DNA Structure and Function

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DNA Structure and Function

• Chapter 13

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What are genes?

• Scientists figured out how heredity worked years
  before they figured out what a gene is
• They knew a few facts. Genes had to be
• able to store information both for initial
  development and to respond to changes over a
  lifetime
• stable enough to be replicated and passed to
  offspring
• fragile enough so that mutations are possible
• The structure, chemical makeup, and method for
  doing these things, however, was still a mystery

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Deoxyribonucleic Acid

• In 1869, six years after Mendels first
  experiment, Swiss chemist Friedrich Meischer
  discovered a new chemical that contained
  phosphorus but not sulfur
• This fact alone established a new molecule
  distinct from carbohydrates or proteins
• Because it had acidic properties and was
  discovered inside of a nucleus, they were called
  nucleic acids
• Later, in the early 1900s, it was discovered
  there were four types of nucleic acids, each with
  a separate nitrogenous base
• These were called nucleotides

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Griffiths Transformation Experiment

• One of the first genetic experiments conducted,
  as usual, had nothing to do with genetics.
• In 1931, Frederick Griffith was working with mice
  and two strains of Streptococcus pneumoniae
• One strain was rough in appearance and
  nonvirulent
• One strain was smooth in appearance and
  virulent
• When injected with the rough strain, mice lived
• When injected with the smooth strain, mice died.
  Both as expected.

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Griffiths Transformation Experiment

• Also as expected, when he heat-killed the
  virulent, smooth strand, the virus did not kill
  the mice
• When injected with heat-killed smooth strands of
  virus AND healthy, nonvirulent rough strands, the
  mice died.
• Although the virulent strand had been
  heat-killed, the nonvirulent strand had taken up
  part of the virulent strand
• The virulent strand had died, but its genetic
  material survived.

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Griffiths Transformation Experiment

• Griffiths experiment proved the concept of
  transformation, which means cells can take parts
  of other cells and use them for themselves
• What it did in the world of genetics was get
  people wondering about what the actual molecule
  is that gets passed from organism to organism

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What are genes? (Dont you love the mystery??)

• In the early 1900s, good money said genes were
  controlled by proteins
• We know genes are passed from cell to cell. So
  are proteins
• Nucleic acids only have four different
  nucleotides. Proteins have 20 different amino
  acids.
• Considering the trillions of genes that must
  exist, is it more likely they are built with 4
  different pieces or 20?

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Hershey and Chase Experiment

• In 1952, Alfred Hershey and Martha Chase used a
  T2 bacteriophage to answer this question.
• They inserted radioactive isotopes of phosphorus
  and sulfur into bacteriophages, knowing they
  would be taken in due to transformation
• Both isotopes would be visible under radioactive
  treatments
• Whatever radiation is found in offspring would
  show which molecule is passed genetically
• Phosphorus is only found in nucleic acids
• Sulfur is only found in proteins
• In the offspring, only phosphorus was detected.

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Deoxyribonucleic Acid

• The structure of a nucleic acid contains three
  sections
• A Phosphate phosphates connect nucleotides
  together
• Ribose sugar the structural backbone
• Nitrogenous base the genetic code
• There are two ribose sugars, one with an extra
  oxygen (ribose) and one missing an oxygen
  (deoxyribose)
• Which sugar the nucleotide has determines whether
  it is DNA or RNA (ribonucleic acid).

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Nucleic Acids

• For DNA, the four nucleotides are
• Adenine (A)
• Cytosine (C)
• Guanine (G)
• Thymine (T)
• For RNA, Thymine is replaced with a fifth
  nucleotide
• Uracil (U)

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Chargoffs Rule

• In the 1940s Erwin Chargoff conducted
  experiments on DNA of various species.
• By measuring the quantities of each nucleotide,
  Chargoff came to some conclusions that he called
  Chargoffs rules.
• 1. The amount of A, T, C, and G in DNA varies
  from species.
• 2. In each species, for DNA, the amount of A T
  and the amount of C G.

