Transcript Chapter 16 - History of DNA

OBJECTIVES
1.Understanding the molecular basis of
inheritance .
2.Describe nucleosomes and the levels of
DNA organization in eukaryotes.
3.Understand the basis of work that was
important in proving that DNA was in
fact the genetic material.
4.Structure and Function of DNA.
One (or more) of the choices for each question here is wrong, can you find which one that is?
1. Erwin Chargaff
a. Chargaff determined that all species have the same numbers of A, T, C, and G.
b. Chargaff used sperm from different species for his research.
c. Chargaff determined that the ratio of A:T and G:C is constant for an organism.
2. Linus Pauling
a. Pauling’s triple helix model acted as a catalyst for Watson and Crick.
b. Pauling worked with (Phoebus) Levene to develop a protein-based model of heredity.
c. Pauling developed a three-chain model of DNA.
3. Rosalind Franklin
a. Franklin’s X-ray patterns showed Watson and Crick that DNA is helical.
b. Franklin’s X-ray patterns showed that the diameter of DNA’s helix is constant.
c. Franklin showed that DNA’s phosphates must be on the outside.
d. Franklin crystallized white blood cells from pus.
e. Franklin and (Maurice) Wilkins both obtained X-ray crystallographic patterns of DNA.
4. Maurice Wilkins
a. Wilkins determined that DNA is crystalline.
b. Wilkins determined that protein carries the genetic code.
5. James Watson and Francis Crick
a. Watson and Crick determined how A and T, G and C, formed base pairs.
b. Watson and Crick flipped a coin to decide whose name went first (on the 1953 Nature paper).
c. Watson and Crick developed a triple helix model of DNA.
1. Erwin Chargaff
a. Chargaff determined that all species have the same numbers of A, T, C, and G. (X)
b. Chargaff used sperm from different species for his research.
c. Chargaff determined that the ratio of A:T and G:C is constant for an organism.
2. Linus Pauling
a. Pauling’s triple helix model acted as a catalyst for Watson and Crick.
b. Pauling worked with (Phoebus) Levene to develop a protein-based model of heredity. (X)
c. Pauling developed a three-chain model of DNA.
3. Rosalind Franklin
a. Franklin’s X-ray patterns showed Watson and Crick that DNA is helical.
b. Franklin’s X-ray patterns showed that the diameter of DNA’s helix is constant.
c. Franklin showed that DNA’s phosphates must be on the outside.
d. Franklin crystallized white blood cells from pus. (X)
e. Franklin and (Maurice) Wilkins both obtained X-ray crystallographic patterns of DNA.
4. Maurice Wilkins
a. Wilkins determined that DNA is crystalline.
b. Wilkins determined that protein carries the genetic code. (X)
5. James Watson and Francis Crick
a. Watson and Crick determined how A and T, G and C, formed base pairs.
b. Watson and Crick flipped a coin to decide whose name went first (on the 1953 Nature paper).
c. Watson and Crick developed a triple helix model of DNA. (X)
The Gift of Internet to
understanding DNA
What makes it?
How are the assembled?
How are they packed?
http://www.dnai.org/a/index.html
What would happen to the base pairing if an A is substituted with a C?
a) Strengthened
b) Weakened
c) No change
Why?
C
What would happen to the base pairing if in the previous question the T
is also substituted with a G?
a) Strengthened
b) Weakened
c) No change
Why?
G
C
Building a model – Match the arrows on the left with the attribute
that matches on the right
1) deoxyribose
to form
2) A, T, G, C (purine/pyrimidine)
associated with
3) Nucleotides
polymerize to make
4) DNA
are localized in
5) Genes
is a constituent of
6) Chromosome
Structure
Building a model (solution)
Behavior
• deoxyribose
• associated with
•
A, T, G, C (purine/pyrimidine)
• to form
•
Nucleotides
• polymerize to make
• DNA
• Genes
• Chromosome
• is a constituent of
• are localized in
Historical perspective
•
When was the model of the DNA doublehelix first proposed?
A.
B.
C.
D.
E.
10,000 BC.
Way back in the 1950’s, dude!
1970’s.
1920’s.
At the Red Dawn.
Size of the prokaryotic genome ranges
from 5,000,000 bases t0 50,000,000
bases
A representation in this
figure can promote
serious misconception
about chromosomes,
can you identify it?
http://www.youtube.com/watch?v=4PKjF7OumYo
http://www.youtube.com/watch?v=Xjq5eEslJhw
The Human Genetic Information
Humans have 23 kinds of chromosomes (n=23)
Chromosomal profile from a normal human cell
How many chromosomes are depicted? Explain.
Scientific History
• The march to understanding that DNA is the
genetic material
– T.H. Morgan (1908)
– Frederick Griffith (1928)
– Avery, McCarty & MacLeod (1944)
– Hershey & Chase (1952)
– Watson & Crick (1953)
– Meselson & Stahl (1958)
Genes are on chromosomes
1908 | 1933
• T.H. Morgan
– working with Drosophila (fruit
flies)
– genes are on chromosomes
– but is it the protein or the DNA
of the chromosomes that are
the genes?
20
What happened here?
live pathogenic
strain of bacteria
A.
mice die
live non-pathogenic
strain of bacteria
B.
mice live
heat-killed
pathogenic bacteria
C.
mice live
2005-2006
mix heat-killed
pathogenic &
non-pathogenic
bacteria
D.
mice die
The “Transforming Factor”
• Frederick Griffith
– Streptococcus pneumonia bacteria
• was working to find cure for pneumonia
– harmless live bacteria mixed with
heat-killed infectious bacteria causes
disease in mice
– substance passed from dead bacteria
to live bacteria = “Transforming
Factor”
1928
THE EXPERIMENT
If you
were to
do this
expt..
