Chapter 12: Molecular Genetics

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• Chapter 12 Molecular Genetics
• Discovery of DNA as genetic material
• Mid-1900s - scientists knew the following about
  chromosomes
• They contained genetic information
• They were made up of DNA (deoxyribonucleic acid)
  and proteins
• They did NOT know whether the DNA or the protein
  was the actual genetic material.
• Several experiments were done to show that DNA
  was the genetic material.

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• Frederick Griffiths experiment 1928
• Worked with 2 strains of bacteria
• S (smooth) strain caused pneumonia (coat protects
  it from hosts immune system, and host dies top
  picture)
• R (rough) strain did not cause pneumonia (no
  coat, is killed by hosts immune system bottom
  picture)

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• Results
• Live S strain mouse died
• Live R strain mouse lived
• Heat-killed S strain mouse lived
• Mixture of heat-killed S strain and live R strain
  mouse died

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• When Griffith isolated live bacteria from the
  dead mice who has been injected with the mixture
  of heat-killed S strain and live R strain, he
  found the smooth trait.
• This suggested that the disease-causing trait had
  been passed to the live R bacteria.
• So live R bacteria were transformed into live S
  bacteria wondered what the transforming
  substance was?

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• Today, we know why this happened.
• The transforming substance was DNA.
• The heat killed the S strain bacteria, but not
  its DNA.
• S strain DNA was taken up by live R strain
  bacteria, allowing it to grow the protective coat
  of the S strain.
• Transformed bacteria caused the mice to die. ?

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• Oswald Averys experiment 1944
• Set out to identify transforming substance from
  Griffiths experiment
• Isolated different molecules (DNA, proteins,
  lipids) from killed S cells
• Exposed live R cells to each molecule separately
• When live cells exposed to DNA, they transformed
  into S cells.

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• Avery concluded that when S cells were killed,
  their DNA was released
• R bacteria took that DNA in, and they transformed
  into S cells
• Averys conclusions were not widely accepted
  (although he was right!!)

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• Hershey and Chase experiment 1952
• Alfred Hershey and Martha Chase showed that DNA
  was the genetic material through two experiments
  with a T2 (type 2) virus.
• They knew the following to be true
• Viruses are made of DNA and protein
• Viruses inject genetic material into bacterium to
  reproduce

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• In first experiment, viral DNA was labeled with
  radioactive phosphorous.
• Virus was allowed to inject genetic material into
  bacterium.

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• In second experiment, viral protein was labeled
  with radioactive sulfur.
• Virus was allowed to inject genetic material into
  bacterium.

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• Because radioactive phosphorous was found inside
  the bacteria, and radioactive sulfur was not,
  they showed that DNA and not protein was the
  genetic material found in chromosomes.

Animation
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• Discovery of the structure of DNA
• The following was known about DNA by the early
  1950s
• DNA is made of nucleotides
• Phosphate
• Sugar deoxyribose
• 1 of 4 bases adenine, cytosine, thymine, and
  guanine
• Chargaffs rules
• Amount of adenine and thymine always equal
• Amount of cytosine and guanine always equal

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• DNA is in the shape of a double helix
  discovered by Franklin Wilkins through X-ray
  diffraction of DNA (a)
• 1953 - Watson Crick used above information to
  construct 1st model of DNA (b)

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Structure of DNA

• DNA is a polynucleotide nucleotides are composed
  of
• Phosphate
• Sugar (deoxyribose)

• 1 of 4 nitrogen-containing bases - adenine (A),
  thymine (T), guanine (G), and cytosine (C)

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• There are 2 strands of nucleotides
• 2 strands are held together by hydrogen bonds
• Two strands twist around each other to form a
  double helix
• A T, C G are complementary base pairs (purine
  to a pyrimidine)
• Purines A G
• Pyrimidines T C

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• DNA strands are anti-parallel run in opposite
  directions orientation of sugars
• 5 pronounced 5 prime
• 3 pronounced 3 prime

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• When double helix is unwound, it resembles a
  ladder
• A T pair with 2 hydrogen bonds
• C G pair with 3 hydrogen bonds

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DNA Replication

• Purpose DNA makes an exact copy of itself prior
  to cell division ensures that each new cell gets
  a complete copy of the DNA

DNA
DNA
DNA DNA
Replication
Cell Division
DNA
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• Steps (enzymes in red)
• Helicase attaches to DNA and breaks H2 bonds
  between bases DNA chain unwinds and unzips
  (Special proteins keep it unzipped)
• RNA primase adds an RNA primer (short segment of
  RNA) to each strand of DNA
• DNA polymerase attaches to separated strand,
  helping add complementary nucleotides to the new
  DNA strand

