Structure
(chapter 10, pages 266 – 278)
and
Replication of DNA
(chapter 12, pages 318 – 334)
Dr. Ravi Palanivelu
Rpalaniv@ag.arizona.edu
http://ag.arizona.edu/research/ravilab/
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Transmission genetics
•
Previous discussions focused on the
individual.
1. Phenotype caused by an individual
genotype
2. Transmission of genes of an individual
organism to the next generation
3. Offspring produced by crossing two
individuals
Molecular genetics
• Focus will now shift to genes
• How are they encoded (Lecture 9)
• How are they replicated (Lecture 9)
• How are they expressed (genotype -> phenotype; (Lectures 10, 11)
• How do we study them (Lectures 12,
13)
What is the
hereditary/genetic material?
• Until the mid-20th century, most
scientists believed that proteins
must be the hereditary (genetic)
material
Why did they believe this?
• Nuclei from male and female reproductive
cells fuse during reproduction
– Nuclei contain chromatin
– Chromatin made-up of nuclein (DNA) and
protein
• Understood chromosome movements, and
that chromosomes were the vehicles of
inheritance
– Chromosomes made-up of nuclein (DNA) and
protein
How did they come to
this conclusion?
• The basic chemistry (the parts) of DNA
had been worked-out, but not the
structure (how the parts fit together)
– Thought it was a very simple repeating
molecule
– Thought DNA was structural in chromosomes
(scaffolding)
• Proteins are much more complex
– Could account for the complexity of cells
Important Dates in Determining the Structure of DNA
Miescher isolates
DNA from nuclei
Experiments that led to
determining the genetic material
• Frederick Griffith, 1928
– British
physician/bacteriololgist
– Streptococcus
pneumoniae
• Pneumonia in humans
• Septicemia in mice (lethal)
Frederick Griffith
Streptococcus pneumoniae
Used two strains
IIIS = smooth, virulent
• Enclosed in a polysaccharide capsule
• The colonies have a smooth appearance
IIR = rough, non-virulent
• Polysaccharide coat is missing
• Colonies have a rough appearance
Frederick Griffith
Injected mice with
1. Live IIIS strain only
2. Live IIR strain only
3. Heat-killed IIIS strain
4. Heat-killed IIIS strain + live IIR strain
Frederick Griffith
• Injected mice with
1. Live IIIS strain
• Mice died
• IIIS strain recovered from blood
Frederick Griffith
• Injected mice with
1. Live IIIS strain
• Mice died
• IIIS strain recovered from blood
2. Live IIR strain
• Mice lived
• Nothing recovered in blood
Frederick Griffith
• Injected mice with
1. Live IIIS strain
• Mice died
• IIIS strain recovered from blood
2. Live IIR strain
• Mice lived
• Nothing recovered in blood
3. Heat-killed IIIS strain
• Mice lived
• Nothing recovered in blood
Frederick Griffith
•
Injected mice with
4. Heat-killed IIIS strain + live IIR strain
– Expect the mice to live
– Recover nothing from the blood
Frederick Griffith
• Injected mice with
4. Heat-killed IIIS strain + live IIR strain
• Found mice died
– Live IIIS strain recovered from blood
Frederick Griffith
• Found
– Something in the dead (heat-killed) IIIS
strain transformed some of the live IIR
cells into live IIIS cells
– The idea was that this transforming
principle is the genetic material
Generalized
Bacterial/Prokaryotic
Cell
Experiments toward determining
the genetic material
• Oswald Avery, Colin MacLeod, and
Maclyn McCarty, 1944
– Wanted to find the transforming
(principle) agent from Griffith’s
experiment
– The transforming agent must be the
genetic (hereditary) material
Avery, MacLeod and McCarty
• Oswald Avery, Colin MacLeod, and
Maclyn McCarty, 1944
– Separated debris from dead S cells into
classes of molecules (DNA, RNA,
proteins, lipids, polysaccharides)
– Added each separately to live R cells to
see what happens
Avery, MacLeod and McCarty
• Oswald Avery, Colin MacLeod, and Maclyn
McCarty, 1944
– DNA was shown to be the transforming agent
– By using proteases and nucleases they were
able to show that there were no contaminating
proteins that could really be the transforming
agent
– Most people assumed that the transforming
agent and the genetic material were one and
the same, thus DNA was the genetic material
Practice problems
(8, structure and replication of DNA)
is due next Monday (4/5/01)
http://highered.mcgrawhill.com/olcweb/cgi/pluginpop.cg
i?it=swf::535::535::/sites/dl/free/
0072437316/120076/micro04.sw
f::DNA%20Replication%20Fork
Experiments toward determining
the genetic material
• Alfred Hershey and Martha Chase,
1952
– The experiment that convinced all the
others, most of whom were virologists,
that DNA is the genetic material
Hershey and Chase
• Alfred Hershey and
Martha Chase,
1952
– Worked with T2
virus
– A bacteriophage of
E. coli
T2 virus is madeup of
•A protein coat
•Surrounding a
DNA molecule
Alfred Hershey and
Martha Chase, 1952
•Knew that viruses
inject something into
the bacteria, then the
bacteria reproduce
new viruses
•Hypothesis: whatever
is injected into the
bacteria is the genetic
material
T2 virus is madeup of
•A protein coat
(contains sulfur)
•Surrounding a
DNA molecule
(contains
phosphorous)
Hershey and Chase
• T2 virus is made-up of a protein coat,
surrounding a DNA molecule
– Proteins contain sulfur
• Labeled the proteins with radioactive 35S
– DNA contains phosphorous
• Labeled DNA with radioactive 32P
Hershey and Chase
• Allowed the virus enough time to
infect the bacteria
– Sheared-off the viruses from the
bacteria in a Warring blender
– Looked to see whether the radioactivity
in the bacteria was 35S (protein) or 32P
(DNA)
Hershey and Chase
• Hershey and Chase found that the
radioactive DNA was injected into the
bacteria, and passed to the phage
progeny
Structure of DNA
• Once it was determined that DNA
was the genetic material, the race
was on to elucidate the structure of
DNA.