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Chargoffs DNA Database
Species A T G C
Bacillus Subtillus (Bacillus bacteria) 28.4 29.0 21.0 21.6
Escherichia coli (E. coli) 24.6 24.3 25.5 25.6
Neurospora crassa (Bread mold) 23.0 23.3 27.1 26.6
Zea mays (Corn) 25.6 25.3 24.5 24.6
Drosophila melanogaster (Fruit fly) 27.3 27.6 22.5 22.5
Homo Sapiens (Human) 31.0 31.5 19.1 18.4
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Watson and Crick (And Franklin)

• In the 1940s, Rosalind Franklin used x-ray beams
  to pass through a crystalline DNA.
• The x-Ray diffracted through the crystal and
  created an atomic ray pattern that showed, among
  other things, a relative shape of DNA molecules.
• Two of Franklins colleagues, James Watson and
  Francis Crick, used this image Franklin created
  to develop one of the most important discoveries
  of the 20th century the DNA model

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Watson and Crick (And Franklin)

• The model showed a double-helix structure. Two
  strands of DNA attached to each other.
• The strands matched nitrogenous base to
  nitrogenous base (A matched with T, C matched
  with G)
• Bases are connected using hydrogen bonds
• This proved Chargoffs rules.
• Each nucleotide was attached using the phosphates
• Each strand faces the opposite direction
• The strand fits the size and shape of Franklins
  photograph

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Complimentary Base Pairing

• Pyramidines (Nitrogenous base is a single ring)
• Thymine and Cytosine
• Purines (Nitrogenous base is a double ring)
• Adenine and Guanine
• Adenine and Thymine are connected using two
  hydrogen bonds
• Guanine and Cytosine are connected using three
  hydrogen bonds
• This is how the two strands of DNA attach to each
  other

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DNA Replication

• DNA Replication is the process of copying a DNA
  molecule
• Watson and Cricks model was so effective and
  accurate that immediately after publishing it
  they were easily able to develop their
  replication hypothesis
• During replication, each strand of DNA is
  separated from the other.
• These strands are then used as a template for
  building a new strand

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DNA Replication

• 1. Unwinding.
• The weak hydrogen bonds holding the strands
  together at the nitrogenous bases are unzipped.
• An enzyme called Helicase unwinds the molecule
• 2 Base Pairing
• New nucleotides are constantly being built and
  present in the nucleus of cells
• An enzyme called DNA polymerase attaches new
  nucleotides to the new strand of DNA
• Polymerase knows which nucleotide comes next
  because of the base pairing rules

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DNA Replication

• After the strands are separated by helicase, the
  first thing that happens is an enzyme called RNA
  polymerase lays down an RNA primer on top of the
  template
• DNA polymerase cannot start a strand. It uses the
  RNA primer as its co-factor.
• Once activated, the DNA polymerase begins
  attaching new nucleotides to the 3 carbon
• Thus, DNA polymerase moves in the 5?3 direction

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DNA Replication

• Because DNA strands face the opposite direction
  from each other, DNA polymerase works well on the
  strand moving in the 5?3 direction
• This is called the leading strand
• For the other strand, polymerase cannot go in the
  opposite direction.
• Instead, small sections of DNA called Okazaki
  fragments are built separately, then attached one
  section at a time.
• This is called the lagging strand.

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Semiconservative Replication

• Replication was finally confirmed by Meselson and
  Stahl in 1958
• Meselson and Stahl took a strand of DNA that
  contained 15N and allowed it to go through
  replication with free nucleotides that contained
  14N nitrogen ions.
• 14N is lighter than 15N.
• When placed in a centrifuge and allowed to sit
  for 2-3 days, the heavier DNA will sink to the
  bottom and the lighter will float toward the top

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Semiconservative Replication

• After replicating DNA, if Watson and Cricks
  model is correct then a centrifuge should show
  both strands of DNA
• If another model is correct, they should only see
  one or the other floating in the centrifuge
• At the end of the experiment, both strands were
  visible in the centrifuge.
• This proved semi conservative replication, which
  means each new copy of DNA has one recycled
  strand and one newly-formed strand.

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Prokaryotic Replication

• Prokaryotic DNA is a single, circular structure
  called a plasmid.
• Replication occurs in either one or both
  directions
• Bacteria takes approximately 40 minutes to copy
  the entire genome at a rate of 106 base
  pairs/minute
• Eukaryotic replication occurs at multiple origins
  and at a much slower rate
• 500-5000 bp/minute.

