History for the Discovery of DNA

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History for the Discovery of DNA

• Chapter 16
• The Molecular Basis of Inheritance

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Next Unit Chapter 16 DNA History,
Structure ReplicationChapter 17 Genetic
Expression (protein synthesis) Chapter 18
Viruses Bacteria (selected parts) Chapter
19 Regulation (selected parts)Chapter 20
Genetic Engineering Biotechnology
3
Overview of Chapter 16

• TOPIC Pgs.
• History Discovery of DNA 293-296
• as Genetic Material
• Structure of DNA 296-298
• DNA Replication 298-307

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Introductory Questions (1)

• What was the significance of Griffiths
  Experiment in 1928?
• Give three reasons why Neurospora was in genetic
  studies to discover the one gene, one enzyme
  principle?
• What did James Sumner purify in 1926?
• How was Avery, MacLoed, and McCarty work
  different from Griffiths?
• Matching
• Garrod A. Urease
• Griffith B. T2 Bacteriophage
• Beadle Tatum C. Alkaptonuria
• Sumner D. Neurospora
• Hershey Chase E. Transformation Principle

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Key Questions Explored in this Next unit

• What are Genes made of?
• How do Genes work?
• How can information be stored, retrieved, and
  modified over time?
• What keeps this molecule so stable?
• Why is DNA and not protein responsible for the
  inheritance of genetic traits?

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Key Discoveries

• Miescher (isolated nuclein from soiled
  bandages) 1869
• Garrod (Proteins inborn errors) 1902
• Sutton (Chromosome structure) 1903
• Morgan (Gene mapping) 1913
• Sumner (Purified Urease, showed it to be an
  enzyme) 1926
• Griffiths Experiment (Transforming
  Principle) 1928
• Avery, McCarty, and Macleod 1944
• Chargaff (Base pairing species specific) 1947
• Hershey and Chase 1952
• Pauling, Wilkins, and Franklin 1950s
• Watson and Crick 1953

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Discovery of DNA

• 1868 Miescher first isolated deoxyribonucleic
  acid, or DNA, from cell nuclei

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Fredrick Griffith (1928)

• First suggestion that about what genes are made
  of.
• Worked with
• 1) Two strains of Pneumococcus bacteria
• Smooth strain (S) Virulent
  (harmful)
• Rough strain (R)
  Non-Virulent
• 2) Mice-were injected with these strains of
  bacteria and watched to see if the survived.
• 3) Four separate experiments were done
• -injected with rough strain (Lived)
• -injected with smooth strain (Died)
• -injected with smooth strain that was heat
  killed (Lived)
• -injected with rough strain heat killed
  smooth (????)

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Griffiths Experiment-1928
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Conclusion of Griffiths Experiment

• Somehow the heat killed smooth bacteria changed
  the rough cells to a virulent form.
• These genetically converted strains were called
  Transformations
• Something (a chemical) must have been transferred
  from the dead bacteria to the living cells which
  caused the transformation
• Griffith called this chemical a Transformation
  Principle

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Avery, MacLeod, and McCarty (1944)

• Chemically identified Griffiths transformation
  principle as DNA
• Separated internal contents of the S cells into
  these fractions
• (lipids, proteins, polysaccharides, and nucleic
  acids)
• They tested each fraction to see if it can cause
  transformation to occur in R cells to become S
  cells.
• Only the nucleic acids caused the transformation
• This was the first concrete evidence that DNA is
  the genetic material.
• Some were not completely convinced because they
  were not sure if this was true for eukaryotes.

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Next Breakthrough came from the use of Viruses

• Viruses provided some of the earliest evidence
  that genes are made of DNA
• Molecular biology studies how DNA serves as the
  molecular basis of heredity
• Only composed of DNA and a protein shell

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Various Types of Viruses
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T2 Bacteriophage
15

• Phage reproductive cycle

Phage attaches to bacterial cell.
Phage injects DNA.
Phage DNA directs host cell to make more phage
DNA and protein parts. New phages assemble.
Cell lyses and releases new phages.
Figure 10.1C
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A Typical Bacteriophage
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Alfred Hershey Martha Chase (1952)

• Worked with T-2 Bacteriophages
• Infected Escherchia coli (E. coli) Host cell
• Used Radioactive Isotopes
• (S35) Sulfur-35
• (P32) Phosphorus-32
• Why did they use these particular isotopes?
• Sulfur is found in proteins and not in DNA
• Phosphorus is found in DNA but not in protein

