Download DNA Structure & Function

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1866- Mendel's Paper
1875- Mitosis worked out
1890's- Meiosis worked out
1902- Sutton, Boveri et. al. connect
chromosomes to Meiosis.
1907- Morgans “fly room” provides support for
chromosomes as the hereditary material
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1928- Griffith discovers transformation
1944- Avery announces DNA is the
transforming material, but no one believes him
1952 Hershey & Chase use radio-labeled
bacteriophages to prove DNA must be the
genetic material
Review of the Experiments
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html
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Identify the process at the center of most
biotechnology research & development
Identify the scientists and describe their
experiments that led understanding the
Structure of DNA
Identify the purpose of the Meselson Stahl
experiment, explain the experimental
procedures, and major conclusions
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Describe the 3 stages of DNA replication
List the enzymes necessary for DNA
replication and identify both their function and
which stage they participate in
Identify similarities in DNA between
organisms of different species
Identify differences in DNA between
organisms of different species
List the 3 sources of DNA
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Explain what a plasmid is and how they’re
transferred to bacteria
Explain the importance of plasmids and
viruses in creating rDNA
Define “virus”
Explain the steps of viral gene therapy and
identify diseases that may be “cured” by this
therapy
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During the last decades of the twentieth
century, a “new” biotechnology industry grew
due to innovative techniques to both transfer
DNA between cells and to manipulate the cells
to manufacture specific proteins
The manipulation of genetic information is at
the center of most biotechnology research and
development
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After the Hershey and Chase experiment,
scientists were convinced that DNA contained
the genetic material…
But how is this information stored
 And how is it passed on?
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To answer these questions, scientists began
studying the structure of DNA
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1950’s arrangement of covalent bonds in
nucleic acids was established, but the 3
dimensional shape was still unknown
People important in elucidating the structure of
DNA
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Erwin Chargaff
Rosalind Franklin
Watson & Crick
 In 1949, Erwin Chargaff observed that for each
organism he studied, the amount of adenine
always equaled the amount of thymine…
 A=T
 Likewise, the amount of guanine always
equaled the amount of cytosine…
 C=G
 However, the amount of each equal pair differs
between different organisms.
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Ultimately her diffraction pictures were used
by Watson & Crick to determine the structure
of DNA
She determined the sugar phosphate
backbones were on the outside of the DNA
molecule *
 In 1952, she took many photographs of
sections of DNA using a method called X-Ray
Diffraction
 In the process, X-ray beams were bounced off of
DNA and the rays were diffracted or scattered onto
a piece of film
 This method is similar to shining a light on an
object and analyzing its shadow
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X-ray crystallography is an extremely precise means
of imaging the exact structure of a given molecule or
macromolecule in a crystal lattice.
A crystal is any regularly repeating arrangement of
unit cells which range in size from less than 100 atoms
— to tens of thousands
is famous for being the tool first used to discover the
structure of DNA. Also used to determine the
structure of
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diamond,
penicillin,
numerous proteins,
entire viruses
In all, over 400,000 structures have
been described using x-ray
crystallography.
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Researchers using X-ray crystallography grow
solid crystals of the molecules they study
crystallographers aim high-powered X-rays at
a tiny crystal containing trillions of identical
molecules.
The crystal scatters the X-rays onto an
electronic detector
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the electronic detector today is the same type used to
capture images in a digital camera.
After each blast of X-rays, the researchers
rotate the crystal enabling the them to capture
in three dimensions how the crystal scatters, or
diffracts, X-rays.
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The intensity of each diffracted ray is fed into a
computer, which uses a mathematical equation
called a Fourier transform to calculate the
position of every atom in the crystallized
molecule
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Watson gets a copy of Rosalind Franklins x-ray
diffraction of DNA and because he is familiar
with the types of patterns helical molecules
produce (thanks to working with Crick), he
immediately knows DNA is helical
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He’s was also able to deduce the width of the helix
and the spacing of the nitrogenous basses along it
The width suggested that it was made up of 2
strands (up till now it was believed to be made of 3
strands)
He also showed DNA has a uniform width the entire
length of the molecule
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Franklin had concluded the sugar-phosphate
backbones were on the outside of the helix
This arrangement is appealing because it put
the hydrophobic nitrogenous bases on the
inside where they would be shielded from the
