Chapter 14 Flashcards
(37 cards)
Frederick Griffith – 1928
-Studied Streptococcus pneumoniae, a pathogenic bacterium causing pneumonia
-2- strains of Streptococcus
S strain is virulent
R strain is nonvirulent
-Griffith infected mice with these strains hoping to understand the difference between the strains
Griffith’s results
Live S strain cells killed the mice
Live R strain cells did not kill the mice
Heat-killed S strain cells did not kill the mice
Heat-killed S strain + live R strain cells killed the mice
Transformation
- Information specifying virulence passed from the dead S strain cells into the live R strain cells
- Our modern interpretation is that genetic material was actually transferred between the cells
Avery, MacLeod, & McCarty – 1944
- Repeated Griffith’s experiment using purified cell extracts
- Removal of all protein from the transforming material did not destroy its ability to transform R strain cells
- DNA-digesting enzymes destroyed all transforming ability
- Supported DNA as the genetic material
Hershey & Chase –1952
- Investigated bacteriophages
- Viruses that infect bacteria
- Bacteriophage was composed of only DNA and protein
- Wanted to determine which of these molecules is the genetic material that is injected into the bacteria
- Bacteriophage DNA was labeled with radioactive phosphorus (32P)
- Bacteriophage protein was labeled with radioactive sulfur (35S)
- Radioactive molecules were tracked
- Only the bacteriophage DNA (as indicated by the 32P) entered the bacteria and was used to produce more bacteriophage
- Conclusion: DNA is the genetic material
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DNA Structure
-DNA is a nucleic acid C-omposed of nucleotides -5-carbon sugar called deoxyribose -Phosphate group (PO4) -Attached to 5′ carbon of sugar -Nitrogenous base -Adenine, thymine, cytosine, guanine -Free hydroxyl group (—OH) -Attached at the 3′ carbon of sugar
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
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)
Rosalind Franklin
- 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
James Watson and Francis Crick – 1953
- Deduced the structure of DNA using evidence from Chargaff, Franklin, and others
- Did not perform a single experiment themselves related to DNA
- Proposed a double helix structure
Double helix
- 2 strands are polymers of nucleotides
- Phosphodiester backbone – repeating sugar and phosphate units joined by phosphodiester bonds
- Wrap around 1 axis
- Antiparallel
Complementary base pairing—critical
for DNA replication and gene expression.
-Complementarity of bases
A forms 2 hydrogen bonds with T
G forms 3 hydrogen bonds with C
Gives consistent diameter
DNA Replication
-3 possible models of replication Conservative model -Semiconservative model-Most important Dispersive model Meselson and Stahl – 1958
Meselson and Stahl – 1958
-Conservative model = rejected 2 densities were not observed after round 1 -Semiconservative model = supported Consistent with all observations 1 band after round 1 2 bands after round 2 -Dispersive model = rejected 1st round results consistent 2nd round – did not observe 1 band
DNA Replication
Requires 3 things Something to copy Parental DNA molecule Something to do the copying Enzymes Building blocks to make copy Nucleotide triphosphates
DNA replication includes
- Initiation – replication begins
- Elongation – new strands of DNA are synthesized by DNA polymerase
- Termination – replication is terminated
RNA polymerase makes primer
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DNA polymerase extends prime
DNA “reads” template strand from 3’5’
Nucleotides are added from 5’3’
-DNA polymerase has to add new bases to 3′
end of an existing strand
-Requires a primer of RNA laid down by RNA
polymerase (primase)
DNA polymerase
-Matches existing DNA bases with complementary nucleotides and links them
-Synthesize in 5′-to-3′ direction
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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
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-Helicases – use energy from ATP to unwind DNA
-Single-strand-binding proteins (SSBs) coat strands to keep them apart
-Unwinding DNA causes torsional strain
-Topoisomerase prevent supercoiling
DNA gyrase is used in replication
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DNA helicase breaks H bonds—unzips DNA
SSBs coat strands to keep them open
DNA primase (RNA polymerase) lays down RNA primer sequence
DNA polymerase III adds complementary nucleotides
DNA polymerase I removes RNA primer and replaces it with DNA nucleotides DNA ligase splices fragments together
Leading strand—single primer— continuous
Lagging strand—not continuous; multiple primers
Okazaki fragment spliced by DNA ligase
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