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Labeling of Virus Structures
19
Details of the Hershey Chase Experiment
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• The Hershey-Chase Experiment

Agitate in a blender to separate phages outside
the bacteria from the cells and their contents.
Centrifuge the mixture so bacteria form a pellet
at the bottom of the test tube.
Measure the radioactivity in the pellet and
liquid.
Mix radioactivelylabeled phages with bacteria.
The phages infect the bacterial cells.
1
2
3
4
Radioactiveprotein
Emptyprotein shell
Radioactivityin liquid
Phage
Bacterium
PhageDNA
DNA
Batch 1Radioactiveprotein
Centrifuge
Pellet
RadioactiveDNA
Batch 2RadioactiveDNA
Centrifuge
Radioactivityin pellet
Pellet
Figure 10.1B
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Video clip of Hershey Chase Experiment

• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter14/animations.html
• Key findings the phage DNA entered in the host
  cell and when these cells were returned to the
  culture medium the infection ran its course
  producing E.coli and other bacteriophages with
  the radioactive phosphorus. (pg. 298)

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DNA is a Double-Stranded Helix

• James Watson and Francis Crick worked out the
  three-dimensional structure of DNA, based on work
  by Rosalind Franklin

Figure 10.3A, B
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Rosalind Franklins Image (pg. 297)

• and Media 

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DNA and RNA are polymers of Nucleotides

• DNA is a nucleic acid, made of long chains of
  nucleotides

Phosphate group
Nitrogenous base
Nitrogenous base(A, G, C, or T)
Sugar
Phosphategroup
Nucleotide
Thymine (T)
Sugar(deoxyribose)
DNA nucleotide
Figure 10.2A
Polynucleotide
Sugar-phosphate backbone
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• DNA has four kinds of bases, A, T, C, and G

Thymine (T)
Cytosine (C)
Adenine (A)
Guanine (G)
Pyrimidines
Purines
Figure 10.2B
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DNA Maintains a Uniform Diameter
See pg. 298
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DNA Bonding

• Purines A G
• Pyrimidines C T (Chargaff rules)
• A H bonds (2) with T and C H bonds (3)
  with G
• Van der Waals attractions between the stacked
  pairs

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• RNA is also a nucleic acid

• RNA has a slightly different sugar
• RNA has U instead of T

Nitrogenous base(A, G, C, or U)
Phosphategroup
Uracil (U)
Sugar(ribose)
Figure 10.2C, D
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• Hydrogen bonds between bases hold the strands
  together

• Each base pairs with a complementary partner
• A pairs with T
• G pairs with C

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DNA Structure

• Chargaff ratio of nucleotide bases (AT CG)
• Watson Crick (Wilkins, Franklin)
• The Double Helix
• v nucleotides nitrogenous base (thymine,
  adenine, cytosine, guanine) sugar deoxyribose
  phosphate group

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• Three representations of DNA

Hydrogen bond
Ribbon model
Partial chemical structure
Computer model
Figure 10.3D
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• Each strand of the double helix is oriented in
  the opposite direction

5? end
3? end
P
P
P
P
P
P
P
P
3? end
5? end
Figure 10.5B
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DNA Replication History Discovery

• First model suggested by Watson Crick
• Three models were proposed
• -Semiconservative (half old half new)
• -Conservative (old strands remain
  together)
• -Dispersive (random mixture)
• Heavy isotopic nitrogen (N-15) was used to label
  the nitrogenous bases in the DNA
• Density gradient centrifugation was used
• DNA was mixed with Cesium chloride (CsCl)

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Three Proposed Models of DNA Replication

• 
  
  

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Meselson Stahls Experiment
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Meselson-Stahl Experiment
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Meselson Stahl Experiment (Pg. 300)

• Grew E. coli on a medium containing isotopic
  Nitrogen (15N) in the form of NH4Cl
• Nitrogenous bases incorporated the isotopic
  nitrogen
• DNA was extracted from the cells
• Density gradient centrifugation was used on the
  DNA to determine the banding region of the heavy
  isotopic nitrogen.
• The rest of the bacteria was then grown on a
  medium containing normal nitrogen and allowed to
  grow.

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Meselson Stahl Experiment contd.

• The newly synthesized strands of DNA were
  expected to have the lighter normal nitrogen in
  their bases.
• The older original strands were labeled with the
  heavier isotopic nitrogen.
• Two generations were grown in order to rule out
  the conservative and dispersion models.

