Download DNA

A molecule that serves as the genetic material must
•
•
•
•
Be able to be exactly copied
Be suitable for storing genetic information
Allow the information stored to be accessible
Allow for enough variation for evolution to occur
But before 1944 it wasn’t known: DNA or Protein?
1
1869: Friedrich Miescher
and a weak acid in the nucleus.
With staining and microscopes, nuclei discovered.
Stains bound to a weakly acidic substance in nucleus.
Later discovered to be polymer of nucleotides =
“nuclein” or nucleic acid. But no one knew what it
was doing in the nucleus.
http://academics.hamilton.edu/biology/kbart
/image/nucleus.jpg
2
1870’s thru 1920’s: more microscopy and chemistry
Threadlike structures in nucleus appear, separate,
and disappear = chromosomes. Somehow involved
in inheritance?
Chromosomes consist of proteins and DNA, but
which is the genetic molecule?
http://europa.eu.int/comm/research/quality
-of-life/image/chromosomes.jpg
3
The Case for Proteins as genetic material
4
Measurement error by Levene in 1910: equal amounts
of adenine, thymine, cytosine, and guanine.
Conclusion: DNA is boring.
Proteins: made of 20 amino acids in a variety of
combinations; surely this explains all the
different possible traits.
Computer technology just being born. If all of it is
possible with just 1001010010101010 why not with
A T C G? No one thinking like that.
Griffith looks for vaccine against Diplococcus
(Streptococcus pneumoniae), gets a surprise.
Smooth = wild type
Rough = mutant
Wild type cells form smooth colonies because of slimy capsule; capsule
protects bacteria against WBC, so they live and cause disease.
5
Griffith looks for vaccine against Diplococcus
(Streptococcus pneumoniae), gets a surprise.
6
• Griffith is experimenting with two strains of bacteria
– Normal (“smooth”) Streptococcus
• Makes smooth, slimy colonies
• Each cell surrounded with a polysaccharide capsule
which protects the cell, helps it cause infections.
– Mutant (‘rough”) Streptococcus
• Makes rough-appearing colonies
• Lacks the capsule, unable to cause lethal infections
– The ability to make a capsule is inherited
• Model system: mice
Griffith treats bacteria, infects mice
mice
Bacteria w/ capsule
Mice die.
mice
Mutant w/o capsule
Mice live.
7
Griffith treats bacteria, infects mice-2
8
mice
Virulent Strep
killed with heat
Mice live.
Neither mutant bacteria nor heat-killed bacteria kill the
mice, yet when the two are injected together into the mice:
+
mice
“transformation”!
9
Furthermore:
Living Strep with capsules are
isolated from the dead mice!
Genetic information (for making a polysaccharide
capsule) must have been transferred from the dead
bacteria to the live mutants.
http://www.cheque.uq.edu.au/research/bioengineering/research/Metaboli
c_Engineering/HA_capsule.gif
http://www.wadsworth.org/databank/images/streptococcuspneum.gif
http://www.abc.net.au/science/news/img/life/white_mouse.jpg
1944 Avery, MacLeod, and McCarty
10
use Griffith’s findings as model system
What molecule from bacteria caused the genetic change?
Bacterial cells broken and extracted to remove various
biochemical components. Extracts were treated with enzymes
which destroyed different types of molecules in the extract,
then combined with mutant bacterial cells.
Do any mutant bacteria change into virulent ones?
One out of every 100 bacterial colonies are “smooth” after
protease treatment and after RNAase treatment.
DNAse removes activity. The genetic molecule must be:
DNA
The Hershey Chase Experiment
What is genetic material: DNA or proteins?
11
A phage is about 50/50
DNA and protein.
After infection, genetic
molecule is inside
directing virus
production.
Put cells in a blender,
knock the virus “coat”
off. Which molecule
goes inside, which stays
outside?
Hershey Chase Experiment-2
12
These radioactive phages used to infect unlabeled E. coli cells.
Hershey and Chase Experiment-3
First: S-35 labeled phage.
Radioactivity found outside in
viral coat.
Second: P-32 labeled phage.
Radioactivity found inside
bacterial cell.
