Griffith’s Experiments (1928) using Streptococcus...
Transcript of Griffith’s Experiments (1928) using Streptococcus...
The Molecular Basis of Inheritance
Griffith’s Experiments (1928) using Streptococcus pneumoniae and mice
1. Mice injected with live, infective S cells (control to show effect of S cells)
RESULT: Mice die. Live, infective S cells in their blood; shows that S cells are virulent.
2. Mice injected with live, noninfective R cells (control to show effect of R cells)
RESULT: Mice live. No live R cells in their blood; shows that R cells are nonvirulent.
Griffith’s Experiments, cont. 3. Mice injected with heat-killed S cells (control to show effect of dead S cells)
RESULT: Mice live. No live S cells in their blood; shows that live S cells are necessary to be virulent to mice.
4. Mice injected with heat-killed S cells plus live R cells
RESULT: Mice die. Live S cells in their blood; shows that living R cells can be converted to virulent S cells with some factor from dead S cells.
Hershey and Chase’s Experiments (1952)
Watson and Crick (1953)
! Deoxyribonucleic acid (DNA) molecule forms the genetic material of all living organisms
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Rosalind Franklin: Discovered DNA Structure through X-ray Diffraction
! Performed X-ray diffraction studies to identify the 3-D structure
• Discovered that DNA is helical • Using Maurice Wilkins’ DNA fibers,
discovered that the molecule has a diameter of 2 nm and makes a complete turn of the helix every 3.4 nm
Chargaff’s Rules
! Erwin Chargaff determined that • Amount of adenine = amount of thymine • Amount of cytosine = amount of guanine • Always an equal proportion of purines (A and G)
and pyrimidines (C and T)
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Nucleotide Subunits of DNA
! DNA is made of subunits called nucleotides
! Each nucleotide consists of a phosphate group, a deoxyribose and a nitrogenous base
! The nucleotides are covalently linked via the sugar-phosphate backbone
! Each strand of DNA has a 5’ and a 3’ end
Purin
es
Pyrim
idin
es
Adenine Guanine NH2 C C N N
N C
H N C
C H O
H
H O C N C
H N C NH2
H C H O
O C N C
H N C H3C
C H H O
O C N C
H N C H
C H
NH2 C C N N
N C
H N C
C H H
Nitrogenous Base
4´ 5´
1´ 3´ 2´
2 8 7 6
3 9 4 5
1 O P O–
–O
Phosphate group
Sugar
Nitrogenous base
O CH2
N N
O
N
NH2
OH in RNA Cytosine
(both DNA and RNA) Thymine
(DNA only) Uracil
(RNA only)
OH H in DNA
DNA Structure
! Phosphodiester bond • Bond between
adjacent nucleotides • Formed between the
phosphate group of one nucleotide and the 3! —OH of the next nucleotide
! The chain of nucleotides has a 5!-to-3! orientation
Base CH2
O
5´
3´
O P O
OH
CH2
–O O
C
Base O
PO4
Phosphodiester bond
DNA Structure, cont.
! Complementarity of bases
! A forms 2 hydrogen bonds with T
! G forms 3 hydrogen bonds with C
! Gives consistent diameter
A
H
Sugar
Sugar Sugar
Sugar
T
G C
N H
N O
H
CH3
H H N
N N H N N
N
H
H H
N O H H
H N
N H N
N H N N
Hydrogen bond
Hydrogen bond
DNA: Complimentary Base Pairing DNA Double Helix 2 nm
5' end
Distance between each pair of bases =0.34 nm
Each full twist of the DNA double helix =3.4 nm
Phosphate group
5-carbon sugar deoxyribose)
Nitrogenous base (guanine)
Hydrogen bond 3' end
! Two molecules of DNA are connected through hydrogen bonds between the nitrogenous bases.
! [A] = [T] and [G] = [C]
DNA Replication
! 3 possible models 1. Conservative model 2. Semiconservative model 3. Dispersive model
Meselson and Stahl Experiment, 1957
3 Replication Models
2nd replication
Parental DNA Replicated DNA
KEY
a. Semiconservative replication
1st replication
b. Conservative replication c. Dispersive replication
Meselson-Stahl Experiment (cont.)
1st replication
15N medium 14N medium
2nd replication
Meselson-Stahl Experiment (cont.)