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Introductory Questions (1)

• What was the significance of Griffiths
  Experiment in 1928?
• How was Avery, MacLoed, and McCarty work
  different from Griffiths?
• How was the dispersive model conservative
  models ruled out as the way in which DNA
  replicates?
• Matching
• Meselson Stahl A. X-ray diffraction
• Griffith B. T2 Bacteriophage
• Franklin Wilkins C. Semiconservative model
• Chargaff D. Base pairing C-G T-A
• Hershey Chase E. Transformation Principle

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Introductory Questions 2

1. Briefly explain what density gradient
   centrifugation is and what it is used for.
2. Name the organism used by Meselson Stahl to
   label the DNA.
3. Name all of the enzymes required for DNA
   replication to occur and what purpose they serve.
4. In what direction is the newly synthesized strand
   made? What end of the old strand do the
   nucleotides add to?
5. What direction is the new strand growing?
   (towards or away from the replication fork)
6. How long ( nucleotides) are the Okasaki
   fragments? How long are the RNA primers?

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• The structure of DNA consists of two
  polynucleotide strands wrapped around each other
  in a double helix

1 chocolate coat, Blind (PRA)
Figure 10.3C
Twist
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DNA replication depends on specific base pairing

• In DNA replication, the strands separate
• Enzymes use each strand as a template to assemble
  the new strands

A
A
Nucleotides
Parental moleculeof DNA
Both parental strands serveas templates
Two identical daughtermolecules of DNA
Figure 10.4A
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• Untwisting and replication of DNA

Figure 10.4B
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Anti-parallel Structure of DNA
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Antiparallel nature

• 5 end corresponds to the Phosphate end
• 3 end corresponds to the OH sugar
• Replication runs in BOTH directions
• One strand runs 5 to 3 while the other runs
  3 to 5
• Nucleotides are added on the 3 end of the
  newly synthesized strand
• The new DNA strand forms and grows in the
  5 ? 3 direction only

46
How a Nucleotides adds to the old Strand
5 end
3 end
5 end
47
Building New Strands of DNA

• Each nucleotide it a triphosphate
• (GTP, TTP, CTP, and ATP)
• Nucleotides only add to the 3 end of the growing
  strand (never on the 5 end)
• Two phosphates are released (exergonic) and the
  energy released drives the polymerization
  process.

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Origin of replication (bubbles) beginning of
replication (pg. 301)
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Key Enzymes Required for DNA Replication (pg.
303-304)

• Helicase - catalyzes the untwisting of the DNA at
  the replication fork
• DNA Polymerase - catalyzes the elongation of new
  DNA and adds new nucleotides on the 3 end the
  growing strand.
• SSBPs - single stranded binding proteins,
  prevents the double helix from reforming
• Topoisomerase Breaks the DNA strands and
  prevents excessive coiling
• RNA primase synthesizes the RNA primers and
  starts the replication first by laying down a few
  nucleotides initially.
• DNA primase will get replaced by DNA polymerase

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RNA Primers

• Initiates the Replication process and begins the
  building of the newly formed strands.
• Laid down by RNA primase
• Consists of 5 to 14 nucleotides
• Synthesized at the point where replication begins
• Will be laid down on both template strands of the
  DNA

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3?
DNA polymerasemolecule
5?

• How DNA daughter strands are synthesized

5? end
Daughter strandsynthesizedcontinuously
Parental DNA
5?
3?
Daughter strandsynthesizedin pieces
3?
P
5?

• The daughter strands are identical to the parent
  molecule

5?
P
3?
DNA ligase
Overall direction of replication
Figure 10.5C
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Laying Down RNA Primers
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DNA Replication-New strand Development

• Leading strand synthesis is toward the
  replication fork
• (only in a 5 to 3 direction from the 3
  to 5 master strand)
• -Continuous
• Lagging strand synthesis is away from the
  replication fork
• -Only short pieces are made called Okazaki
  fragments
• - Okazaki fragments are 100 to 2000 nucleotides
  long
• -Each piece requires a separate RNA primer
• -DNA ligase joins the small segments together
• (must wait for 3 end to open again in a
  5 to 3 direction)
• View video clip
• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter14/animations.html

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DNA Replication Fork
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Video Clip of DNA Replication

• http//highered.mcgraw-hill.com/sites/0072437316/s
  tudent_view0/chapter14/animations.html

