Download DNA - anisam2

DNA: The Genetic Material
• Search for genetic material---is it composed of
protein/DNA or RNA?
•
•
•
•
•
Griffith’s Transformation Experiment
Avery’s Transformation Experiment
Hershey-Chase Bacteriophage Experiment
Tobacco Mosaic Virus (TMV) Experiment
Lederberg and Tautum conjugation expt.
• Chemistry of DNA: composition and structure
• Double-helix model of DNA - Watson & Crick
Search for the genetic material:
1. Biologically useful and stable source of information
2. Ability to replicate accurately and transmitted across generations
3. Capable of change
4. Express itself to make other biomolecules
Timeline of events:
•
1900
Chromosomes shown to contain hereditary information,
later shown to be composed of protein & nucleic acids.
•
1928
Griffith’s Transformation Experiment
•
•
•
1944
1946
1952
Avery’s Transformation Experiment
Lederberg and Tautum’s conjugation experiment
Hershey-Chase Bacteriophage Experiment
•
1953
Watson & Crick propose double-helix model of DNA
•
1956
First demonstration that RNA is viral genetic material.
How do we know that all of our
genetic information comes from
DNA?
• What type of experiment would you
design to determine that DNA is the
source of all genetic information?
Griffith’s Experiment with S. pneumoniae and the
accidental discovery of transformation (1928)
Rough
smooth
Streptococcus pneumoniae
Gram postive bacteria
Commonly Exist as diplococci
Frederick Griffith
Griffith’s Experiment with S. pneumoniae and the
accidental discovery of transformation
Smooth colonies
Rough colonies
Griffith’s Experiment did not prove that
DNA was responsible for transformation
How would you design an
experiment to prove that DNA was
responsible for transformation?
Avery, McCarty, and MacLeod
Repeated Griffith’s Experiment
(1944)
Oswald Avery
Maclyn McCarty
Colin MacLeod
Heat kill Smooth Type cells—break---isolate RNA, DNA, etc
Carbohydrates
Lipids
Proteins
RNA
DNA
Add each to Rough type bacteria-----only DNA could transform them to S
type
To the Heat-Killed Smooth Type, added enzymes that
destroyed…
Carbohydrates
Lipids
Proteins
RNA
DNA
Add to R-cells…only DNase inactivated
transformation
Conclusion:
DNA was the
transforming factor!
Hershey-Chase Bacteriophage Experiment - 1953
Bacteriophage = Virus that attacks
bacteria and replicates by
invading a living cell and using
the cell’s molecular machinery.
Fig. 2.4
Structure of T2 phage
Bacteriophages
are composed of
DNA & protein
Fig. 2.5: Life cycle of virulent T2 phage:
Fig. 2.6: Hershey-Chase Bacteriophage Blender Experiment - 1953
1.
T2 bacteriophage is composed
of DNA and proteins:
2.
Set-up two replicates:
•
•
Label DNA with 32P
Label Protein with 35S
3.
Infected E. coli bacteria with
two types of labeled T2
4.
32P
is discovered within the
bacteria and progeny phages,
whereas 35S is not found within
the bacteria but released with
phage ghosts.
Alfred Hershey
The Hershey-Chase results
reinforced the Avery, McCarty,
and MacLeod conclusion:
DNA carries the genetic code!
Gierer & Schramm Tobacco Mosaic Virus (TMV) Experiment - 1956
Fraenkel-Conrat & Singer - 1957
Demonstrated that RNA is the genetic material of TMV.
Conclusions about these early experiments:
Griffith 1928 & Avery 1944:
DNA (not RNA) is transforming agent.
Hershey-Chase 1953:
DNA (not protein) is the genetic material.
Gierer & Schramm 1956/Fraenkel-Conrat & Singer 1957:
RNA (not protein) is genetic material of some viruses, but no known
prokaryotes or eukaryotes use RNA as their genetic material.
