DNA Structure and Analysis

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Chapter 9

• DNA Structure and Analysis

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Characteristics of Genetic Material

• Replication double the amount of DNA and
  separate into new daughter cells
• Storage of information may have all DNA but
  only part is used at any time
• Expression of information information flow
  transcription and translation
• Variation by mutation introduce changes in
  proteins and pass on to future generations
• central dogma of molecular genetics

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Central Dogma
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Protein as Genetic Material

• Knew there was both protein and nucleic acids in
  cells as options for genetic material
• Due to the abundance of proteins it was thought
  to be the material that ran the cell
• Felt DNA lacked the diversity of that would be
  needed to control cellular processes
• only 4 nucleotides that were in equal amounts in
  DNA, repeated over and over tetra-nucleotide
• Chargaff in 1944 proved not equal amount of the 4

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Evidence for DNA Genetic Material

• Avery, MacLeod and McCarty reported the
  transforming principle that is responsible for
  our understanding of DNA as source of genetic
  material
• 1 of 3 great revolutions in biology
• Darwins work
• rediscovery of Mendels work
• DNA as genetic material

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Transformation Studies

• Used Diplococcus pneumoniae (Streptococcus)
• virulent strain causes pneumonia in some
  vertebrates
• avirulent strain no illness caused
• Virulence is caused by the polysaccharide capsule
  not easily dealt with by the immune system
• capsule present makes a smooth colony
• no capsule makes a rough colony

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• Various strains are around and vary by
  composition of the capsule identify by using
  serology and immunology
• Identify with Roman numerals and colony morphology

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Transformation

• Heat killed IIIS somehow converted IIR to IIIS
  which is the transforming principle
• somehow got IIR to make a virulent polysaccharide
  coat
• Got the same results in a culture tube
• Were not sure what was the cause of this
  phenomenon
• not seen as a genetic event

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MacLeod, Avery and McCarty
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DNA as Genetic Material

• Found that DNA was responsible for how the IIR
  strain became IIIS
• Also recognized the genetic and biochemical
  importance DNA gives rise to enzymes in type
  IIR to eventually make the type IIIS
  polysaccharide capsule
• Transformation is heritable all subsequent
  generations have the capability to make capsule
• Take advantage of this in the lab to make DNA and
  proteins to study

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Bacteriophage T2 and E. coli

• Bacteriophage T2 has a protein coat with a DNA
  core
• Life cycle
• attach to bacteria and inject the DNA into the
  cell which then makes more virus particles that
  are released by rupturing the bacterial cell

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Hershey-Chase Experiment

• Knew 3 things about the virus
• it is 50 protein and 50 DNA
• infection by absorption of phage by tail fibers
• new viruses are made and released
• Question is it the protein or the DNA
• Labeled the protein with 35S and the DNA with 32P
  and followed where they ended up

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Transfection Experiments

• Treat cells with lysozyme to remove cell wall to
  create protoplasts or spheroplast
• Can initiate phage multiplication with disrupted
  particles
• Use DNA in transfection add to protoplasts
  get out virus

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Indirect Evidence Distribution of DNA

• DNA found only in the nucleus and protein is in
  both nucleus and cytoplasm
• small amount of DNA in mitochondria and
  chloroplasts
• DNA is where genetic activities are located
• Should also see a correlation with amount of DNA
  in gametes and somatic cells, do not see that
  with protein

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Indirect Evidence - Mutagenesis

• UV light can cause mutations in the DNA and you
  can measure the number of mutations formed in
  yeast cells
• Make an action spectrum by plotting the mutation
  vs the wavelength of UV
• Look at the absorption spectrum of DNA and
  protein
• see that the wavelength that caused mutation is
  the wavelength that is absorbed by DNA

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Direct Evidence Recombinant Studies

• Recombinant studies take YFG and incorporate it
  in a bacterial chromosome, this is then put back
  in the bacteria and look for YFP
• used to make insulin and interferon
• Direct evidence as you put in DNA and get protein
  out
• Create transgenic mouse by adding the gene for
  human ?-globin into a fertilized mouse egg
• found human ?-globin in all the mouse cells and
  in the offspring

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RNA as Genetic Material

• Some viruses use RNA as their genetic material
• In 1956 found RNA in the tobacco mosaic virus
  that infects and damages tobacco plants
• Isolated a RNA replicase from Q? virus
  replicates RNA in vitro add this RNA to a cell
  and get new viruses
• Retroviruses are a special group
• replicate in an unusual fashion by making a DNA
  copy and inserting it into the chromosome of the
  host
• found a RNA dependent DNA polymerase called
  reverse transcriptase used now to convert
  isolated RNA into cDNA
• HIV and RNA tumor viruses

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Structure of DNA

• Key to understanding the function of DNA
• 2 sources of info used by Watson and Crick to
  figure out the structure of DNA
• base composition of hydrolyzed DNA
• X-ray crystalography
• Used both to build a model

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Purines, Pyrimidines and Sugars

• Nucleotide is the building block of nucleic acids
• Composed of a nitrogenous base, 5 C sugar and PO4
  groups
• Nitrogenous base
• purine 9 member ring A and G
• pyrimidine 6 member ring C, U and T
• Sugars ribose and deoxyribose

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• Nucleoside base sugar
• Nucleotide base sugar PO4

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Building Nucleotides

• Add bases to 1 C N-9 if purine and N-1 if
  pyrimidine
• PO4 added to either 2, 3 or 5 C but mostly on
  5
• Nucleoside mono-, di- and tri- phosphate with 1,
  2 or 3 PO4 groups
• Use the energy in the bond between 2nd and 3rd
  PO4 to couple to another reaction see in many
  cellular activities

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Phosphodiester Bond

• Formed by adding a PO4 between 2 sugars
• 5C on one sugar and 3 C on the other sugar
• get and ester bond between the 2 OH groups of the
  sugars
• Oligonucleotides 20 nt
• Polynucleotide long chains of nt
• Possible arrangenments 1000 nt and then 41000
  ways to arrange