Download Lecture #7 Date ______

Summary of Results –Due Fri 11/30
• Title
• Introduction/Background (historical context)
• Separation of DNA: Explain the process
-Why does this technique work?
-Why do you need salt, shampoo, & alcohol?
• Results: Explain what you observed
– Describe what you saw
– How was the Banana DNA different from your DNA?
– How could you verify that what you have is DNA?
• Conclusion/Evaluation:
– How could this lab activity be improved? (Sources of error)
– What else could be used? Other techniques?
History for the Discovery of
DNA
• Chapter 16
The Molecular
Basis of Inheritance
Next Unit:
**Chapter 16: DNA: History, Structure & Replication
**Chapter 17: Genetic Expression (protein synthesis)
Chapter 18: Viruses & Bacteria (selected parts)
Chapter 19: Regulation (selected parts)
**Chapter 20: Genetic Engineering & Biotechnology
Overview of Chapter 16:
TOPIC
History & Discovery of DNA
as Genetic Material
Pgs.
293-296
Structure of DNA
DNA Replication
296-298
298-307
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?
Introductory Questions (#1)
1) How long have we known about the existence of
DNA? Who was the first to isolate it?
2) Why are bacteria and viruses so important to our
discovery of identifying DNA as our genetic
material?
3) What was the significance of Griffith’s Experiment
in 1928?
4) What did James Sumner purify in 1926?
5) How was Avery, MacLeod, and McCarty work
different from Griffith’s? Why was their work still
met with criticism? See pg. 294
Key Discoveries
•
•
•
•
•
•
•
•
•
•
•
Miescher (isolated “nuclein” from soiled bandages)
Garrod (Proteins & inborn errors)
Sutton (Chromosome structure)
Morgan (Gene mapping)
Sumner (Purified Urease, showed it to be an enzyme)
Griffith’s Experiment (Transforming Principle)
Avery, McCarty, and Macleod
Chargaff (Base pairing & species specific)
Hershey and Chase
Pauling, Wilkins, and Franklin
Watson and Crick
1869
1902
1903
1913
1926
1928
1944
1947
1952
1950’s
1953
Discovery of DNA
• 1868: Miescher first isolated
deoxyribonucleic acid, or DNA, from cell
nuclei
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
-injected with smooth strain
-injected with smooth strain that was heat killed
-injected with rough strain & heat killed smooth
(Lived)
(Died)
(Lived)
(????)
Griffith’s Experiment-1928
Conclusion of Griffith’s 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”
Avery, MacLeod, and McCarty
(1944)
• Chemically identified Griffith’s 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.
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
Various Types of Viruses
T2 Bacteriophage
• 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
A Typical Bacteriophage
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
Labeling of Virus Structures
Details of the Hershey & Chase Experiment
• The Hershey-Chase Experiment
1
Mix radioactively
labeled phages with
bacteria. The phages
infect the bacterial
cells.
Phage
2
Agitate in a blender
to separate phages
outside the bacteria
from the cells and
their contents.
Radioactive
protein
Bacterium
3
Centrifuge the mixture
so bacteria form a
pellet at the bottom of
the test tube.
4
Empty
protein shell
Measure the
radioactivity in
the pellet and
liquid.
Radioactivity
in liquid
Phage
DNA
DNA
Batch 1
Radioactive
protein
Centrifuge
Pellet
Batch 2
Radioactive
DNA
Figure 10.1B
Radioactive
DNA
Centrifuge
Pellet
Radioactivity
in pellet
Video clip of Hershey Chase
Experiment
•
http://highered.mcgraw-hill.com/sites/0072437316/student_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)
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
Rosalind Franklin’s Image (pg. 297)
• and Media
Video #1 DNA: The Blueprint of Life
1. Name the technology used in the movie Jurassic
Park.
2. Where did Meissner extract the “nuclein” material
that later was identified as DNA?
3. How did Hershey & Chase separate the virus from
its bacterial host? How did they trace (track) the
DNA and protein?
4. What did x-ray crystallography reveal about DNA?
5. What purpose do enzymes serve in the replication
process?
Segment #2:
**Need Five key Statements for the segment
Introductory Questions (#1)
1) How long have we known about the existence of
DNA? Who was the first to isolate it?
2) Why are bacteria and viruses so important to our
discovery of identifying DNA as our genetic
material?
3) What was the significance of Griffith’s Experiment
in 1928?
4) What did James Sumner purify in 1926?