aqueous environment
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They still thought like paired with like, A-A, T-T,
C-C, G-G
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If adenine
paired with
adenine, and
thymine paired
with thymine,
how would that
affect the
diameter of the
helix?
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Purine-purine pairs are too
wide, and pyrimidinepyrimidine pairs are too
narrow
Since adenine is always found
in the same amount as
thymine, it was determined
that adenine would pair with
thymine
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Coincidentally, adenine can only
form hydrogen bonds with
thymine, and cytosine can only
form H-bonds with guanine
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Watson & Crick ended their 1 page paper on the structure of DNA
by saying: “It has not escaped our notice that the specific pairing we
have postulated immediately suggests a possible copying mechanism for
the genetic material.”
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Semi-conservative: DNA strands separate 
each strand acts as a template to build a new
strand on
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Each daughter DNA molecule is composed of a
parent strand and a new strand
Conservative: DNA strands separate  each
strand acts as a template to build a new strand
on parent strands reassociate with each
other, and new strands associate with each
other
Dispersive: all 4 strands of DNA following
replication are a mixture of old and new DNA
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1st culture E. coli on a media containing a heavy
isotope of nitrogen 15N
Bacteria incorporate the 15N into the
nitrogenous bases of their DNA
Next, transfer bacteria into culture containing
only 14N
Any new DNA that the bacteria synthesized
would be lighter than the parent DNA made in
the 15N
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Meselson & Stahl could then distinguish DNA
of different densities by centrifuging the DNA
strands
Heavier Strands would travel farther down
then lighter strands
http://highered.mcgraw-hill.com/olc/dl/120076/bio22.swf
 The complementary structure of DNA is also
used as a basis to make exact copies of the
DNA each time a cell divides
 Cell division allows an organism to grow,
develop, and to replace old cells
 Enzymes, biological catalysts, are responsible
for synthesizing new strands of DNA
 DNA replication is also called DNA synthesis
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Initiation
Synthesis
Termination
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ORI: origins of replication
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Specific sites along a DNA molecule where
replication begins
 Bacteria have 1 circular chromosome, and 1 origin of
replication
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Proteins can recognize the DNA sequence in the
ORI, and use this sequence recognition to bind and
open up a replication bubble
Replication of DNA then proceeds in both directions
until the entire molecule has been copied
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May have hundreds or even thousands of
ORI’s
Multiple replication bubbles form and
eventually fuse, thus speeding up the
replication process
Replication proceeds in both directions as it
does in prokaryotes
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At the end of each replication bubble is a
replication fork, the Y-shaped region where the
parental DNA is being unwound
Several Kinds of proteins participate in the
unwinding
Helicase: enzymes that break the hydrogen bonds
holding the 2 strands together
 Single strand binding proteins (SSBP’s): bind the now
single stranded DNA to keep the 2 strands from
reassociating
 Topoisomerases: relieves the strain caused by the
untwisting of the DNA molecule by breaking the covalent
bonds within the DNA backbone, swiveling, and
rejoining the strands
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The unwound sections of DNA can now act as
a template to build a new strand on
Primase: an enzyme that comes in and builds
an RNA primer on the parental DNA strand
This step is necessary because DNA polymerase
can’t initiate synthesis of a new polynucleotide,
rather, it can only add nucleotides to a preexisting
strand
 DNA Polymerase is the enzyme that builds the new
DNA strand
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DNA Polymerases polymerize the synthesis of
a new strand of DNA by adding nucleotides to
a preexisting strand
SSBP’s
Topoisomerase
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The direction of
DNA affects
elongation
DNA polymerase
can only add
nucleotides to a
free 3’ end
Thus a new DNA
strand can only
elongate in the
5’3’ direction
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Because DNA polymerase can only add to the
free 3’ end of a growing polynucleotide chain,
one strand of DNA will be synthesized
continuously, and the other will be synthesized
discontinuously
The strand synthesized continuously is called
the leading strand
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Grows in the direction towards the replication fork
The strand synthesized discontinuously is
called the lagging strand
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The lagging strand grows in the direction away
from the replication fork
The lagging strand is synthesized
discontinuously in a series of fragments called
Okazaki Fragments
Okazaki Fragments must eventually be joined
together to form 1 continuous DNA stand
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Remember, DNA polymerase can only add
nucleotides to an already existing 3’end
This means every time a new Okazaki
fragment is started, a new RNA primer must 1st
be created
Before two okazaki fragments can be joined, all
the RNA must be excised and replaced with
