated  small part
lost with each cycle
Fig. 16-19
5
Ends of parental
DNA strands
Leading strand
Lagging strand
3
Last fragment
Previous fragment
RNA primer
Lagging strand
5
3
Parental strand
Removal of primers and
replacement with DNA
where a 3 end is available
5
3
Second round
of replication
5
New leading strand 3
New lagging strand 5
3
Further rounds
of replication
Shorter and shorter daughter molecules