DNA: The Genetic Material

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DNA The Genetic Material

• A Short history of DNA

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Classic Genetics

• When Mendel came out with his laws in 1865 , he
  used the term factors to describe what we today
  call genes. Genes were not known at that time
  .Actually not much about cell was known even when
  his work was rediscovered in 1900.
• Then when scientists started to explore more
  about genetic factors they thought the genes
  were located in the cytoplasm. ( cytoplasmic
  theory of inheritance) Chromosomes were known but
  most scientists belived genes were located
  somewhere in cytoplasm. Not much about
  chromosomes cell division was known which added
  to the strength of that theory.
• Then Sutton (1902) came out with his chromosomal
  theory of inheritance which stated that genes
  were located on the chromosomes. Many scientists
  followed with many experiments which established
  the chromosomal theory of inheritance as a
  irrefutable fact.

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Suttons Reasoning

• Sutton made two very important points in favour
  of his theory.
• It was known from breeding experiments that both
  parents contributed equally to the offspring. If
  that was the case cytoplasm couldnot have the
  genes as the egg and the sperms have very
  different amount of cytoplasm. Eggs are very
  large and mostly cytoplasm. Sperms are very
  small with almost no cytoplasm. ( very minimal
  cytoplasm). However the nucleus of both had the
  same amount of materials. He reasoned that if
  both parents contributed the same it the genes
  had to come from the nucleus.

Parallelism between meiosis and the behavior of
the genes. Genes are in pairs as do the
chromosomes. Half genes come from one parent the
other half from the other parent as in
fertilization- half chromosomes come from egg the
other half from the sperm . There are many other
parallelisms between the behavior of genes and
chromosomes during meiosis.
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Road to DNA Discovery

• Hammerling's experiment with the single celled
  green algae, Acetabularia, showed that the
  nucleus of a cell contains the genetic
  information that directs cellular development.
• A. mediterranea has a smooth, disc shaped cap,
  while A. crenulata has a branched, flower-like
  cap. Each Acetabularia cell is composed of three
  segments the "foot" or base which contains the
  nucleus, the "stalk," and the "cap."
• In his experiments, Hammerling grafted the stalk
  of one species of Acetabularia onto the foot of
  another species. In all cases, the cap that
  eventually developed on the grafted cell matched
  the species of the foot rather than that of the
  stalk. In this example, the cap that is allowed
  to grow on the grafted stalk looks like the base
  species one... A. mediterranea.
• This experiment shows that the base is
  responsible for the type of cap that grows. The
  nucleus that contains genetic information is in
  the base, so the nucleus directs cellular
  development.

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Hammerling's Acetabularia
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The Genetic Material

• Frederick Griffith, 1928
• studied Streptococcus pneumoniae, a pathogenic
  bacterium causing pneumonia
• there are 2 strains of Streptococcus
• - S strain is virulent
• - R strain is nonvirulent
• Griffith infected mice with these strains hoping
  to understand the difference between the strains

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The Genetic Material

• Griffiths results
• - live S strain cells killed the mice
• - live R strain cells did not kill the mice
• - heat-killed S strain cells did not kill the
  mice
• - heat-killed S strain live R strain cells
  killed the mice

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The Genetic Material

• Griffiths conclusion
• - information specifying virulence passed from
  the dead S strain cells into the live R strain
  cells
• - Griffith called the transfer of this
  information transformation

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The Genetic Material

• Avery, MacLeod, McCarty, 1944
• repeated Griffiths experiment using purified
  cell extracts and discovered
• - removal of all protein from the transforming
  material did not destroy its ability to transform
  R strain cells
• - DNA-digesting enzymes destroyed all
  transforming ability
• - the transforming material is DNA

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The Genetic Material

• Hershey Chase, 1952
• - investigated bacteriophages viruses that
  infect bacteria
• - the bacteriophage was composed of only DNA and
  protein
• - they wanted to determine which of these
  molecules is the genetic material that is
  injected into the bacteria

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The Genetic Material

• - Bacteriophage DNA was labeled with radioactive
  phosphorus (32P)
• - Bacteriophage protein was labeled with
  radioactive sulfur (35S)
• - radioactive molecules were tracked
• - only the bacteriophage DNA (as indicated by the
  32P) entered the bacteria and was used to produce
  more bacteriophage
• - conclusion DNA is the genetic material

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DNA Structure

• DNA is a nucleic acid.
• The building blocks of DNA are nucleotides, each
  composed of
• a 5-carbon sugar called deoxyribose
• a phosphate group (PO4)
• a nitrogenous base
• adenine, thymine, cytosine, guanine

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DNA Structure

• The nucleotide structure consists of
• the nitrogenous base attached to the 1 carbon of
  deoxyribose
• the phosphate group attached to the 5 carbon of
  deoxyribose
• a free hydroxyl group (-OH) at the 3 carbon of
  deoxyribose

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DNA Structure

• Nucleotides are connected to each other to form a
  long chain
• phosphodiester bond bond between adjacent
  nucleotides
• formed between the phosphate group of one
  nucleotide and the 3 OH of the next nucleotide
• The chain of nucleotides has a 5 to 3
  orientation.

