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1A 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?
21869: 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
31870’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
4The Case for Proteins as genetic material
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 withA T C G? No one thinking like that.
5Griffith 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.
6Griffith looks for vaccine against Diplococcus
(Streptococcus pneumoniae), gets a surprise.• 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
7Griffith treats bacteria, infects mice
mice
Bacteria w/ capsuleMice die.
mice
Mutant w/o capsule
Mice live.
8Griffith treats bacteria, infects mice-2
Virulent Strep killed with heat
mice
Mice live.
Neither mutant bacteria nor heat-killed bacteria kill the mice, yet when the two are injected together into the mice:
+ mice
9“transformation”!
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/Metabolic_Engineering/HA_capsule.gif
http://www.wadsworth.org/databank/images/streptococcuspneum.gifhttp://www.abc.net.au/science/news/img/life/white_mouse.jpg
101944 Avery, MacLeod, and McCarty 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
11The Hershey Chase Experiment What is genetic material: DNA or proteins?
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?
12Hershey Chase Experiment-2
These radioactive phages used to infect unlabeled E. coli cells.
13Hershey and Chase Experiment-3
The genetic material: it’s
DNA
First: S-35 labeled phage.
Radioactivity found outside in viral coat.
Second: P-32 labeled phage.
Radioactivity found inside bacterial cell.
141953, Watson and Crick
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.
15DNA is genetic material in prokaryotes and viruses, but what about eukaryotes?
• Indirect evidence– 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.
16DNA is not always the genetic molecule
• 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.
17
Copyright © Ramaswamy H. Sarma 1996
DNA has a dynamic structure
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.
18DNA and fragments of DNA can be separated
and identified using electrophoresis
• 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.
19
www.biology.arizona.edu/ .../03t.html
Principles of Electrophoresis
Wells in which DNAsamples are added.
‘Gel’ is typically agarose and contains ~98% water and salts to carry the current.
DNA is negatively charged, moves toward positive pole.
Separation is by SIZE, smaller pieces wiggle thru gel more easily, travel farther.
20
www.biology.arizona.edu/ .../03t.html
Principles of Electrophoresis (continued)
Wells in which DNAsamples are added.
A “band” is comprised of many DNA molecules of the same size.
A band is visualized using radioactivity or some type of staining.
21DNA structure and synthesis• DNA is a polymer of nucleotides
22Nucleotide base terminology
Base Nucleoside Nucleotide
Adenine Adenosine Adenosine ___ Phosphate
Guanine Guanosine Guanosine____Phosphate
Cytosine Cytidine Cytidine_____Phosphate
Thymine Thymidine 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.
23
DNA chains are antiparallel.
DNA “backbones”: alternating sugar-phosphates.
Interior: Complemen-tary bases. T always pairs with A, C with G.
http://courses.bio.psu.edu/fall2005/biol230weve/tutorials/tutorial2_files/figure_16_5_part2.gif
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/
images/BasePairs.gif
25Dimensions of DNA10 bp per turn
3.4 nm per turn
Right-handed helix
Central axis
26Structure/Function Relationships in DNA
• 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.
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