– It was felt that by understanding the
structure it would explain much about
inheritance and function of DNA
What I appreciated was that genetics
was the key part of biology – and that
one had to explain genetics in
structural terms.
Francis Crick
Structure of DNA
Players (College of
Physicians and
Surgeons, Columbia
University, New York)
– Erwin Chargaff
(biochemist)
• Worked with nucleic acids
• Found the fundamental
ratio of bases
Structure of DNA
Players (Cambridge University)
– Cavendish Laboratory (Sir Lord
Rutherford, found neutrons and
electrons)
• Sir Lawrence Bragg (physicist)
– X-ray crystallography
• Max Perutz (chemist)
– Director of Watson and Crick’s unit
– Nobel prize for structure of hemoglobin
X-ray crystallography
Crystallized substance
Structure of DNA
Players (Kings
College – London)
– Maurice Wilkins
(physicist)
• X-ray studies of
DNA
– Rosalind Franklin
(chemist turned
crystallographer)
Structure of DNA
Players (California Institute of
Technology, Pasadena)
– Linus Pauling (Chemist)
• Structure of chemical bonds
• One of three people to receive
two Nobel Prizes [Marie Curie
(Physics, Chemistry); John
Burden (Physics)]
– Chemistry – chemical bonds –
1954
– Peace – campaign against above
ground atomic testing - 1962
Structure of DNA
Players (Cambridge University, the
Cavendish Laboratory)
– Francis Crick (physicist turned biologist)
– Ph.D. candidate
– Mid-thirties
– James Watson (biologist, American)
–
–
–
–
–
–
Young postdoc
Early 20s
University of Chicago
Quiz Kids
University of Indiana
Studied bacteriophage with Luria
Structure of DNA
Players
– Watson and Crick produced the
structure by model-building
• The basis was:
– Erwin Chargaff’s data
– Rosiland Franklin’s x-ray photographs
Structure of DNA
• Chargaff’s data
Amount of A = amount of T; A/T ratio = 1.0
Amount of G = amount of C; G/C ratio = 1.0
– One purine and one pyrimidine
Structure of DNA from
X-ray Diffraction
• Consistent 2.0 nm diameter
• Very long repetitive molecule – 1,000
nm
• Helical, with a complete turn every
3.4 nm
– 0.34 nm between nucleotides
– 10 nucleotides per turn
• Could not tell if two or three strands.
Solved by model building
We wish to suggest a structure for the salt
of deoxyribonucleic acid (D.N.A.). The
structure has novel features which are of
considerable biological interest.
J. D. Watson and F. H. C. Crick, 1953
Structure of DNA
• DNA is composed of four basic
molecules; nucleotides
– Nucleotides contain:
• Phosphate
• Sugar, deoxyribose
– Ribose in RNA
• One of four nitrogenous bases (two purines
and two pyrimidines)
RNA
Pentose sugar
DNA
Structure of DNA
• Designate the Nucleotides
– Purines
• Guanine = G
• Adenine = A
– Pyrimidines
• Thymine = T
• Cytosine = C
Structure of DNA
• Nucleotides join together, forming a
polynucleotide chain, by
phosphodiester bonds
– The phosphate attached to the 5’ carbon
on one sugar
– Attaches to the 3’ hydroxyl (OH) group
on the previous nucleotide
5’-phosphate of
last nucleotide
chemically
bonded to the
3’-hydroxyl of
the next-to-last
nucleotide
A phosphodiester bond
Structure of DNA
• DNA is a double helix (two strands)
held together by hydrogen bonds
– Adenine (A) and thymine (T) are paired
– Guanine (G) and cytosine (C) are paired
– Always a purine with a pyrimidine
The two stands
twist around
each other
forming a righthanded helix
•The DNA double helix is 2.0
nm in diameter
•The bases are spaced 0.34
nm apart
•Each chain makes one
complete turn every 3.4 nm
•So there are 10 bases per
turn of the double helix
5’-end
3’-end
(free 3’-OH)
The two polynucleotide
strands (the
backbones) in the
double helix run in
opposite directions,
and are said to be antiparallel
3’-end
5’-end
(free 5’phosphate)
5’-end
3’-end
(free 3’-OH)
Because of the pairing
(A-T; G-C), one
polynucleotide chain is
always complementary
to the base sequence of
the other strand
3’-end
5’-end
(free 5’phosphate)
It has not escaped our notice that
the specific pairing we have
postulated immediately suggests a
possible copying mechanism for the
genetic material.
J. D. Watson and F. H. C. Crick, 1953
I have said many times that I regard the
working out of the detailed structure of DNA
one of the great achievements of biology in
the twentieth century, comparable in
importance to the achievements of Darwin
and Mendel in the nineteenth century. I say
this because the Watson-Crick structure
immediately suggested how it replicates or
copies itself with each cell generation, how it
is used in development and function, and how
it undergoes the mutational changes that are
the basis of organic evolution.
George Beadle