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Chapter 16: The Molecular Basis of Inheritance. Chapter 14 – traits carried in gametes Chapter 15 – genes found on chromosomes Chapter 16 – DNA is the molecule of inheritance How did we learn this?. - PowerPoint PPT Presentation
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Chapter 16: The Molecular Basis of Inheritance
Chapter 14 – traits carried in gametes
Chapter 15 – genes found on chromosomes
Chapter 16 – DNA is the molecule of inheritance
How did we learn this?
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?
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:
Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells.
EXPERIMENT
RESULTS
CONCLUSION
Living S(control) cells
Living R(control) cells
Heat-killed(control) S cells
Mixture of heat-killed S cellsand living R cells
Mouse dies Mouse healthy Mouse healthy Mouse dies
Living S cellsare found inblood sample.
Transformation – external DNA assimilated by a cell which changes the cell’s genotype and phenotype
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?
- Bacteriophage – viruses that infect bacteria
Phagehead
Tail
Tail fiber
DNA
Bacterialcell
100
nm
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?
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.
EXPERIMENT
Radioactivity(phage protein)in liquid
Phage
Bacterial cell
Radioactiveprotein
Emptyprotein shell
PhageDNA
DNA
Centrifuge
Pellet (bacterialcells and contents)
RadioactiveDNA
Centrifuge
PelletRadioactivity(phage DNA)in pellet
Batch 1: Phages weregrown with radioactivesulfur (35S), which wasincorporated into phageprotein (pink).
Batch 2: Phages weregrown with radioactivephosphorus (32P), which was incorporated into phage DNA (blue).
1 2 3 4Agitated in a blender toseparate phages outsidethe bacteria from thebacterial cells.
Mixed radioactivelylabeled phages withbacteria. The phagesinfected the bacterial cells.
Centrifuged the mixtureso that bacteria formeda pellet at the bottom ofthe test tube.
Measured theradioactivity inthe pellet and the liquid
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?
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.
Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material.
RESULTS
CONCLUSION
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?
- Nucleotide composition- Human
- A – 30.3%- T – 30.3%- C – 19.9%- G – 19.5%
Chargaff’s rulesA=TC=G
Sugar-phosphatebackbone
Nitrogenousbases
5 endO–
O P O CH2
5
4O–
HH
OH
HH
3
1H O
CH3
N
O
NH
Thymine (T)
O
O P O
O–
CH2
HH
OH
HH
HN
N
N
H
NH
H
Adenine (A)O
O P O
O–
CH2
HH
OH
HH
HH H
HN
NN
OCytosine (C)
O
O P O CH2
5
4O–
H
O
HH
3
1
OH2
H
N
NN H
ON
N HH
H H
Sugar (deoxyribose)3 end
Phosphate
Guanine (G)
DNA nucleotide
2
N
Note: 5’ end – phosphate group3’ end - -OH
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?4. What experiment did Watson & Crick do?
- NONE – compiled everyone else’s data
(a) Rosalind Franklin Franklin’s X-ray diffractionPhotograph of DNA
(b)Double helix
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?4. What experiment did Watson & Crick do?
- NONE – compiled everyone else’s data- Determined double helix from Rosalind Franklin’s data
Purine + Purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: widthConsistent with X-ray data
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?4. What experiment did Watson & Crick do?
- NONE – compiled everyone else’s data- Determined double helix from Rosalind Franklin’s data
N H O CH3
N
N
O
N
N
N
N H
Sugar
Sugar
Adenine (A) Thymine (T)
N
N
N
NSugar
O H N
H
NH
N OHH
N
Sugar
Guanine (G) Cytosine (C)
O
–O O
OHP
H2C
O
–O O
OP
H2C
O
–O O
OP
H2C
O
–OO
OP
O
OH
O
O
OT A
C
GC
A T
O
O
O
O
OH
CH2
O O–
OOP
CH2
OO–
OOP
CH2
OO–
OO
P
CH2
OO–
OOP
5 end
Hydrogen bond3 end
5 end
3 end
C
T
A
A
T
CG
GC
A T
C G
AT
AT
A T
TA
C
TA0.34 nm
3.4 nm
(a) Key features of DNA structure (b) Partial chemical structure (c) Space-filling model
G
1 nm
G
H2C
G
Figure 16.7 The double helix
DNA is antiparallelHydrogen bonds hold double helix togetherNow that we know how DNA is shaped & how it fits together…..
