Molecular Genetics

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Molecular Genetics
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BACKGROUND

• 1860 - Gregor Mendel determined patterns of
  inheritance
• 1868 Friedrich Miescher discovered material
  inside the cell nucleus (chromosomes) is half
  protein and half something else

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• - other half later discovered to be DNA
  (deoxyribonucleic acid)
• 1902 - Walter Sutton genetic material is found
  on chromosomes

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Conclusion

• 1. Chromosomes are made up of DNA Protein
• 2. Which one makes up the GENES?

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Experiments to Determine DNA or Protein

• 1. Frederick Griffith (1928) was attempting to
  develop a vaccine against pneumonia. He never
  succeeded but did make some important discoveries
  concerning DNA .

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Griffiths Experiment

• Took 2 strains of bacteria Streptococcus
  pnuemoniae inject them into mice in 4 different
  experiments
• 1) Bacteria Enclosed in a smooth mucous coat
  (smooth S strain) kill mice
• 2) Bacteria with Coat absent (rough R strain)
  mice live
• 3) Heated strain S bacteria made harmless,
  mice lived
• 4) Mixed heated S strain with R strain MICE
  DIED!!

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Conclusion

• Transformation had taken place. Transformation
  process by which bacterial cells incorporate DNA
  from dead bacterial cells (transfer of genetic
  information). The question remains Is DNA or
  protein portion of the chromosome responsible for
  transformation?

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Avery, McCarty, Macleod

• 2. Oswald Avery, Colin Macleod, Maclyn McCarty
  (1944)
• Strong evidence for DNA as the transforming
  principle.
• Used Enzymes (repeated Griffith experiment)
• Use a Protein destroying enzyme transformation
  still occurs
• Use a DNA NO
  transformation!

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Hershey Chase

• 3. Martha Chase Alfred Hershey (1952) Proved
  DNA is the hereditary material
• Used a Bacteriophage a virus that infects a
  bacteria cell made of a DNA core protein coat
  attached radioactive labels (32P to DNA 35S to
  Protein) in two different batches.

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• Viruses given time to attach to bacteria and
  inject their genetic material
• Separated the mixture using a high speed
  centrifuge, this removes any viral material
  remaining on the outside
• 35S radioactivity found only in liquid
• 32P radioactivity found only in bacteria

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ALL NEW VIRUSES produced in future generations
contained only radioactive 32P

• CONCLUSION DNA and NOT protein must be the
  genetic material!

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The Structure of a DNA Molecule

• Nucleotides subunits of DNA made up of 3
  components
• 1. 5 - Carbon Sugar molecule (deoxyribose)
• 2. Phosphate group
• 3. Nitrogen Base (4)

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Nitrogenous Bases

• Purines
• - Adenine Guanine
• - Double ring structure
• Pyrimidines

- Cytosine Thymine
- Single ring structure
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Hydrogen bonds hold the nitrogen bases together
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Determining the Structure of a DNA Molecule

• Erwin Chargaff (1950) discovered in cells that
  equal amounts of A T and G C always exist.
• Chargaffs Rule AT CG (Purine always bonded
  to a pyrimidine)

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Determining the Structure of a DNA Molecule

• Rosalind Franklin (1954) used X-ray diffraction
  to determine that DNA is a long, thin molecule.
  She interpreted the shape of a DNA molecule to be
  in the shape of a helix (single coil)

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Determining the Structure of a DNA Molecule

• James Watson Francis Crick (1962) determined
  the structure of a DNA molecule to be in the
  shape of a Double Helix (twisted ladder)

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DNA STRUCTURE

• DNA molecule is made of COMPLEMENTARY strands
• one strand A T T G C A T
• Complement T A A C G T A

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DNA STRUCTURE

• Twisted ladder structure
• Sugar - Phosphate backbone outside rails of the
  ladder, held together by strong covalent bonds
• Nitrogen Base Pairs make up the inside rungs
  (steps) held together by weak hydrogen bonds

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DNA ReplicationHow does DNA make a copy of
itself before Mitosis?

• Replication process by which genetic information
  gets copied such as during Interphase of the cell
  cycle
• Involves separating unzipping the DNA molecule
  into 2 strands
• Each strand serves as a template for making a new
  complementary strand
• The process is SEMI CONSERVATIVE each new
  molecule consists of one new and one old strand
  of DNA
• the sequence of bases gets preserved

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Steps in the process of Replication

• 1. Enzyme Helicase unwinds the DNA helix (1A)
• 2. A Y-shaped Replication Fork results (1B)
• 3. Single stranded DNA binding proteins prevent
  the strands from recombining (1C)

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Steps in the Process of Replication (cont.)

• 4. Topoisomerase removes any twists or knots that
  form (1D)
• 5. RNA Primase initiates DNA replication at
  special nucleotide sequences called origins of
  replication using RNA Primers

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Steps in the Process of Replication (cont.)

• 6. DNA Polymerase attaches to the RNA primers and
  begins elongation adding DNA nucleotides to the
  complement strand DNA polymerase moves in the
  3 ? 5 direction along each template (3)

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Steps in the Process of Replication (cont.)

• 7. The Leading Complementary Strand ( 5 ?3 )
  is assembled continuously (4)
• 8. The Lagging Complementary Strand ( 3 ?5 ) is
  assembled in short Okazaki fragments which are
  joined by DNA Ligase (5A, 5B)

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Steps in the Process of Replication (cont.)

