DNA, The Genetic Material
The Hammerling Experiment – Where is the hereditary information stored in a the cell?
A Danish biologist Joachim Hammerling in the 1930’s did some experimentation with a plant
Acetabularia to find this out. This plant grows up to 5 cm. and has distinct foot, stalk and cap
regions. The nucleus is located in the foot. He found this out by doing some graphing exchange
on 2 types of Acetabularia that grows different looking caps.
Robert Briggs & Thomas King did an experiment circa 1952 by removing the nucleus from a
tad pole egg and finding that it did not develop. After they replaced the nucleus, the egg
developed into a frog.
John Gordon took the experiment a step further by trying to find what part of the cell held
the genetic material. Through some difficulty he found out the nuclei held the genetic material.
F.C. Steward did some experimentation circa 1958 and found that all cells contain the
genetic material to generate an individual. The term given for this is TOTIPOTENT.
The Griffith Experiment – Which of the two, proteins or DNA, held the genes for heredity?
British biologist started work in 1928 experimenting with a pathogenic bacteria,
Streptococcus pneumoniae, and continued for 30 years. He injected mice with the disease
causing bacteria and the mice died. He later heat treated the bacteria, injecting into mice and they
lived. He removed the polysaccharide coating surrounding bacteria, and injected it into the mice
and they lived. He then injected the heat treated bacteria without the coating, and the mice died.
He concluded that something other than the coating held the genetic information that had
transformed the bacteria.
The Avery Experiment – The agent for transforming the Streptococcus however went undiscovered
until 1944. Oswald Avery, and his coworkers Colin Macleod, Maclyn McCarty characterized what
they referred to as the “transforming principle”. They prepared the same mixtures of bacteria that
Griffith had used. They removed 99.98% of the protein and found that the bacteria still
experienced the transformation. They concluded that a nucleic acid of deoxyribose type was the
fundamental unit of the transforming principle; in essence, DNA is the hereditary material.
The Hershey-Chase Experiment – Avery’s result was not widely accepted at first; many still
believed that proteins were responsible for the hereditary information.
Alfred Hershey and Martha Chase experimented with bacteriophages, viruses that attack
bacteria, and provided support for Avery’s theory circa 1952. These scientists put radioactive tags
on the virus, 32P on the DNA and 35S on the protein. After the viruses were permitted to infect the
bacteria, they agitated the container which caused the proteins to fall off the bacteria. Once this
was done they waited and checked the bacterial cells and found that they were infected with the
viruses and the radioactive tags. They concluded that DNA was the molecule of hereditary and not
the protein.
The Fraenkel-Conrat Experiment – Some viruses contain RNA instead of DNA and yet they
manage to reproduce quite satisfactorily. What genetic material do they use?
In 1957, Heinz Fraenkel-Conrat answered this question for two RNA-containing viruses:
tobacco mosaic virus (TMV) & Holmes ribgrass virus (HRV). The protein was stripped of the RNA
and it was determined once again that the RNA was infective and the protein was not. Further
investigations were done by taking the HRV nucleic acid and mixing it with TMV proteins, then
infecting a tobacco plant. The tobacco plant was infected with the hybrid and developed lesions
characteristic of HRV. Once again this demonstrated that the nucleic acid carried the hereditary
material and not the protein.
Retroviruses – RNA infects the cell and has to make a DNA strand. Because this process is
reverse of the normal way things are transcribed these viruses are called retroviruses.
DNA REPLICATION
SEMICONSERVATIVE replication means that while the DNA is unzipped, and being duplicated on
both strands, the duplicated strands are not going to be together. They will remain separated,
hence the term semi-conservative.
