The Molecular Basis of Inheritance

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The Molecular Basis of Inheritance

• Chapter 16

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I. DNA as the Genetic Material

• A. The Search for the Genetic Material
• 1. TH Morgans group linked genes to chromosomes
• Is it the DNA of chromsomes
• or the Protein of chromsomes?
• This would become the debate
• The diversity of proteins brought them to the
  forefront of opinion

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• 2. Frederick Griffith
• Began in 1928
• Studied strains of
• Streptococcus pneumoniae
• R strain - harmless
• S strain - pathogenic

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• Experiment
• mixed heat-killed S strain with
• live R strain
• injected into mouse
• Mouse died
• He recovered the S strain from the mouse

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• Experiment
• Called Transformation
• assimilation of a foreign substance by a cell
  changing its genotype and phenotype
• What is this transforming substance??
• This question would occupy geneticists for
  the next 14 years

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• 3. Avery, McCarty and MaCleod, 1944
• Announced it was DNA, not protein
• Many remained skeptical

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• 4. Hershey and Chase, 1952
• Finally provided key proof
• DNA is the genetic material

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• 4. Hershey and Chase, 1952
• The Experiment
• T2 phage virus
• Made of protein and DNA
• Attacks bacteria
• Injects genetic material into bacteria,
  turning it into a virus factory
• Question - what is that genetic material?
  DNA or Protein?

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• 4. Hershey and Chase, 1952
• The Experiment
• Grew T2 phages with the proteins marked with
  radioactive sulfur
• Grew T2 phages with DNA marked with
  radioactive phosphorus
• Allowed each to infect separate cultures
• Spun infected cells in a blender
• this shook loose viral parts left outside
  the bacteria
• Centrifuged - separated heavy bacteria cells
  from lighter free viral parts

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Hershey Chase Experiment
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• 4. Hershey and Chase, 1952
• The Experiment
• When tested, radioactive phosphorus was in the
  bacteria - hence DNA
• radioactive sulfur remained outside
• the protein
• Conclusion
• DNA must the the genetic material allowing
  the assembly of new viruses

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• 5. More Circumstantial Evidence
• During mitosis, cells double DNA and
• distribute it equally to daughter cells
• Diploid chromosome sets
• double the DNA as haploid sets

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• 6. Erwin Chargaffs Rules, 1947
• He already knew
• DNA a polymer of nucleotide
• Nucleotide
• Deoxyribose sugar
• Phosphate
• Nitrogenous base
• Four possible bases
• Adenine, Thymine
• Cytosine, Guanine

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• 6. Erwin Chargaffs Rules, 1947
• Chargaffs Rules
• of Adenine of Thymine
• of Guanine of Cytosine

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I. DNA as the Genetic Material

• B. The Structure of DNA
• Race to determine DNA structure - 1950s
• 1. Wilkins and Franklin
• X-ray crystallography methods to find its shape

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• B. The Structure of DNA
• Race to determine DNA structure - 1950s
• 2. Watson and Crick - 1953
• From Franklins photo
• DNA must be helical
• Measured width of helix
• Worked with wire models
• Came up with the double helix
• Side rails - sugar and phosphate
• Steps - base pairs
• A to T, C to G

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• B. The Structure of DNA
• 2. Watson and Crick - 1953
• Explained Chargaffs rules
• Linear Sequence of bases
• unlimited possibilities

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• B. The Structure of DNA
• 2. Watson and Crick - 1953
• 1953 - published the structure called the Double
  Helix Published in Nature

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II. DNA Replication and Repair

• A. DNA method of replication became clear to
  Watson and Crick due to base pairing
• Existing strands serve
• as template for new
• complimentary strands.
• DNA Replication -
• Watson and Cricks
• second published paper.

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• B. Various
• models
• proposed

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II. DNA Replication and Repair

• C. Watson and Crick proposed the
• Semi-conservative model
• Confirmed in late 1950s by
• Meselson and Stahl
• Labeled old strands with heavy N isotope.
• Were able to track their pathway

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II. DNA Replication and Repair

• D. The Process of Replication
• 1.Very fast and accurate
• 2. Replication begins at special sites
• Origins of Replication
• In prokaryotes
• Enzymes recognize a single seq.
• Strands separate here
• Replication bubble
• Replication goes both directions

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• In Eukaryotes
• There are 100s of origin sites per
  chromosome.
• Many bubbles form with replication forks
  at both ends

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• 3. Action of DNA Polymerase
• This enzyme adds the new nucleotides on to the
  growing strand
• The raw nucleotides are
• Nucleoside triphosphates
• nitrogen base
• deoxyribose sugar
• triphosphate tail

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• 3. Action of DNA Polymerase
• As nucleoside is added
• last two phosphate groups are hydrolyzed and
  broken off as
• pyrophosphate
• Only 1 phosphate remains in chain
• Pyrophosphate breaks exergonically
• back to two phosphates
• energy to drive the polymerization

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• 4. The two DNA strands are
• Anti-parallel - The sugar phosphate backbones
  run in opposite directions
• Each strand has
• 3 end
• 5 end
• phosphate
• end
• They run opposite

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• 4. The two DNA strands are
• Anti-parallel
• Implications
• DNA polymerase can only add
• nucleotides onto 3 end
• The new strand can only grow
• 5 to 3 direction
• At replication fork, the two strands are
  opposite