Announcements- Test corrections
- Stapled behind- Place in box
- Canned food – no later than Thursday- Pie Festival – for Ch 16 – 18 test (instead of cans)
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?4. What experiment did Watson & Crick do? 5. How does DNA replicate itself? The basic concept
(a) The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.
(b) The first step in replication is separation of the two DNA strands.
(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.
(d) The nucleotides are connected to form the sugar-phosphate backbones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.
ACTAG
ACTAG
ACTAG
ACTAG
TGATC
TGATC
ACTAG
AC
T
A
G
TGATC
TGATC
TGATC
T
G
A
TC
But is replication done in a conservative, semi-conservative or dispersive manner?
Conservativemodel. The twoparental strandsreassociate after acting astemplates fornew strands,thus restoringthe parentaldouble helix.
(a)
Semiconserva-tive model.The two strandsof the parentalmolecule separate, and each functionsas a templatefor synthesis ofa new, comple-mentary strand.
(b)
Dispersivemodel. Eachstrand of bothdaughter mol-ecules containsa mixture ofold and newlysynthesizedDNA.
(c)
Parent cellFirstreplication
Secondreplication
Figure 16.10 Three alternative models of DNA replication
Conservative- Original strands return together
Semi-conservative- One original & one new strand
Dispersive- Original strands are dispersed through new strands
The Meselson – Stahl Experiment showed how DNA replicates.
Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15N. The bacteria incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with only 14N, the lighter, more common isotope of nitrogen. Any new DNA that the bacteria synthesized would be lighter than the parental DNA made in the 15N medium. Meselson and Stahl could distinguish DNA of different densities by centrifuging DNA extracted from the bacteria.
EXPERIMENT
The bands in these two centrifuge tubes represent the results of centrifuging two DNA samples from the flask in step 2, one sample taken after 20 minutes and one after 40 minutes.
RESULTS
Bacteriacultured inmediumcontaining15N
Bacteriatransferred tomediumcontaining14N
21
DNA samplecentrifugedafter 20 min(after firstreplication)
3 DNA samplecentrifugedafter 40 min(after secondreplication)
4Lessdense
Moredense
Figure 16.11 Does DNA replication follow the conservative, semiconservative, or dispersive model?
CONCLUSION Meselson and Stahl concluded that DNA replication follows the semiconservative model by comparing their result to the results predicted by each of the three models in Figure 16.10. The first replication in the 14N medium produced a band of hybrid (15N–14N) DNA. This result eliminated the conservative model. A second replication produced both light and hybrid DNA, a result that eliminated the dispersive model and supported the semiconservative model.
First replication Second replication
Conservativemodel
Semiconservativemodel
Dispersivemodel
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?4. What experiment did Watson & Crick do? 5. How does DNA replicate itself? 6. How is DNA replicated?
- Origin of replication - sequence of DNA
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?4. What experiment did Watson & Crick do? 5. How does DNA replicate itself? 6. How is DNA replicated?
- Origin- Elongation
- DNA polymerase- 500 bp/sec bacteria, 50 bp/sec in us- New base added to 3’end (-OH)- dNTPs provide energy
New strand Template strand5’ end 3’ end
Sugar A TBase
C
G
G
C
A
C
T
PP
P
OH
P P
5’ end 3’ end
5’ end 5’ end
A T
C
G
G
C
A
C
T
3’ end
Nucleosidetriphosphate
Pyrophosphate
2 P
OH
Phosphate
Figure 16.13 Incorporation of a nucleotide into a DNA strand
DNA polymeraseOH
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?4. What experiment did Watson & Crick do? 5. How does DNA replicate itself? 6. How is DNA replicated?
- Origin- Elongation- Dealing with antiparallel strands
Parental DNA
DNA pol Ill elongatesDNA strands only in the5 3 direction. 3
5
5
3
35
21
Okazakifragments
DNA pol III
Templatestrand
Leading strandLagging strand
32 1
Templatestrand DNA ligase
Overall direction of replication
One new strand, the leading strand,can elongate continuously 5 3as the replication fork progresses.