DNA has two strands that are COMPLEMENTARY to each other, meaning that one side matches
the other side with its complementary base. If the DNA is going to duplicate itself, it must “unzip” it
self in order to be read, and then duplicated. The enzyme responsible for adding nucleotides
together to match the original strand is called DNA POLYMERASE III
DNA Polymerase III is 10X larger than the DNA Polymerase I. (DNA Polymerase II found in prokaryotes only)
- contains 10 different kinds of polypeptide chains
- has 2 similar multi-subunit complexes (in order to synthesize both strands simultaneously)
- Each complex catalyzes replication of 1 strand
The subunits include:
- large catalytic alpha, catalyzes 5’  3’
- smaller subunit that proofreads 3’  5’ for mistakes
- ring shaped beta2 that clamps the polymerase III around the DNA double helix
This Polymerase moves at a fast rate (1000 nucleotides/second or 100 full turns of the helix, 0.34
micrometers). This enzyme can ONLY link nucleotides to the parent strand to the 3 ’ end (-OH
group). Replication always proceeds in the 5’  3’ direction. This is known as the LEADING
strand.
There is another enzyme that links on the LAGGING strand and it is an RNA polymerase called
PRIMASE. It does so by adding 10 RNA nucleotides complementary to the DNA parent template.
DNA polymerase recognizes the primer and adds DNA nucleotides to it in order to construct the
new DNA strands. Because this strand is synthesized away from the fork of the DNA, the
synthesis is broken into fragments, called Okazaki fragments (~ 100 to 200 nucleotides in
eukaryotes or 1000 to 2000 in prokaryotes).
There is a 3rd enzyme that is called LIGASE. Its job is to attach the fragments together.
This process of DNA replication is said to be SEMIDISCONTINUOUS.
The process of DNA replicating has 5 interlocking steps:
1. Opening up the DNA double helix.
a. binding of initiator proteins – to open the helix
b. enzymes called “helicases” move along a strand and opens the helix
c. a stabilizing protein binds to exposed single strand to protect them and to prevent
rewinding of the helix.
d. Enzymes known as topisomerases or gyrases cleave a strand of the helix (to relieve
the resulting twisting or torque) and allow it to swivel around the intact strand and
then reseals the broken strand.
2. Building a primer
a. DNA cannot be synthesized until a primer is constructed.
b. RNA primer so that it is temporary – easy to excise later.
3. Assembling complementary strands
4. Removing the primer
a. DNA polymerase I removes the RNA primer and fills in the Okazaki fragments.
5. Joining the Okazaki fragments
a. Ligase enzyme joins the fragments
The (History) Chemical Nature of DNA
1. DNA was discovered and named by Friedrich Miescher (German chemist) in 1869, only four
years after Mendels work was published. He extracted a white substance form the nuclei of
a human cell and fish sperm. He found a high proportion of nitrogen and phosphorus in the
substance. He termed the substance he found as “Nuclein”, later changed to nucleic acid
because it was slightly acidic.
2. P.A. Levine – 1920’s – determines primary structure of DNA to be composed of smaller
molecule: a. phosphate, b. 5-carbon sugar, c. nitrogen base. One phosphate + one sugar +
one nitrogen base = nucleotide. DNA & RNA are composed of nucleotides strung together.
There were four base molecules (adenine, thymine, guanine and cytosine). Thus there
were four different nucleotides. The phosphates bonded to the sugars to create a “chain” of
nucleotides.
Levine erroneously reported the same amounts of each kind of nucleotide in DNA.
Scientists originally though DNA as a polymer composed of GCAT sequences repeated
over and over. Scientists couldn’t see a coding system in this structure and thus it was
difficult to accept DNA as Avery’s transforming principle
3. Erwin Chargaff – 1940’s – demonstrated that DNA from different cells had different amounts
of the four bases. No matter what the source of DNA, the amount of adenine always equals
the amount of thymine (A = T). The amount of guanine always equals the amount of
cytosine (G = C)
4. Beadle & Tatum – 1941 – one gene/ one polypeptide hypothesis. The information encoded
within DNA of chromosomes acts to specify particular enzymes. They set out to create
Mendelian mutations in chromosomes and then study the effects of these mutations on an
organism ,Neurospora (fungus)
5. Maurice Wilkins and Rosalind Franklin – late 1940’s – Wilkins produced purified DNA fibers
and Franklin produced X-rays diffraction pictures of DNA. These images showed DNA to be
a helix and regular in width.