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• Each side is done differently, since new
  nucleotides can be added to the 3 end of the new
  strand only
• Leading strand built continuously
• Lagging strand elongates away from elongation
  fork. Made in small sections called Okazaki
  fragments
• Okazaki fragments are later connected by the
  enzyme DNA ligase

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Overview of DNA replication
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Ladder configuration and DNA replication
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• Each old strand of nucleotides serves as a
  template for each new strand.
• The process is semiconservative because each new
  double helix is composed of an old strand of
  nucleotides from the parent strand and one
  newly-formed strand (daughter strand).
• Proofreading and repair limits error rate to less
  than 1 per billion nucleotides.

http//www.stolaf.edu/people/giannini/flashanimat/
molgenetics/dna-rna2.swf
Replication fork
DNA replication song
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DNA (Deoxyribonucleic acid) RNA (Ribonucleic acid)
Sugar Deoxyribose Ribose
Bases Adenine, thymine, guanine, cytosine Adenine, uracil, guanine, cytosine
Strands Double-stranded with base pairing Single-stranded
Helix Yes No
Location Nucleus Nucleus, cytoplasm
Types XXXXXXXXX Messenger, transfer, ribosomal
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RNA vs. DNA
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• Messenger RNA - carries genetic information to
  the ribosomes
• Ribosomes - part of the cell where proteins are
  made
• Ribosomal RNA - found in the ribosomes
• Transfer RNA - transfers amino acids to the
  ribosomes

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• Making Proteins
• A gene is a segment of DNA that specifies the
  amino acid sequence of a protein.
• DNA is found in the nucleus of a cell proteins
  are made outside the nucleus at the ribosomes.

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Overview of gene expression
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• Two processes are involved in the synthesis of
  proteins in the cell
• Transcription DNA is copied into mRNA, which
  will take a copy of the DNA code to the ribosome
  to direct the making of protein occurs in
  nucleus
• Translation - the process of building proteins,
  the sequence of bases of mRNA is translated
  into a sequence of amino acids occurs in
  ribosome
• These processes are the same in all organisms

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The Genetic Code

• DNA holds instructions to make a protein
• Instructions are copied into mRNA, which will be
  used to make a protein
• Codon - each three-letter unit of an mRNA
  molecule
• Each codon represents 1 amino acid
• There are 64 possible codons, and only 20 amino
  acids, so most amino acids have more than one
  codon

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Messenger RNA codons
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• Transcription
• Purpose Makes a copy of the DNA code that can
  leave the nucleus and travel to the ribosome to
  direct protein synthesis mRNA
• Occurs in the nucleus
• Occurs at only 1 gene at a time
• Adenine in DNA pairs with uracil in RNA, not
  thymine
• Thymine in DNA pairs with adenine in RNA

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• Steps
• Starting at promoter (signals the start of a
  gene), segment of DNA unwinds and unzips
• ½ of DNA will serve as a template (DNA template
  strand is in the 3 to 5 direction RNA in 5 to
  3)
• RNA polymerase joins the RNA nucleotides so that
  the codons in mRNA are complementary to the code
  in DNA.
• Termination signal (signals end of gene) is
  reached, process ends, and DNA closes back up

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Animation
Animation
From video shown in class
http//www.youtube.com/watch?v41_Ne5mS2ls
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Transcription and mRNA synthesis
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• RNA Processing
• DNA contains exons (parts of a gene that are
  expressed) and introns (intragene segments not
  expressed)
• Before mRNA leaves the nucleus, the introns are
  removed so that only the exons remain
• The splicing of mRNA is done by ribozymes,
  enzymes composed of RNA.
• Primary mRNA/pre-mRNA (with introns exons) is
  processed into mature mRNA (without introns).

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• Translation
• Protein constructed during this process
• Occurs at the ribosomes
• Key players in translation
• mRNA (messenger RNA)
• Made during transcription, has codons
• Travels from nucleus to ribosome
• Contains copy of DNA code to make protein
• tRNA (transfer RNA)
• rRNA (ribosomal RNA)

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• Transfer RNA (tRNA)
• tRNA molecules bring amino acids to the ribosomes
• Free-floating in the cytoplasm of the cell

• Each tRNA has a sequence of nucleotides called an
  anticodon it is this sequence that determines
  which amino acid each tRNA has

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• Complementary base pairing occurs between
  anticodons of tRNA and codons of mRNA
  determines the sequence of amino acids to
  construct the polypeptide.

• If mRNA codon is AUG, tRNA anticodon would be UAC

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• Ribosomal RNA (rRNA)
• rRNA is made in the nucleolus (a cell structure
  found inside the nucleus)
• Ribosome made of a large subunit and small
  subunit that join just prior to protein synthesis

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• Ribosome has a binding site for mRNA and binding
  sites for two tRNA molecules at a time.
• Several ribosomes may attach and translate the
  same mRNA, therefore the name polyribosome
  (letter c below).