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Mutations

• DNA polymerase not only attaches nucleotides
  together, but proofreads its work.
• Mismatched nucleotides causes a kink in the
  strand which is identified by the polymerase
• The enzyme then excises the incorrect nucleotide
  and replaces it with the correct one.
• Still, polymerase is so accurate that this is
  only necessary once per 100,000 base pairs.
• After proofreading, the likelihood of a mutation
  is only one mistake every 1 billion base pairs

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Mutations

• Other mutations occur due to mutagens, or
  environmental factors.
• UV, radiation, organic chemicals such as tobacco
  smoke, pesticides, etc.
• For these, the cell has DNA repair enzymes that
  go around and look for errors after replication
  is over.
• Mutations may cause harm (cancer) or have no
  affect at all.
• Throughout history, they also serve as the
  possibility for evolutionary changes

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Gene Activity

• Chapter 14

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Nucleic Acids to Proteins

• Enzymes perform the reactions that make up your
  individual traits. But nucleic acids make up the
  genes that code for traits.
• How do we get from a strand of nucleic acids to a
  strand of proteins?
• One of the first experiments to test this issue
  also revealed one of the strangest examples of a
  mutation in the human genome

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Pauling/Itano Experiment

• Linus Pauling and Harvey Itano knew that
  hemoglobin, a molecule in red blood cells,
  contained a charge.
• They wanted to see if the hemoglobin in normal
  RBCs is different than the hemoglobin in sickle
  RBCs.
• To do this, they compared an electrophoresis
  experiment of the two hemoglobin structures with
  known hemoglobin samples

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Pauling/Itano Experiment

• Based on this experiment, they proved that RBCs
  contain a type of hemoglobin called HBA, while
  Sickle RBCs contain HBB.
• Later, they compared the polypeptide chains of
  each strand of hemoglobin
• Believing the two chains would be completely
  different, they were surprised to find the
  difference between a normal and sickle RBC gene
  sequence is only one nucleotide out of 438.

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RNA

• RNA is a polymer of nucleotides. Only a few
  differences exist between DNA and RNA though
• RNA contains uracil as a replacement for thymine
• RNA is generally single-stranded
• RNA freely leaves the nucleus of cells
• RNA stands can pair with DNA strands according to
  the same base-pairing rules. So RNA serves as an
  excellent copying system for DNA.

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RNA

• There are hundreds of classes of RNA, but we care
  about three of them
• Messenger RNA (mRNA) takes a message from the
  nucleus to the ribosome
• Transfer RNA (tRNA) transfers amino acids to
  ribosomes
• Ribosomal RNA (rRNA) makes up a portion of
  ribosomes

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Transcription

• Transcription is the process of forming an mRNA
  strand as a copy of a DNA strand
• This occurs separately and independently from
  replication
• 1. The DNA strand unwinds and unzips similarly to
  replication.
• The location this occurs is called a promoter.
  Promoters are sequences of DNA that identify the
  origin of a gene sequence.
• The strand will continue to unwind until it
  reaches a sequence called the terminator.

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Transcription

• 2. Once the strand unwinds, an enzyme called RNA
  polymerase attaches RNA nucleotides in the 5?3
  direction only.
• 3. When the polymerase reaches the terminator, it
  stops and releases an RNA transcript called mRNA.
• 4. The mRNA then exits the nucleus to be picked
  up by a ribosome.
• The cell will have multiple RNA polymerases
  working simultaneously to ensure the maximum
  output of mRNA sequences.

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Introns and Exons

• To enhance the integrity of the mRNA strand when
  it leaves the cell, a couple steps occur
• First, a cap is put on the 5 and 3 ends of the
  strand so that nothing can accidentally break off
  or be added to the strand.
• Second, sections of the mRNA are removed by a
  spliceosome
• The sections that are removed are called introns.
• The remaining sections, called exons, are what
  get expressed (read) by ribosomes

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Introns and Exons

• Why introns and exons?
• The short answer is, who knows?
• Introns create redundancy, which helps reduce the
  likelihood that a mutation will cause a problem
• If 100 of the nucleotides are expressed, then a
  mutation will cause a problem 100 of the time.
• Introns allow for more variety of gene sequences
• Take the word hearth.
• From this word, you get he, ear, art,
  heart, and earth, depending on which letters
  you cut out.