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• 4. The two DNA strands are
• Anti-parallel
• Result
• One strand can be added to continuously
  (3 to 5 into fork)
• The Leading Strand
• The other must be pulled out in segments
  and added to the other way. (5 to 3 out from
  fork)
• The Lagging Strand
• The segments - Okazaki fragments

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• 4. The two DNA strands are
• Anti-parallel
• Result
• The Okazaki fragments (100-200 bases)
• are joined by DNA Ligase

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• 5. The Initiation of Replication
• DNA Polymerase can only add to an existing
  chain.
• Starting a new chain needs a Primer
• A short RNA segment (10 bases)
• Primase builds the primer onto a DNA strand by
  adding Ribonucleotides
• Once the primer is built,
• DNA Polymerase can add to the 3 end of the
  primer.
• Later, DNA Polymerase replaces the primer
  with normal nucleotides

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• 5. The Initiation of Replication
• The Leading strand
• Needs a single primer
• The lagging strand
• New primer for each
• Okazaki fragment
• Primer is converted to
• DNA before DNA ligase
• Seals it

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• 6. Action of Helicase - untwists and unzips DNA
  at the replication fork
• 7. Single-strand binding proteins - keep the
  strands apart during replication

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• E. DNA Proofreading and repair
• One error per 10,000 bases
• 1. DNA polymerase proofreads immediately
• If incorrect, wrong nucleotide is
• removed, correct one added.
• Final error rate, 1/ 1billion
• 2. Further mistakes can occur
• Each cell monitors and seeks to repair
• 130 different repair enzymes found in
  humans

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• E. DNA Proofreading and repair
• One error per 10,000 bases
• 3. Types of repairs
• Mismatch repair - fix incorrectly paired
  nucleotides
• Nucleotide Excision repair - nuclease cuts
  out segment of damaged strand
• The gap filled by DNA polymerase and ligase
• When repair fails - disorders result
• example - Xeroderma pigmentosum

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• F. Replicating the Ends of a DNA molecule
• DNA Polymerase has trouble at the final ends -
  No way to complete the ends
• Repeated replication shortens the DNA

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• F. Replicating the Ends of a DNA molecule
• Defense against this?
• Telomeres
• Special sequences at the ends
• Typically TTAGGG repeated 100 to 1000x
• These slowly become eroded and protect the
  actual code section of a gene

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• F. Replicating the Ends of a DNA molecule
• Restoring Telomeres possible in eukaryotes
• Telomerase
• Extends the 3 end of telomere
• Lengthens telomere
• Most multicelled organism cells do not have
  telomerase however
• Telomeres do shorten
• may limit lifespans of certain tissues
• Telomerase present in
• germ-line cells
• cancerous somatic cells

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III. DNA Organization in Chromosomes

• A. In Prokaryotes
• 1. DNA associated with only a small
• amount of protein
• 2. Double stranded circular molecule
• 3. Packed into a DNA dense region
• called a Nucleoid

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III. DNA Organization in Chromosomes

• B. In Eurkaryotes
• 1. DNA associated with a large amount of
  protein
• 2. Long linear chromosomes
• 3. DNA its protein Chromatin

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III. DNA Organization in Chromosomes

• B. In Eurkaryotes
• 4. DNA Packing in eukaryotes
• a. The Double Helix sides are negative
• charged (due to phosphates)

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• 4. DNA Packing in eukaryotes
• b. Double helix chain wraps around
• positive proteins Histones
• amino acids lysine or arginine
• 4 key types H2A, H2B, H3, H4
• Total mass of histones
• total mass of DNA in chromatin

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• 4. DNA Packing in eukaryotes
• c. DNA wraps around a cluster of 8 histones
• creates a bead appearance
• each bead Nucleosome
• made of 2 of each of the 4 types
• of histones
• Each histone has a tail (N terminus) that
• hangs out from the nucleosome

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• 4. DNA Packing in eukaryotes
• d. Chain of nucleosomes with a length of
• linker DNA between them.
• A 5th histone type H1
• causes this chain to coil or fold
• into a thicker fiber (30nm)

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• 4. DNA Packing in eukaryotes
• e. The 30nm fiber forms looped domains
• These loops attach to a protein scaffold

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• 4. DNA Packing in eukaryotes
• f. During mitosis, this length of looped domains
  coils and folds further
• makes the familiar condensed chromosome
  700nm thick

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• 4. DNA Packing in eukaryotes
• g. Temporal changes in packing density
• diffuse during interphase
• condenses during prophase metaphase

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• 4. DNA Packing in eukaryotes
• h. Normal interphase condition
• Some parts 10mn fiber
• Other parts 30mn string of looped domains
• Looped domains appear attached to
• nuclear lamina and to fibers of a
• nuclear matrix
• Keeps chromatin organized for use
• Chromatin of each chromosome occupies a
  specific region

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• 4. DNA Packing in eukaryotes
• i. Some clumps highly packed
• (especially centromeres telomeres)
• Called Heterochromatin
• Inaccessible for use
• Most areas loosely packed
• Called Euchromatin
• Accessible for use

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• 4. DNA Packing in eukaryotes
• j. Degree of packing can undergo modification
  during the activities of the cell
• Affected by access needs
• Histones may play a role here

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