The genetic material: it’s
DNA
13
1953, Watson and Crick
14
DNA is the genetic material, but how does it work? What is its
chemical structure?
Chargaff (1949-1953) determines that there are NOT equal
numbers of all four nucleotides: A=T and G=C
Rosalind Franklin does x-ray diffraction studies, provides key
data and almost solves structure herself.
Watson and Crick build model that works. Double helix with
sugar-phosphate chains on outside, bases on inside.
DNA is genetic material in prokaryotes and
viruses, but what about eukaryotes?
• Indirect evidence
15
– Cytological evidence says genetic material should be in
the nucleus. DNA is in the nucleus.
– What molecule doubles in amount during the cell cycle,
then halves in amount during mitosis or meiosis? DNA.
– An egg cell or sperm should have half as much genetic
material. Each contains half as much DNA.
– A mutation is an inheritable change in the genetic
information. UV light at 260 nm causes the most
mutations; DNA absorbs light best at 260 nm.
• Direct evidence: recombinant DNA technology.
DNA is not always the genetic molecule
16
• Various viruses have RNA as their genetic molecule
– Actually, many rather familiar causes of disease:
• Measles
• Mumps
• Rabies
• Influenza
• HIV
• Hypothesis: RNA came before DNA or proteins
– Self splicing RNAs (ribozymes) made be remnant of
earliest forms of life.
Copyright © Ramaswamy H. Sarma 1996
DNA has a
dynamic structure
17
Many forms of DNA are
possible under different
conditions. B DNA is
presumed to be normal
in cells. However, A
DNA, P DNA, and even
Z DNA is possible.
Z DNA is a left-handed
double helix.
DNA and fragments of DNA can be separated
and identified using electrophoresis
18
• Physical separation by movement in electric field.
– May be preceded by fragmentation of DNA, by random
breakage or by site specific cleavage (restriction).
– DNA applied to wells at one end of a gel.
Principles of Electrophoresis
19
‘Gel’ is typically agarose and
contains ~98% water and
salts to carry the current.
DNA is negatively charged,
moves toward positive pole.
Wells in which DNA
samples are added.
Separation is by SIZE,
smaller pieces wiggle thru
gel more easily, travel farther.
www.biology.arizona.edu/ .../03t.html
Principles of Electrophoresis (continued)
A “band” is comprised of
many DNA molecules of
the same size.
Wells in which DNA
samples are added.
A band is visualized using
radioactivity or some type
of staining.
www.biology.arizona.edu/ .../03t.html
20
DNA structure and synthesis
• DNA is a polymer of nucleotides
21
Nucleotide base terminology
Base
Nucleoside
Nucleotide
Adenine
Adenosine
Adenosine ___ Phosphate
Guanine
Guanosine
Guanosine____Phosphate
Cytosine
Thymine
Cytidine
Thymidine
Cytidine_____Phosphate
Thymidine____Phosphate
Nucleotides generally have 1 (mono), 2 (di) or 3 (tri)
phosphate groups.
Nucleosides that make up DNA have deoxyribose instead of
ribose as sugar; have deoxy as a prefix in the name.
22
23
DNA
“backbones”:
alternating
sugarphosphates.
http://courses.bio.psu.edu/fall2005/
biol230weve/tutorials/tutorial2_fil
es/figure_16_5_part2.gif
Interior:
Complementary bases.
T always pairs
with A,
C with G.
DNA chains are antiparallel.
24
DNA chains held
together with H
bonds.
A-T pairs: 2
G-C pairs: 3.
Bases are flat, planar; they stack on the inside of the
molecule. Hydrophobic interactions stabilize DNA.
DNA chains twist together around a
central axis, not around each other.
http://genetics.nbii.gov/i
mages/BasePairs.gif
25
Dimensions of DNA
10 bp per turn
3.4 nm per turn
Right-handed helix
Central axis
Structure/Function Relationships in DNA
26
• Note how the structure presents a mechanism for
exact replication, needed for the genetic molecule.
• Bases can be arranged in any sequence; provides
info for specifying 20 amino acids.
• Mispairing due to mistakes, damage, lead to
mutation, lead to individual variation and evolution.