The other new strand, thelagging strand must grow in an overall3 5 direction by addition of shortsegments, Okazaki fragments, that grow5 3 (numbered here in the orderthey were made).
DNA ligase joins Okazakifragments by forming a bond betweentheir free ends. This results in a continuous strand.
2
3
1
4
Figure 16.14 Synthesis of leading and lagging strands during DNA replication
Overall direction of replication
3
3
3
35
35
35
35
35
35
35
3 5
5
1
1
21
12
5
5
12
35
DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1.
6 The lagging strand in this region is nowcomplete.
7
DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2.
5
After the second fragment is primed. DNA pol III adds DNAnucleotides until it reaches the first primer and falls off.
4
After reaching the next RNA primer (not shown), DNA pol III falls off.
3
DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment.
2
Primase joins RNA nucleotides into a primer.
1
Templatestrand
RNA primer
Okazakifragment
Figure 16.15 Synthesis of the lagging strand
Announcements- Learning log – available – greater percentage of “critical thinking” questions- People are hungry!!!!! – tardies????
Figure 16.16 A summary of bacterial DNA replicationOverall direction of replication
Helicase unwinds theparental double helix.
Molecules of single-strand binding proteinstabilize the unwoundtemplate strands.
The leading strand issynthesized continuously in the5 3 direction by DNA pol III.
Leadingstrand Origin of replication
Laggingstrand
Laggingstrand
LeadingstrandOVERVIEW
Leadingstrand
Replication fork
DNA pol III
Primase
PrimerDNA pol III Lagging
strand
DNA pol I DNA ligase
1
2 3
Primase begins synthesisof RNA primer for fifthOkazaki fragment.
4
DNA pol III is completing synthesis ofthe fourth fragment, when it reaches theRNA primer on the third fragment, it willdissociate, move to the replication fork,and add DNA nucleotides to the 3 endof the fifth fragment primer.
5 DNA pol I removes the primer from the 5 endof the second fragment, replacing it with DNAnucleotides that it adds one by one to the 3’ endof the third fragment. The replacement of thelast RNA nucleotide with DNA leaves the sugar-phosphate backbone with a free 3 end.
6 DNA ligase bondsthe 3 end of thesecond fragment tothe 5 end of the firstfragment.
7
Parental DNA
5
3
43
21
5
3
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?4. What experiment did Watson & Crick do? 5. How does DNA replicate itself? 6. How is DNA replicated?7. How is DNA repaired?
- Proofreading – mismatch repair – DNA polymerase fixes mistakes made during copying
- Excision repair
Nuclease
DNApolymerase
DNAligase
A thymine dimerdistorts the DNA molecule.1
Repair synthesis bya DNA polymerasefills in the missingnucleotides.
3
DNA ligase seals theFree end of the new DNATo the old DNA, making thestrand complete.
4
A nuclease enzyme cutsthe damaged DNA strandat two points and thedamaged section isremoved.
2
Figure 16.17 Nucleotide excision repair of DNA damage
Chapter 16: The Molecular Basis of Inheritance1. What evidence did Frederick Griffith have that DNA was the genetic material?2. How did Hershey & Chase determine that DNA was the genetic material?3. What was Chargaff’s contribution?4. What experiment did Watson & Crick do? 5. How does DNA replicate itself? 6. How is DNA replicated?7. How is DNA repaired?8. What happens at the end of chromosomes (telomere)?
- Recall DNA polymerase works 5’ → 3’ direction- After RNA primer is removed, chromosome is shortened
End of parentalDNA strands
Leading strandLagging strand
Last fragment Previous fragment
RNA primer
Lagging strand
Removal of primers andreplacement with DNAwhere a 3 end is available
Primer removed butcannot be replacedwith DNA because
no 3 end availablefor DNA polymerase
Second roundof replication
New leading strandNew lagging strand 5
Further roundsof replication
Shorter and shorterdaughter molecules
5
3
5
3
5
3
5
3
3
Figure 16.18 Shortening of the ends of linear DNA molecules
2009 Nobel Prize in Medicine - for the discovery of how chromosomes are protected by telomeres and the enzyme telomerase
Telomerase - active in germ line cells & newborns
to prevent shortening-extends telomere a bit-has its own RNA
- complementary to telomere- serves as primer