6. Jim Watson (American) and Francis Crick (Englishman) – 1953 – discovered the physical
structure of DNA. They’re model proposed:
a. DNA is a double helix – 2 strands of nucleotides wrapped around each other.
b. The bases were hydrophobic and pointed inward.
c. 2 bases were double-ringed compounds (adenine & guanine). 2 bases were singleringed compounds (thymine & cytosine). A & G are purines and C & T are
pyrimidines. A purine nucleotide always pairs with a pyrimidine nucleotide. This
keeps the diameter of the molecule regular in shape.
d. A bonds to T because of 2 hydrogen bonding sites. G bonds to C because of 3
hydrogen bonding sites.
e. The molecule is a “double-helix” – 2 strands of complimentary nucleotides wrapped
around each other like a twisted ladder.
7. Meselson – Stahl – 1958 – proved that DNA is semi-conservative, meaning that the strands
of DNA goes in opposite directions to each other. ex. 5’ATTGCAT 3’ and 3’ TAACGTA 5’.
They grew bacteria in medium containing heavy isotopes of N15 (nitrogen) which became
incorporated into the bases of bacterial DNA. At zero generation, all DNA is heavy, after
one replication all DNA has a hybrid density & after 2 replicated all DNA is hybrid or light.
8. Sanger, Frederick – 1953 – during the same year Watson & Crick unraveled the structure of
DNA, he announced the complete sequence of amino acids in the protein – insulin. His
achievement was extremely significant because it demonstrates for the first time that
proteins consisted of definable sequences of amino acids – every molecule has the same
amino acid sequence.
DNA REPLICATION
DNA strands “unzip” down the middle between the hydrogen bonds. Each half reconstructs
its complimentary half from free floating nucleotides. The two new DNA strands each contain ½
of the original “double helix” – semiconservative.
DNA unzips – origins of replication – multiple sites on DNA strand where replication begins
– a specific base sequence is necessary. Enzyme helicase causes DNA ot uncoil and
hydrogen bonds between bases to break, resulting in “replication bubbles”. The bonding of
new nucleotides during replication occurs at the replication forks (the spots where the DNA is
separating). DNA polymerase (enzyme) causes new nucleotides to bond on to original DNA
strands.
RNA primer – a sequence of RNA nucleotides bonded to DNA. DNA polymerase then
bonds DNA nucleotides to complete the complimentary half to the original DNA half. Eventually
the RNA primer nucleotides are replaced by DNA nucleotides.
The two sides of the original DNA molecule are replicated in opposite directions.
Replication on one side – leading strand – occurs as DNA nucleotides are bonded one at a
time moving towards the replication forks.
Replication on the other side moves away form the replication fork as DNA polymerase
assembles nucleotide segments called Okazaki fragments. The nucleotides are added as
segments rather than individually. Okazaki fragments are usually 100 – 200 nucleotides long in
eukaryotes.
DNA replication is said to be semidiscontinuous, since one half of the DNA strands
continuously adds nucleotides and other side adds nucleotide strands periodically.
Replication in prokaryotic cells – one replication bubble on plasmid (circular DNA).
Replication of strands is continuous in opposite directions. The same type of replication occurs
in the circular DNA of mitochondria and chloroplasts.
The EUKARYOTIC CHROMOSOME
Replication units – zones on chromosomes where replication starts. By having many
replication units the replication process is more rapid.
Human chromosomes have an average of 200 million nucleotide pairs. To compact the
DNA, it is wrapped around proteins called histones (they’re positive in charge & DNA is
negative). Eight histones are joined to form a cluster called a nucleosome and they form a
“string of beads” called chromatin. Heterochromatin is so tightly wound that RNA transcription
can’t occur. Euchromatin is less tightly coiled and transcribes RNA.
The chromosome represents the most tightly coiled chromatin. Karyotype is the visual
representation of a cells chromosome in metaphase.
GENE – length of DNA (nucleotides) that codes for production of polypeptide or complete
protein. Mutation is the change in the nucleotide sequence of a DNA strand.