5) How was Avery, MacLeod, and McCarty work
different from Griffith’s? Why was their work still
met with criticism? See pg. 294
DNA and RNA are polymers of
Nucleotides
• DNA is a nucleic acid, made of long chains
of nucleotides
Phosphate
group
Nitrogenous
base
Sugar
Phosphate
group
Nitrogenous base
(A, G, C, or T)
Nucleotide
Thymine (T)
Sugar
(deoxyribose)
DNA nucleotide
Polynucleotide
Sugar-phosphate backbone
Figure 10.2A
• DNA has four kinds of bases, A, T, C, and G
Thymine (T)
Cytosine (C)
Pyrimidines
Adenine (A)
Guanine (G)
Purines
Figure 10.2B
DNA Maintains a Uniform Diameter
See pg. 298
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
• 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)
Phosphate
group
Uracil (U)
Sugar
(ribose)
Figure 10.2C, D
• Hydrogen bonds between bases hold the
strands together
– Each base pairs with a complementary partner
– A pairs with T
– G pairs with C
DNA Structure
• Chargaff
ratio of nucleotide
bases (A=T; C=G)
• Watson & Crick
(Wilkins, Franklin)
• The Double Helix
√ nucleotides: nitrogenous
base (thymine, adenine,
cytosine, guanine); sugar
deoxyribose; phosphate group
• Three representations of DNA
Hydrogen bond
Ribbon model
Partial chemical structure
Computer model
Figure 10.3D
3 end
P
P
P
P
P
P
P
• Each strand of the
double helix is
oriented in the
opposite direction
5 end
P
Figure 10.5B
3 end
5 end
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)
Video #1 DNA: The Blueprint of Life
Name the technology used in the movie Jurassic Park.
Where did Meissner extract the “nuclein” material that later was identified as
DNA?
How did Hershey & Chase separate the virus from its bacterial host? How
did they trace (track) the DNA and protein?
What did x-ray crystallography reveal about DNA?
What purpose do enzymes serve in the replication process?
Segment #2:
Name the disorder that Andrew and his sister inherited. What were the
major symptoms of this disorder?
How can this genetic defect be treated? Name the gene that is defective.
How can a gene be transported and carried to a cell?
What is a vector? Give an example.
What purpose do restriction enzymes serve? What about ligase?
What does PCR stand for?
Segment #3:
What is the first step of gene therapy?
How long would all of the DNA contained in all of the chromosomes in a
human cell be if they were connected end to end?
Which chromosome consists of 5% of all the genes in the human genome?
Introductory Questions (#1)
1)
2)
What was the significance of Griffith’s Experiment in
1928?
Give three reasons why Neurospora was in genetic
studies to discover the “one gene, one enzyme” principle?
(See Chapter 17 also)
3)
4)
5)
What did James Sumner purify in 1926?
How was Avery, MacLoed, and McCarty work different
from Griffith’s?
Matching:
Garrod (ch. 17)
A. Urease
Griffith
B. T2 Bacteriophage
Beadle & Tatum (ch. 17)
C. Alkaptonuria
Sumner
D. Neurospora
Hershey & Chase
E. Transformation Principle
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?
Three Proposed Models of DNA Replication
Meselson & Stahl’s Experiment
Meselson-Stahl Experiment
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.
Meselson & Stahl Experiment cont’d.
• 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.
• The structure of DNA consists of two
polynucleotide strands wrapped around
each other in a double helix
1 chocolate coat,
Blind (PRA)
Twist
Figure 10.3C
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
Nucleotides
Parental molecule
of DNA
Both parental strands serve
as templates
Two identical daughter
molecules of DNA
Figure 10.4A
• Untwisting and replication of DNA
Figure 10.4B
Anti-parallel Structure of DNA
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
How a Nucleotides adds to the old
Strand
5’ end
3’ end
5’ end
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.
Origin of replication (“bubbles”):
beginning of replication (pg. 301)
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.
• SSBP’s - 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
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
• How DNA
daughter
strands are
synthesized
• The
daughter
strands are
identical to
the parent
molecule
DNA polymerase
molecule
5 end
Daughter strand
synthesized
continuously
Parental DNA
5
3
Daughter
strand
synthesized
in pieces
3
5
P
5
3
3
5
P
DNA ligase
Overall direction of replication
Figure 10.5C
Laying Down RNA Primers
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/student_view0/chapter14/animations.html#
DNA Replication Fork
Video Clip of DNA Replication
•
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#
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
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)
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 person’s cells divide approx. 20-30X vs an infant
which will divide 80-90X
Telomeres
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?
Chapter 17
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.
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”
Metabolic Pathway for the
breakdown of Tyrosine
Tyrosine
↓
Hydroxyphenylpyruvate
↓
Homogentisic acid
Alkaptonuria
(Inactive enzyme)
Maleyacetoacetate
(active ↓ enzyme)
CO2 & H2O
Garrod’s 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 & Tatum’s work
in the 1940’s with Neurospora (breadmold)
George Beadle & EdwardTatum
(1940’s)
• 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
won’t be expressed
• Neurospora can grow easily in only: salt, sugar, &
Biotin
George Beadle & EdwardTatum
(1940’s) cont’d
• 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.
Beadle & Tatum’s Conclusion
“One Gene affects One Enzyme”
Later  Revised
“One Gene affects One Protein”
Later

Revised
“One Gene affects One Polypeptide Chain”
Suggestions on how to Review
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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