• 9. RNA primers get replaced by DNA nucleotides

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Mutations any sequence of nucleotides that does
not match the original DNA molecule from which it
was made

• Mutagen anything that causes a mutation to
  occur (UV light, radiation, drugs, chemicals
  etc.)
• DNA can proof read itself
• DNA polymerase often does this
• Excision repair enzymes can fix mistakes

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Types of Mutations

• Original DNA MESSAGE
• THE DOG RAN AND THE FOX DID TOO
• Dna is read by the cell 3 base letters (CODON) at
  a time, this is called a Reading Frame

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Point (substitution) an incorrect nucleotide

• THE HOG RAN AND THE FOX DID TOO

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Deletion missing nucleotide

• THE DOG RAN AND THE FOX DID TO

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Insertion additional nucleotide is added

• THE DOG RAA NAN DTH EFO XDI DTO O
• Frameshift mutation reading frame is every 3
  bases (Codon)

What the ...
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Duplication section of nucleotides gets
repeated

• THE DOG THE DOG THE DOG RAN AND THE FOX DID TOO

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Inversion sequence of nucleotides gets turned
around

• THE GOD RAN AND THE FOX DID TOO

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Translocation sequence of nucleotides gets
moved to another chromosome

• THE DOG RAN AND THE CAT HAS FUN ALL DAY

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Protein Synthesis

• DNA in chromosomes contains genetic instructions
• Those instructions regulate development, growth,
  and metabolic activities.
• They also determine cell type and characteristics
• DNA controls the cell by using codes of
  Polypeptides (Proteins)
• Polypeptides (Proteins) enzymes that regulate
  chemical reactions or structural components

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• GENE (genotype) genetic information for a
  particular trait
• From a molecular viewpoint traits are the end
  product of metabolic processes regulated by
  enzymes!
• The GENE is the DNA segment that codes for a
  particular polypeptide (protein)
  One-gene-one-polypeptide hypothesis

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Protein Synthesis process by which enzymes and
other proteins are manufactured from the
information contained in DNA

• Consists of three steps
• 1. Transcription transfer of information from a
  strand of DNA to a strand of RNA
• 2. RNA Processing modifies the RNA molecule
  with deletions and additions
• 3. Translation processed RNA used to assemble
  amino acids into a polypeptide

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3 types of RNA are involved in the process

• 1. Messenger RNA (mRNA) carries protein
  building instructions out of the nucleus
• 2. Transfer RNA (tRNA) carries amino acids to
  ribosomes
• 3. Ribosomal RNA (rRNA) building blocks of
  ribosomes which coordinate the activities of mRNA
  and tRNA

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How is RNA Different from DNA?

• RNA
• Is single stranded
• Bases A, G, C and U (Uracil) replaces T
• Sugar Ribose

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CODON vs. ANTICODON

• Codon a triplet group of 3 adjacent nucleotides
  in mRNA codes for one specific amino acid
• Anticodon a triplet group of 3 adjacent
  nucleotides in tRNA complementary to mRNA

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PROTEIN SYNTHESIS

• Transcription
• .Initiation RNA polymerase attaches to promoter
  regions on DNA and begins to unzip the DNA into 2
  strands. Promoter region contains the sequence
  T-A-T-A (called the TATA box)
• .Elongation RNA nucleotides are assembled using
  one side of the DNA molecule as a template (5
  ?3)
• Termination RNA polymerase reaches a special
  sequence of nucleotides that serve as a stop
  point Usually AAAAAAA

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PROTEIN SYNTHESIS

• Alterations take place before the mRNA leaves the
  nucleus
• A 5 Cap is added to the 5 end of the molecule
• 5 Cap GTP (guanosine triphosphate)
• This provides stability to the mRNA
• Provides a point of attachment for the ribosome
  (small unit)

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• Poly-A Tail added to the 3 end
• A sequence of 150 to 200 adenine nucleotides
• The tail provides stability
• Controls the movement of the mRNA across the
  nuclear membrane
• Some mRNA segments get removed
• Exons sequences that express a code for a
  protein
• Introns intervening sequences that are
  noncoding
• SnRNPs (small nuclear ribonucleoproteins)
  delete out the introns and splice the exons

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PROTEIN SYNTHESIS

• Translation
• 1. Initiation small ribosomal subunit attaches
  to a special region near the 5 end of the mRNA
• 2. A tRNA with the anticodon UAC attaches to the
  mRNA start codon AUG
• 3. Large ribosomal subunit now attaches to the
  mRNA

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• 4. Elongation tRNAs deliver their amino acids
  to the growing polypeptide
• 5. Ribosome moves over to the next codon and
  repeats the process
• 6. Polypeptide chain elongates one amino acid at
  a time
• 7. Termination occurs when ribosome encounters
  a stop codon
• The completed protein can now be used by the cell
  as a structural unit or as an enzyme!!

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DNA Organization

• DNA packaged with proteins forms a matrix called
  Chromatin
• During cell division DNA compact Chromosomes
• Transposons segments of DNA able to move to new
  locations on the same chromosome or to a
  different chromosome altogether
• Transposons have the effect of a mutation

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Control of Gene Expression

• Every cell in a human contains the exact same
  sequences of DNA
• Cells obviously have different functions however
• Gene expression is regulated by the activation
  then of only certain genes

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• Example gene regulation in E. coli (well
  understood)
• OPERONS sequence of DNA that direct particular
  biosynthetic pathways.

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4 Major Parts of an Operon

• 1. A regulatory gene produces a repressor
  protein that prevents gene expression by blocking
  the action of RNA polymerase
• 2. Promoter region of DNA attaches to RNA
  polymerase to begin transcription
• 3. Operator region blocks the action of RNA
  polymerase
• 4. Structural Genes contain DNA that codes for
  several related enzymes that direct the
  production of a product

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Lac Operon in E. coli controls the breakdown of
Lactose

• Lactose is required to turn on the operon that
  codes for the enzymes that break down lactose.
• If lactose is not present the enzymes are not
  made.