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Translation

• Translation is the process of going from a strand
  of mRNA to a strand of amino acids.
• For this to occur, you need a tRNA molecule.
• tRNA is a molecule built from an RNA strand.
• Bound to one end of the tRNA is a specific amino
  acid.
• Bound to the opposite end of tRNA is a sequence
  of three nucleotides called an anticodon.
• Each tRNA with a particular anticodon (ex. GAA)
  will always carry a specific amino acid (ex.
  Leucine)

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Translation

• Meanwhile, proteins and rRNA have come together
  to build a ribosome.
• Ribosomes come in two sections called subunits.
• The two subunits sandwich themselves over a
  strand of mRNA and a series of tRNAs.
• When this happens, translation is ready to begin
• Eukaryotic cells contain 100,ooos of ribosomes,
  all working simultaneously if necessary.

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Translation

• 1. Initiation
• mRNA strands are organized by codons, or
  combinations of three RNA nucleotides
• The first codon is always the same AUG
• A tRNA with the anticodon that matches with this
  codon (UAC) then enters the ribosome and matches
  with the mRNA, codon-to-anticodon

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Translation

• 2. Elongation
• A second tRNA then enters the ribosome and
  matches anticodon-to-codon.
• When it attaches to the mRNA, the shape of the
  ribosome forces tRNAs to line up in a specific
  orientation.
• That orientation allows the amino acids that each
  tRNA are holding to break from their tRNAs and
  attach to each other.

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Translation

• The ribosome has three sites which are able to
  hold tRNAs.
• The A site, which is where tRNAs enter the
  ribosome and wait their turn.
• The P site, which is where the attachment of
  amino acids will occur (or, the formation of a
  peptide bond)
• The E site, which is where tRNAs will exit,
  leaving their amino acids behind.

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Translation

• During elongation, only one amino acid is
  attached at a time.
• One tRNA must leave the ribosome before the next
  one can enter.
• The entire process to build and form a single
  protein and repeat takes around 15 minutes.
• 3. Termination
• When the ribosome reaches a stop codon (UAA, UAG,
  UGA) the mRNA attaches to a release factor.
• The release factor breaks the final amino acid
  from the final tRNA, thus completing the protein
  chain.

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Translation

• Once the amino acid sequence is released from the
  ribosome, it undergoes folding and modification.
• The endoplasmic reticulum adds lipids,
  carbohydrates, or other proteins to the new
  protein
• The ER also handles the specific folding of the
  protein
• The golgi then wraps a vessicle around the
  protein for transport to its destination.
• Meanwhile, the ER reattaches amino acids to the
  tRNAs that have just given theirs up.
• The mRNA then is re-translated, or is returned to
  the nucleus to be used for spare parts.

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Codon Pattern

• Translation relies on the fact that
• 1) Every codon will match with a specific
  anticodon
• 2) Ever tRNA with that specific anticodon will
  have a specific amino acid.
• Thus, each of the 20 amino acids in organisms
  must have at least one codon that tells ribosomes
  to attach their specific amino acid to the
  sequence.
• As far as we know, the code is the same for every
  species on the planet

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Mutations

• The human genome contains (best guess)
  24,000-26,000 genes. The sequence of DNA to make
  these genes is around 3.3 billion nucleotides.
• Although ribosomes are able to correct mistakes
  in the moment, some do escape notice.
• There are two types of transcription/translation
  mutations you should be aware of.

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Mutation 1 Point Mutations

• A point mutation is when only one nucleotide is
  incorrect.
• Even though it is only 1 nucleotide out of 3.3
  billion, this one mistake has the potential to
  completely change an organisms health.
• Example I have a pet cat. This could be I
  gave a pet cat I wave a pet cat I have a
  wet cat. I have a pet rat.
• As discussed, sickle cell anemia is a disease
  caused by a single point mutation that tells the
  cell to replace a glutamine with a valine

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Mutation2 Frameshift Mutation

• Frameshift mutations are when at least one
  nucleotide is added or deleted from the DNA or
  RNA sequence
• The correct sequence of amino acids is dependent
  on maintaining the 3-nucleotide codon pattern.
• If one nucleotide is added or deleted, the codon
  pattern does not start at the right spot
• This results in an entire sequence of amino acids
  being incorrect.

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