Structure (chapter 10, pages 266

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Structure (chapter 10, pages 266 278)and
Replication of DNA (chapter 12, pages 318
334)
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Dr. Ravi Palanivelu Rpalaniv_at_ag.arizona.edu http
//ag.arizona.edu/research/ravilab/
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Transmission genetics

• Previous discussions focused on the individual.
• Phenotype caused by an individual genotype
• Transmission of genes of an individual organism
  to the next generation
• Offspring produced by crossing two individuals

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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 --gt phenotype
  (Lectures 10, 11)
• How do we study them (Lectures 12, 13)

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What is the hereditary/genetic material?

• Until the mid-20th century, most scientists
  believed that proteins must be the hereditary
  (genetic) material

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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

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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

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Important Dates in Determining the Structure of
DNA
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Experiments that led to determining the genetic
material

• Frederick Griffith, 1928
• British physician/bacteriololgist
• Streptococcus pneumoniae
• Pneumonia in humans
• Septicemia in mice (lethal)

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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

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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

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Frederick Griffith

• Injected mice with
• 1. Live IIIS strain
• Mice died
• IIIS strain recovered from blood

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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

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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

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Frederick Griffith

• Injected mice with
• Heat-killed IIIS strain live IIR strain
• Expect the mice to live
• Recover nothing from the blood

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Frederick Griffith

• Injected mice with
• 4. Heat-killed IIIS strain live IIR strain
• Found mice died
• Live IIIS strain recovered from blood

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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

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Generalized Bacterial/Prokaryotic Cell
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Experiments toward determining the genetic
material

• Oswald Avery, Colin MacLeod, and Maclyn McCarty,
  1944
• Wanted to find the transforming (principle) agent
  from Griffiths experiment
• The transforming agent must be the genetic
  (hereditary) material

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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

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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

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Practice problems (8, structure and replication
of DNA) is due next Monday (4/5/01)
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http//highered.mcgraw-hill.com/olcweb/cgi/pluginp
op.cgi?itswf535535/sites/dl/free/0072437316
/120076/micro04.swfDNA20Replication20Fork
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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

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Hershey and Chase

• Alfred Hershey and Martha Chase, 1952
• Worked with T2 virus
• A bacteriophage of E. coli

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• T2 virus is made-up of
• A protein coat
• Surrounding a DNA molecule

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• 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

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• T2 virus is made-up of
• A protein coat
• (contains sulfur)
• Surrounding a DNA molecule
• (contains phosphorous)

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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

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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)

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Hershey and Chase

• Hershey and Chase found that the radioactive DNA
  was injected into the bacteria, and passed to the
  phage progeny

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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

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What I appreciated was that genetics was the key
part of biology and that one had to explain
genetics in structural terms. Francis Crick
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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

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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 Cricks unit
• Nobel prize for structure of hemoglobin

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X-ray crystallography
Crystallized substance
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Structure of DNA

• Players (Kings College London)
• Maurice Wilkins (physicist)
• X-ray studies of DNA
• Rosalind Franklin (chemist turned
  crystallographer)

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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

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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

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Structure of DNA

• Players
• Watson and Crick produced the structure by
  model-building
• The basis was
• Erwin Chargaffs data
• Rosiland Franklins x-ray photographs

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Structure of DNA

• Chargaffs 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

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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.

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Solved by model building
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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
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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)

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DNA
RNA
Pentose sugar
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Structure of DNA

• Designate the Nucleotides
• Purines
• Guanine G
• Adenine A
• Pyrimidines
• Thymine T
• Cytosine C

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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

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5-phosphate of last nucleotide chemically bonded
to the 3-hydroxyl of the next-to-last nucleotide
A phosphodiester bond
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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

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The two stands twist around each other forming a
right-handed 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

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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 anti-parallel
5-end (free 5- phosphate)
3-end
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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
5-end (free 5- phosphate)
3-end
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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
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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