DNA
DNA Polymerase I is responsible for removing
RNA primers
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Ligase: joins the sugar phosphate backbones of
al Okazaki Fragments into 1 continuous strand
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We tend to think of DNA replication looking
something akin to a train moving along a rain
track
This is INCORRECT in 2 important ways
First: all of the before mentioned proteins involved
in replication do not act individually, but rather
together as one large “DNA replication Machine”
Second: this DNA replication complex does not
move along the DNA strands, rather the DNA
moves through the complex
http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html#
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All DNA molecules are composed of 4
nucleotide monomers, each containing 1 of the
4 nitrogenous bases
Adenine
 Cytosine
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Thymine
Guanine
Virtually all DNA molecules form a double
helix
Nucleotide connect to each other through
strong phosphodiester bonds between sugars
and phosphate groups
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Hydrogen bonds between complementary
nitrogenous bases hold two DNA strands
together
Within a double stranded segment of DNA, the
2 strands are always antiparallel to each other
DNA always undergoes semiconservative
replication
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The number of chromosomes (DNA molecules)
per cell differes between organisms of different
species
The # of base pairs per strand can differ
The number and types of genes can differ
The number of noncoding sequences
The shape of the chromosomes
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Eukaryotes = linear
Prokaryotes = circular
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Because DNA is the molecule responsible for
heredity, all living things have DNA
There are 3 main categories of organisms that
we get DNA from, what do you think they are?
Prokaryotes
Eukaryotes
Viruses
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Prokaryotic DNA is usually contained in a
single circular chromosome that is found free
floating in the cytoplasm in a region of the cell
known as the nucleoid
This chromosome is usually supercoiled
folding over on itself like a twisted rubberband
Prokaryotes DO NOT have introns, which we
will discuss in greater detail later
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Some bacteria contain extra small rings of DNA
floating in the cytoplasm called a plasmid
Plasmids contain non-essential genes that
usually give the bacteria some additional
characteristic or phenotype that allows it to
survive under extreme conditions
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R plasmids contain genes for antibiotic resistance
 What do you think the R stands for?
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Bacteria can transfer plasmids, and thus genetic
information, between members of the same
species, and sometimes members of different
species
Plasmid transfer occurs through 2 main
processes
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Conjugation with other bacteria via the sex pilus
Transformation: when a cell takes up foreign DNA
from the environment
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The ability to transfer plasmids and thus
impart new phenotypes on bacteria provides a
mechanism to drive evolution and thus create
“superbugs” that are resistant to antibiotics
This is a leading cause of emerging infectious
diseases
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Because plasmids are small and easily
extracted from cells, they are often used as
rDNA vectors
Foreign DNA fragments (genes) can be cut and
pasted into the plasmids, and then introduced
to a new host organism
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Any cell can take up a plasmid, not just bacterial
cells
What enzyme (s) is/are necessary for cutting and
pasting DNA
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From protists, plants, animals, and fungi
Eukaryotes typically have several linear
chromosomes, with each species having a
characteristic chromosome number
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Note: every cell within a multicellular organism has the
same number and type of chromosomes
Eukaryotic genomes are typically larger than
prokaryotes and contain more non-coding
sequences of DAN (introns) than prokaryotes do
Total amount of DNA per cell is NOT related to
organism complexity
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Viruses are currently considered non-living,
obligate, intracellular, parasites
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This means they absolutely have to (obligate) infect a
cell (intracellular) and hijack its enzymes, machinery,
and other organism monomers to propagate
(parasite)
DON’T CALL THEM ORGANISMS
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An organism by definition is alive, and because
viruses don’t meet the characteristics of life they ar
enot
Call them “infectious particles”
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Viruses are often used in biotechnology
research as vectors to carry DNA into a target
cell
Viruses do not have a cellular structure
All viruses have a thick protein coat( capsid)
surrounding a nucleic acid core (DNA or RNA,
not both)
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Like plasmid DNA, viral DNA is small and often
used create rDNA which in turn is used to creat a
GMO
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Recombinant virus technology is one technique
used to do gene therapy
In this technique, a virus is used to insert
corrective genes (therapy) into cells that
contain defective genes
Viral gene therapies are currently being
researched in hopes of curing