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DNA Structure

• Determining the 3-dimmensional structure of DNA
  involved the work of a few scientists
• Erwin Chargaff determined that
• amount of adenine amount of thymine
• amount of cytosine amount of guanine
• This is known as Chargaffs Rules

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DNA Structure

• Rosalind Franklin and Maurice Wilkins
• Franklin performed X-ray diffraction studies to
  identify the 3-D structure
• discovered that DNA is helical
• discovered that the molecule has a diameter of
  2nm and makes a complete turn of the helix every
  3.4 nm

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DNA Structure

• James Watson and Francis Crick, 1953
• deduced the structure of DNA using evidence from
  Chargaff, Franklin, and others
• proposed a double helix structure

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DNA Structure

• The double helix consists of
• 2 sugar-phosphate backbones
• nitrogenous bases toward the interior of the
  molecule
• bases form hydrogen bonds with complementary
  bases on the opposite sugar-phosphate backbone

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DNA Structure

• The two strands of nucleotides are antiparallel
  to each other
• one is oriented 5 to 3, the other 3 to 5
• The two strands wrap around each other to create
  the helical shape of the molecule.

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DNA Replication

• Matthew Meselson Franklin Stahl, 1958
• investigated the process of DNA replication
• considered 3 possible mechanisms
• conservative model
• semiconservative model
• dispersive model

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DNA Replication

• Bacterial cells were grown in a heavy isotope of
  nitrogen, 15N
• all the DNA incorporated 15N
• cells were switched to media containing lighter
  14N
• DNA was extracted from the cells at various time
  intervals

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DNA Replication

• The DNA from different time points was analyzed
  for ratio of 15N to 14N it contained
• After 1 round of DNA replication, the DNA
  consisted of a 14N-15N hybrid molecule
• After 2 rounds of replication, the DNA contained
  2 types of molecules
• half the DNA was 14N-15N hybrid
• half the DNA was composed of 14N

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DNA Replication

• Meselson and Stahl concluded that the mechanism
  of DNA replication is the semiconservative model.
• Each strand of DNA acts as a template for the
  synthesis of a new strand.

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DNA Replication

• DNA replication includes
• initiation replication begins at an origin of
  replication
• elongation new strands of DNA are synthesized
  by DNA polymerase
• termination replication is terminated
  differently in prokaryotes and eukaryotes

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Prokaryotic DNA Replication

• The chromosome of a prokaryote is a circular
  molecule of DNA.
• Replication begins at one origin of replication
  and proceeds in both directions around the
  chromosome.

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Prokaryotic DNA Replication

• The double helix is unwound by the enzyme
  helicase
• DNA polymerase III (pol III) is the main
  polymerase responsible for the majority of DNA
  synthesis
• DNA polymerase III adds nucleotides to the 3 end
  of the daughter strand of DNA

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Prokaryotic DNA Replication

• DNA replication is semidiscontinuous.
• pol III can only add nucleotides to the 3 end of
  the newly synthesized strand
• DNA strands are antiparallel to each other
• leading strand is synthesized continuously (in
  the same direction as the replication fork)
• lagging strand is synthesized discontinuously
  creating Okazaki fragments

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Prokaryotic DNA Replication

• The enzymes for DNA replication are contained
  within the replisome.
• The replisome consists of
• the primosome - composed of primase and helicase
• 2 DNA polymerase III molecules
• The replication fork moves in 1 direction,
  synthesizing both strands simultaneously.

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Eukaryotic DNA Replication

• The larger size and complex packaging of
  eukaryotic chromosomes means they must be
  replicated from multiple origins of replication.
• The enzymes of eukaryotic DNA replication are
  more complex than those of prokaryotic cells.

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Eukaryotic DNA Replication

• Synthesizing the ends of the chromosomes is
  difficult because of the lack of a primer.
• With each round of DNA replication, the linear
  eukaryotic chromosome becomes shorter.

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Eukaryotic DNA Replication

• telomeres repeated DNA sequence on the ends of
  eukaryotic chromosomes
• produced by telomerase
• telomerase contains an RNA region that is used as
  a template so a DNA primer can be produced

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DNA Repair

• - DNA-damaging agents
• - repair mechanisms
• - specific vs. nonspecific mechanisms

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DNA Repair

• Mistakes during DNA replication can lead to
  changes in the DNA sequence and DNA damage.
• DNA can also be damaged by chemical or physical
  agents called mutagens.
• Repair mechanisms may be used to correct these
  problems.

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DNA Repair

• DNA repair mechanisms can be
• specific targeting a particular type of DNA
  damage
• photorepair of thymine dimers
• non-specific able to repair many different
  kinds of DNA damage
• excision repair to correct damaged or mismatched
  nitrogenous bases

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