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• Three Steps of Translation
• Chain initiation
• Chain elongation
• Chain termination.
• Enzymes are required for each step, and the first
  two steps require energy.

Animation
Animation
http//www.youtube.com/watch?vD5vH4Q_tAkY
From video shown in class
http//www.youtube.com/watch?v41_Ne5mS2ls
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• Chain Initiation
• Small ribosomal subunit attaches to the mRNA near
  the start codon.
• The anticodon of tRNA, called the initiator RNA,
  pairs with the start codon at the P site on
  ribosome.
• Large ribosomal subunit joins.

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• Chain Elongation
• The initiator tRNA passes its amino acid to a
  tRNA-amino acid complex that has come to the
  second binding site, the A site.
• The ribosome moves forward and the tRNA at the
  second binding site is now at the first site, a
  sequence called translocation.
• The previous tRNA leaves the ribosome at the E
  site of the ribosome

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• Chain Termination
• A stop-codon is reached.
• A release factor (an enzyme) breaks the
  polypeptide from the last tRNA
• The ribosome falls away from the mRNA molecule
  and separates into its two subunits
• A newly synthesized polypeptide may function
  alone or become part of a protein.

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Review of Gene Expression

• DNA in the nucleus contains a triplet code each
  group of three bases stands for one amino acid.
• During transcription, an mRNA copy of the DNA
  template is made.
• The mRNA is processed before leaving the nucleus.
• The mRNA joins with a ribosome, where tRNA
  carries the amino acids into position during
  translation.

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Control of Gene Expression in Prokaryotes The lac
operon

• Regulator gene codes for active repressor, which
  automatically attaches to the operator.
• RNA polymerase cannot attach to promoter, and
  transcription does not occur

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• When lactose attaches to repressor, it becomes
  inactive and cannot attach to the operator. Now
  RNA polymerase can attach to the promoter,
  transcription occurs, and the genes are expressed

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• Control of Gene Expression in Eukaryotes
• In eukaryotes, cells differ in which genes are
  being expressed based on cell function ex.
  nerve vs. muscle.
• One way that eukaryotes can control gene
  expression is through proteins called
  transcription factors
• Two types
• Those that guide and stabilize the binding of RNA
  polymerase to a promotor
• Those that control the rate of transcription (by
  controlling how DNA is folded or preventing
  activators from binding)

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• During development, cells become specialized.
  This differentiation is controlled by a set of
  genes called Homeobox (Hox) genes.
• They code for transcription factors and are
  active in specific parts of the DNA corresponding
  to specific parts of the body that is developing.
  They control what body part will develop in a
  specific location.

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• RNA interference
• Some viruses have double-stranded RNA
• An enzyme called dicer can cut this RNA into
  small segments
• When they attach to protein complexes in the
  cell, one of the strands breaks down.
• The remaining section attaches to molecules of
  mRNA, causing them to break and preventing
  translation

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• Gene Mutations
• Definition - a change in the sequence of bases
  within a gene
• Causes
• Mutations can be spontaneous or caused by
  environmental influences called mutagens.
• Cancer causing mutagens are called carcinogens
• Mutagens include radiation (X-rays, UV
  radiation), and organic chemicals (in cigarette
  smoke and pesticides).

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• Types
• Frameshift mutations
• one or more bases are inserted or deleted from a
  sequence of DNA
• can result in nonfunctional proteins
• can result in no protein at all stop codon
  where there shouldnt be one
• Point mutations
• One base is substituted for another
• May result in change of amino acid sequence
• May not affect protein at all

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• Types of point mutations
• Silent mutation - the change in the codon results
  in the same amino acid
• Ex UAU ? UAC both code for tyrosine
• Nonsense mutation - a codon is changed to a stop
  codon resulting protein may be too short to
  function
• Ex UAC ? UAG (a stop codon)

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• Missense mutation - involves the substitution of
  a different amino acid, the result may be a
  protein that cannot reach its final shape
• Ex Hbs which causes sickle-cell disease

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• In general, mutations can have any of the
  following effects
• No change in proteins made or appearance
• Wrong protein is made
• No protein in made
• New appearance may result

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• Repair of Mutations
• DNA polymerase proofreads the new strand against
  the old strand and detects mismatched pairs,
  reducing mistakes to one in a billion nucleotide
  pairs replicated.
• If errors occur in sex cells mutation may be
  passed onto offspring
• If errors occur in body cells - cancer may result

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Transposons Jumping Genes

• Transposons are specific DNA sequences that move
  from place to place within and between
  chromosomes.
• These jumping genes can cause a mutation to
  occur by altering gene expression.
• It is likely all organisms, including humans,
  have transposons.