Alfred Hershey
Nobel Prize in Physiology or Medicine
1969
Chemistry of DNA
1869: Fred Miescher isolated nuclein
(acidic with high phosphorous content)
1880: Emil Fischer identified purines and pyrimidines
1910: Kossel identified nucleotides (base+sugar+phosphate)
Got the Nobel Prize
1950: Alexander Todd discovered that 2 nucleotides are linked
by 3’ to 5’ phosphodiester bond
1953: Watson and Crick propose double-helical model of DNA
The Race to Discover DNA’s
Structure
The Race to Discover DNA’s
Structure
Linus Pauling
1940s
Discovered the alphahelical structure of
proteins.
“Chargaff’s rule”
A=T & C=G
1.
Base composition studies
indicated double-stranded
DNA consists of
~50% purines (A,G) and
~50% pyrimidines (T, C)
2. amount of A = amount of T
and amount of G = amount
of C
3. %GC content varies from
organism to organism
Erwin Chargaff
Examples:
Homo sapiens
Zea mays
Drosophila
Aythya americana
%A
%T
%G
%C
%GC
31.0
25.6
27.3
25.8
31.5
25.3
27.6
25.8
19.1
24.5
22.5
24.2
18.4
24.6
22.5
24.2
37.5
49.1
45.0
48.4
The Race to Discover DNA’s
Conclusion-DNA
Structure
is a helical
structure with
distinctive
regularities, 0.34
nm & 3.4 nm.
X-Ray diffraction image of DNA
Maurice Wilkins
taken by Franklin in 1951
Rosalind Franklin
X-ray diffraction studies by Rosalind Franklin & Maurice Wilkins
The Race to Discover DNA’s
Structure
1950 Purine + Purine = Too wide
Why do you think
Chargaff
’s Rule:
the bases
match up
Equal amounts
of
this
way?
Pyrimidine + Pyrimidine = Too Narrow
Adenine and Thymine,
and equal amounts of
Guanine and Cytosine
Purine + Pyrimidine = Perfect Fit from X-ray data
James Watson and Francis Crick propose double-helical model of DNA
Double Helix Model of DNA: Six main features
1.
Two polynucleotide chains wound in a right-handed (clockwise)
double-helix.
2.
Nucleotide chains are anti-parallel:
3.
Sugar-phosphate backbones are on the outside of the double
helix, and the bases are oriented towards the central axis.
4.
Complementary base pairs from opposite strands are bound
together by weak hydrogen bonds.
5’  3’
3’  5’
A pairs with T (2 H-bonds), and G pairs with C (3 H-bonds).
5’-TATTCCGA-3’
3’-ATAAGGCT-5’
5.
Base pairs are 0.34 nm apart. One complete turn of the helix
requires 3.4 nm (10 bases/turn).
6.
Sugar-phosphate backbones are not equally-spaced, resulting
in major and minor grooves.
Nucleotide = monomers that make up DNA and RNA (Figs. 2.8)
Three components
1. Pentose (5-carbon) sugar
DNA = deoxyribose
RNA = ribose
(compare 2’ carbons)
2. Nitrogenous base
Purines
Adenine
Guanine
Pyrimidines
Cytosine
Thymine (DNA)
Uracil (RNA)
3. Phosphate group attached to 5’ carbon
Nucleotides are linked by phosphodiester bonds to form polynucleotides.
Phosphodiester bond
Covalent bond between the phosphate group (attached to 5’ carbon) of
one nucleotide and the 3’ carbon of the sugar of another nucleotide.
This bond is very strong, and for this reason DNA is remarkably stable.
DNA can be boiled and even autoclaved without degrading!
5’ and 3’
The ends of the DNA or RNA chain are not the same. One end of the
chain has a 5’ carbon and the other end has a 3’ carbon.
5’ end
3’ end
Type B-DNA
Other DNA forms
include:
A-DNA:
Right-handed double
helix with 11 bases
per turn; shorter and
wider at 2.2 nm
diameter. Exists in
some DNA-protein
complexes.
Z-DNA:
Left-handed double
helix with 12 bases
per turn; longer and
thinner at 1.8 nm
diameter.
Type A, B, and Z conformations of DNA
Fig. 2.14
1962: Nobel Prize in Physiology and Medicine
James D.
Watson
Francis H.
Crick
Maurice H. F.
Wilkins
What about?
Rosalind Franklin