Certain cancers
Some types of diabetes
Cystic fibrosis

The manipulation of genetic information
through innovative techniques to both transfer
DNA between cells and to manipulate the cells
to manufacture specific proteins is at the center
of most biotechnology research and
development



Erwin Chargaff: Adenine and thymine are
always present in equal amounts, and
cytosione & guanine are always present in
equal amounts within a molecule of DNA
Rosalind Franklin & Wilson: used X-ray
crystallography to image DNA
Watson & Crick: discovered that DNA is in the
form of a double helix and that a purine always
pairs with a pyrimidine (more specifically,
Apairs with T and C with G)
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Purpose: to determine if the method of DNA
replication was conservative, dispersive, or
semiconservative
Procedures:
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Grow bacteria in heavy N15 media
Transfer bacteria to lighter N14 media for 2 rounds
of replication
Centrifuge and see where the bands form
Conclusions: DNA replication is
semiconservative
Stage
Enzymes
Initiation
Helicase: unwinds DNA
Topoisomerase: relieves strain ahead of the replication fork by making
cuts through one of the DNA strands and
Termination
Elongation
SSBP’s: hold the 2 opened strands apart
Primase: puts down an RNA primer
DNA Polymerase III: builds the new strand of DNA
DNA Polymerase I: removes RNA primers and replaces them with
deoxynucleotides
Ligase: catalyzes the formation of the phosphodiester bond between the
sugar of one nucleotide and the phosphate group of another to connect
short sequences of DNA (like okazaki fragments)
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All DNA molecules are composed of 4 nucleotide
monomers, each containing 1 of the 4 nitrogenous
bases
double helix
Nucleotide connect to each other through strong
phosphodiester bonds between sugars and
phosphate groups
Hydrogen bonds between complementary
nitrogenous bases hold two DNA strands together
DNA is always antiparallel to each other
DNA undergoes semiconservative replication




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The number of chromosomes (DNA molecules)
per cell differes between organisms of different
species
The # of base pairs per strand can differ
The number and types of genes can differ
The number of noncoding sequences
The shape of the chromosomes


Eukaryotes = linear
Prokaryotes = circular
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Most Cells
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Eukaryotes
 Plants
 Animals
 Fungi
 protists
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Prokaryotes
 Archeabacteria
 Eubacteria
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Viruses
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Small circular pieces of extrachromosomal
DNA that contain non-essential genes that
usually give the bacteria some additional
characteristic or phenotype that allows it to
survive under extreme conditions
Plasmids are transferred through
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conjugation and
transformation

Foreign DNA fragments (genes) can be cut and
pasted into the plasmids, and then introduced
to a new host organism because plasmids are
small and easy to manipulate



non-living obligate intracellular parasites also
called infectious particles
Recombinant virus technology is a technique
used to do gene therapy in which a virus is
used to insert corrective genes into cells that
contain defective genes
Viral gene therapies are currently being
researched in hopes of curing



Certain cancers
Some types of diabetes
Cystic fibrosis