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Prokaryotic vs Eukaryotic Replication

• Prokaryotes
• Circular DNA (no free ends)
• Contains 4 x 106 base pairs (1.35 mm)
• Only one origination point
• Eukaryotes
• -Have free ends
• -Contains 3 x 109 base pairs (haploid cells) 1
  meter
• -Lagging strand is not completely replicated
• -Small pieces of DNA are lost with every cell
  cycle
• -End caps (Telomeres) protect and help to retain
  the genetic information

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Issues with Replication

• Prokaryotes (ex. E. coli)
• Have one singular loop of DNA
• E. coli has approx. 4.6 million Nucleotide base
  pairs
• Rate for replication 500 nucleotides per second
• Eukaryotes w/Chromosomes
• Each chromosome is one DNA molecule
• Humans (46) has approx. billion base pairs
• Rate for replication 50 per second (humans)
• Errors
• Rate is one every 10 billion nucleotides copied
• Proofreading is achieved by DNA polymerase (pg.
  305)

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Telomeres

• Short, non-coding pieces of DNA
• Contains repeated sequences (ie. TTGGGG 20
  times)
• Can lengthen with an enzyme called Telomerase
• Lengthening telomeres will allow more
  replications to occur.
• Telomerase is found in cells that have an
  unlimited number of cell cycles (commonly
  observed in cancer cells)
• Artificially giving cells telemerase can induce
  cells to become cancerous
• Shortening of these telomeres may contribute to
  cell aging and Apotosis (programmed cell death)
• Ex. A 70 yr old persons cells divide approx.
  20-30X vs an infant which will divide 80-90X

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Telomeres
60
Chapter 17
61
James Sumner (1926)

• Isolated the enzyme Urease
• First to identify an enzyme as a protein
• First to crystallize an enzyme
• Awarded the Nobel prize in 1946 in chemistry for
  his crystallization of an enzyme.

62
Archibald Garrod (1902-1908)

• Studied a rare genetic disorder Alkaptonuria
• Thought to be a recessive disorder
• Tyrosine is not broken down properly into carbon
  dioxide and water.
• An Intermediate substance Homogentisic acid
  accumulates in the urine turning it BLACK when
  exposed to air.
• An enzyme was thought to be lacking
• A genetic mutation was thought to be the cause
  An Inborn Error of Metabolism

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Metabolic Pathway for the breakdown of Tyrosine

• Tyrosine
• ?
• Hydroxyphenylpyruvate
• ?
• Homogentisic acid
• Alkaptonuria Maleyacetoacetate
• (Inactive enzyme) (active ? enzyme)
• CO2 H2O

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Garrods Conclusion

• A mutation in a specific gene is associated with
  the absence of a specific enzyme.
• Led to the idea of
• One gene, One Enzyme
• Not validated until Beadle Tatums work in the
  1940s with Neurospora (breadmold)

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George Beadle EdwardTatum (1940s)

• Discovered the One Gene, One Enzyme Principle
• Analyzed mutations that interfered with a known
  metabolic pathway
• Organism they chose to work with Neurospora
  (breadmold)
• -Grows easily
• -Grows as a haploid (no homologs)
• -Mutants are easily identified Dominant allele
  wont be expressed
• Neurospora can grow easily in only salt, sugar,
  Biotin

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George Beadle EdwardTatum (1940s) contd

• Mutants-are unable to make certain organic
  molecules amino acids, lipids, etc.
• These substances are added to the media which
  will allow mutants to grow successfully
• Exposed the haploid spores to x rays UV to
  induce mutations
• Haploid spores were crossed, grown in a variety
  of media to determine what kind of mutation was
  occurring
• They examined the effect of the mutation
  instead of identifying the enzyme.

67
Beadle Tatums Conclusion

• One Gene affects One Enzyme
• Later ? Revised
• One Gene affects One Protein
• Later ? Revised
• One Gene affects One Polypeptide Chain

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Suggestions on how to Review

• Make a List of all Bold Terms (See summaries)
• Make a list of key people generate a timeline
• Answer all MC questions at end of each chapter
• Review all your Quizzes from textbook website
• Review all the MC Questions from your study
  guides
• Look at all the key figures diagrams discussed
• Review all Tables from the four chapters
• Re-Look at the Powerpoint Pres. From my website.
  Think back to what was emphasized
• Anticipate questions to be asked
• Make an outline of all chapters connect the
  concepts discussed