What
would
you need
to
consider?
And the conclusion of the experiment is……..
Avery, McCarty & MacLeod
Oswald Avery
Colin MacLeod
Maclyn McCarty
What is
labeled?
What is
labeled?
1952 | 1969
Confirmation of DNA as Transforming Agent
Martha Chase
Alfred Hershey
Chargaff Rules - http://www.youtube.com/watch?v=HvJlnujmYcg
• DNA composition
– varies from species to species
– all 4 bases not in equal quantity
– bases present in characteristic ratio
• humans:
A = 30.9%
T = 29.4%
G = 19.9%
C = 19.8%
1947
Thought experiment
Cytosine makes up 38% of the nucleotides in a sample
of DNA from an organism. What percent of the
nucleotides in this sample will be thymine?
A.
B.
C.
D.
E.
12
24
31
38
It cannot be determined from the information
provided.
1953 | 1962
Structure of DNA
• Watson & Crick
– developed double helix model of DNA
• other scientists working on question:
– Rosalind Franklin
– Maurice Wilkins
– Linus Pauling
Franklin
2005-2006
Wilkins
Pauling
Watson and Crick
2005-2006
Rosalind Franklin (1920-1958)
2005-2006
Models of DNA Replication
1958
• Meselson & Stahl
– label nucleotides of “parent” DNA strands with
heavy nitrogen = 15N
– label new nucleotides with lighter isotope = 14N
– Would this work if they had used any other isotope? If
so, what and why? Would it be better or worse?
parent
replication
1958
A space probe returns with a culture of a microorganism found
on a distant planet. Analysis shows that it is a carbonbased life form that has DNA. You grow the cells in 15N
medium for several generations and then transfer it to 14N
medium. Which pattern in this figure would you expect if
the DNA were replicated in a conservative manner?
a.
b.
c.
d.
e.
DNA Replication
Replication
• Unwind DNA
– helicase enzyme
• unwinds part of DNA helix
• stabilized by single-stranded binding proteins
single-stranded binding proteins
Energy of Replication
• The nucleotides arrive as nucleosides
– DNA bases with P–P–P
– DNA bases arrive with their own energy source for
bonding
– bonded by DNA polymerase III
ATP
GTP
TTP
2005-2006
CTP
3'
Adding bases
– can only add
nucleotides to
3 end of a
growing DNA
strand
– strand grow
5'3’
DNA
P III
3'
leading strand
5'
Leading & Lagging strands
Leading strand
- continuous synthesis
Okazaki
Lagging strand
- Okazaki fragments
- joined by ligase
- “spot welder” enzyme
Okazaki fragments
Priming DNA synthesis
• DNA polymerase III can
only extend an existing
DNA molecule
– cannot start new one
• cannot place first base
– short RNA primer is built
first by primase
• starter sequences
• DNA polymerase III can
now add nucleotides to
RNA primer
Cleaning up primers
DNA polymerase I
removes sections of
RNA primer and
replaces with DNA
nucleotides
Replication fork
DNA
polymerase I
DNA
polymerase III
lagging strand
Okazaki
fragments
5’
3’
ligase
primase
5’
SSB
3’
DNA
polymerase III
5’
3’
leading strand
direction of replication
3’
5’
helicase
And in the end…
• Ends of
chromosomes are
eroded with each
replication
– an issue in aging?
– ends of
chromosomes are
protected by
telomeres
Telomeres
• Expendable,
non-coding sequences at
ends of DNA
– short sequence of bases
repeated 1000s times
– TTAGGG in humans
• Telomerase enzyme in
certain cells
– enzyme extends telomeres
– prevalent in cancers
• Why?
DNA polymerases
• DNA polymerase III
– 1000 bases/second
– main DNA building enzyme
• DNA polymerase I
– 20 bases/second
– editing, repair & primer removal
DNA polymerase III enzyme
Editing & proofreading DNA
• 1000 bases/second =
lots of typos!
• DNA polymerase I
– proofreads & corrects typos
– repairs mismatched bases
– excises abnormal bases
• repairs damage throughout
life
– reduces error rate from
1 in 10,000 to
1 in 100 million bases
Fast & accurate! – How and Why?
• It takes E. coli <1 hour to copy
5 million base pairs in its single
chromosome
– divide to form 2 identical daughter cells
• Human cell copies its 6 billion bases &
divide into daughter cells in only few hours
– remarkably accurate
– only ~1 error per 100 million bases
– ~30 errors per cell cycle
Damages can result in the inclusion of an
incorrect nucleotide to produce a mutation.
Mutagens (either chemical or radiation)
include
1) base analogues (similar in structure to the
normal bases and can become
incorporated into DNA);
2) base-modifying agents (which can change
a base) and
3) intercalating agents (cause insertions and
deletions).
Ultraviolet (UV) radiation (sunlight) can cause
pyrimidine dimer formation (such as
covalently linked thymines) block replication
and transcription.
Ionizing radiation (such as X-rays) knock
electrons off of biomolecules to generate
highly reactive intermediates that causes all
sorts of DNA damage.
How
• Three major
DNA repairing
mechanisms:
– base excision,
– nucleotide
excision and
– mismatch
repair
Base
excision
Nucleotide
excision
Mismatch
repair
CAN ANYBODY NOTICE ANY PROBLEMS HERE???????????