15N–14N hybrid DNA
14N–14N (light) DNA
15N–15N (heavy) DNA
CsCl forms a density gradient during centrifugation, with the highest density at the bottom of the tube.
DNA molecules move to positions where their density equals that of the CsCl solution and form bands. Shown are the positions of differently labeled DNA molecules. Experimentally the bonds are detected by absorbance of UV light.
Meselson-Stahl Experiment (cont.)
15N–14N hybrid DNA
15N–15N (heavy) DNA
DNA from 15N medium
DNA after one replication in 14N
15N–14N hybrid DNA
DNA after two replications in 14N
14N–14N (light) DNA
Meselson-Stahl Experiment (cont.)
Semiconservative
Conservative
Dispersive
The results support the semiconservative model.
Matches results
Does not match results
Does not match results
Two replications in 14N 15N medium One replication in 14N
Model for semiconservative Replication
Direction of replication
Old
New
Complementary base pairing in the DNA double helix: A pairs with T, G pairs with C.
The two chains unwind and separate.
Each “old” strand is a template for the addition of bases according to the base-pairing rules.
The result is two DNA helices that are exact copies of the parental DNA molecule with one “old” strand and one “new” strand
Enzymes of DNA Replication
! Helicase unwinds the DNA
! Primase synthesizes RNA primer (starting point for nucleotide assembly by DNA polymerases)
! DNA polymerases assemble nucleotides into a chain, remove primers, and fill resulting gaps
! DNA ligase closes remaining single-chain nicks
Assembling a Complementary Chain DNA Replication: Leading and Lagging Strand Assembly
DNA Replication: Enzyme Activities
Unwinding enzyme (helicase)
Primase RNA primers
Replication fork
Overall direction of replication
1 Helicase unwinds the DNA, and primases synthesize short RNA primers.
RNA
Leading strand
Lagging strand DNA polymerase
DNA polymerase
RNA DNA
2 RNA primers are used as starting points for the addition of DNA nucleotides by DNA polymerases.
DNA Replication: The Lagging Strand
Primer being extended by DNA polymerase
DNA polymerase
Newly synthesized primer
DNA unwinds further, and leading strand synthesis proceeds continuously, while a new primer is synthesized on the lagging strand template and extended by DNA polymerase III.
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DNA polymerase
Nick Another type of DNA polymerase (I) removes the RNA primer, replacing it with DNA, leaving a nick between the newly synthesized segments.
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DNA Replication: The Lagging Strand (Okazaki Fragments)
DNA ligase Nick is closed by DNA ligase. 5
Lagging strand Leading strand
Newly synthesized primer
DNA polymerase
Primer being extended by DNA polymerase III
6 DNA continues to unwind, and the synthesis cycle repeats as before: continuous synthesis of leading strand and synthesis of a new segment to be added to the lagging strand.
Telomers shorten with each Replication
Telomerase adds Telomere Repeats Replication From Multiple Origins
Proofreading By DNA Polymerase Photorepair: A Specific Repair Mechanism
! For one particular form of damage caused by UV light
! Thymine dimers • Covalent link of adjacent
thymine bases in DNA
! Photolyase • Absorbs light in visible range • Uses this energy to cleave
thymine dimer
Excision Repair: A Non-Specific Repair Mechanism
! Damaged region is removed and replaced by DNA synthesis
! 3 steps 1. Recognition of damage 2. Removal of the
damaged region 3. Resynthesis using the
information on the undamaged strand as a template 32
! E. coli has 3 DNA polymerases • DNA polymerase I (pol I)
• Acts on lagging strand to remove primers and replace them with DNA
• DNA polymerase II (pol II) • Involved in DNA repair processes
• DNA polymerase III (pol III) • Main replication enzyme
• All 3 have 3!-to-5! exonuclease activity – proofreading • DNA pol I has 5!-to-3! exonuclase activity
Different DNA Polymerase Have Different Functions
Nucleosomes and Chromatin Fiber
Chromosome in
metaphase
DNA
2 nm
Nucleosome and DNA wound around core of 2 molecules each of H2A, H2B, H3, H4
Linker
10-nm chromatin fiber
Nucleosomes Linkers
H1
30-nm chromatin fiber
Chromatin fiber
Solenoid
Prokaryotic DNA Replication