Transcript Part Two – Lecture I

Part Two – Lecture I
Forms of DNA
A DNA
 Rosalind Franklin focused on this form
 Prevalent under high salt
concentrations
 More compact
 Modification of major and minor
grooves
Z DNA discovered
 1979 – Andrew Wang – synthetic
oligonucleotide
 1.8 nm in diameter
 12 base pairs per turn
 G-C base pairs
Ultracentrifugation and the
Svedburg coefficient
 DNA and RNA may be analyzed by
ultracentrifugation
 RNAs are differentiated according to
their sedimentation behavior when
centrifuged at high speeds in a
concentration gradient
Sedimentation Behavior
Sedimentation behavior depends upon
the molecule’s
1. Density
2. Mass
3. Shape

Sedimentation equilibrium
centrifugation
 A density gradient is created that
overlaps the densities of the individual
components of a mixture of molecules.
 The gradient is usually made of a heavy
metal salt such as CsCl
 During centrifugation, the molecules
migrate until they reach a point of
neutral buoyant density
Sedimentation equilibrium
centrifugation
 Can also be used to study the GC
content
 The number of GC pairs in the DNA
molecule is proportional to the
molecule’s buoyant density
Denaturation and Renaturation of
DNA Molecules
 When denaturation of the double
stranded DNA occurs, the hydrogen
bonds open, the duplex unwinds, and
the strand separate
 No covalent bonds break so that the
strands stay intact
 Strand separation can be induced by
heat
Denaturation and uv
spectrophotometry
 Nucleic acids absorb ultraviolet light
most strongly at wavelengths of 254260 nm due to the interaction of the
UV light and the rings of the purines
and pyrimidines
UV spectrophotometry
 The increase of UV absorption of
heated DNA is referred to as the
hyperchromic shift and is easiest to
measure
Renaturation
 Denaturation can be reversed – by slowly
cooling the DNA
 Single strands of DNA can randomly find
their complementary strands and
reassociate
 The hydrogen bonds will form slowly and
then more and more duplexes or double
helixes will form
Molecular Hybridization
 This technique is based upon the
denaturation and renaturation of DNA
 In this case DNA from two different
sources can be mixed
 DNA and RNA and be mixed together –
a transcript can find its
complementary sequence in DNA
Molecular
Hybridization
 Used to determine
the amount of
complementarity or
similarity between
two different
species
Proteins are polymers
 Proteins are polymers of amino acids.
They are molecules with diverse
structures and functions.
 Polymers are made up of units called
monomers
 The monomers in proteins are the 20
amino acids
Blotting Procedures
Autoradiograph
Fluorescent in situ hybridization - FISH
 In this procedure mitotic or
interphase cells are fixed to
slides and subjected to
hybridization conditions.

Biotin is complexed with
the DNA and then bound to
a fluorescent molecule such
as fluorescein
Examples of fluorescence
Reassociation kinetics - Britten
 Used with small fragments of DNA
 DNA is then denatured
 Temperature is lowered and reassociation
monitored
 Used to compare different organisms
 Originally uncovered repetitive DNA
sequences due to a greater than anticipated
complmentarity
Reassociation kinetics and repetitive
DNA
Electrophoresis
 Separates molecules ina mixture by
causing them to migrate under the
influence of an electric field
 A sample is placed in a porous media
such as agarose or polyacrylamide gel
 They are then placed in a solution
(buffer) which conducts an electric
current
Separation of DNA
 DNA has a strong negative charge due
to the phosphate groups
 When the DNA is placed in the gel, it
will migrate toward the positive
electrode
Agarose Gel Electrophoresis
Staining
SDS Polyacrylamide Gels
 Vertical gel
 SDS used to
denature proteins
 Proteins run or
separate according
to their molecular
mass
Native Protein Gels
Native Gels
 In native gels, the proteins migrate
according to a mass/charge ratio
 In the case of hemoglobin the variant
forms are able to be separated based
upon a difference of charge due to the
substitution of amino acids from the
Beta globin chain
Protein Facts
 Proteins: Polymers of Amino Acids
 Proteins are polymers of amino acids. They are
molecules with diverse structures and functions.
 Each different type of protein has a characteristic
amino acid composition and order.
 Proteins range in size from a few amino acids to
thousands of them.
 Folding is crucial to the function of a protein and is
influenced largely by the sequence of amino acids.
Proteins: Polymers of Amino
Acids
 Each different type of protein has a
characteristic amino acid composition
and order.
 Proteins range in size from a few amino
acids to thousands of them.
 Folding is crucial to the function of a
protein and is influenced largely by the
sequence of amino acids.
Proteins are complex molecules
 They have levels of structure
 Structure based upon the sequence of
the amino acids
Polar side chains
Non Polar Hydrophobic side
chains
Electrical charged hydrophilic
Function of Proteins - continued
 Enzymes – Biological catalysts
 Transport of small molecules – Albumin and
haptoglobin
 Transport of oxygen – hemoglobin and
myoglobin
 Membrane proteins – to assist in support
 Channels in membranes – to allow the
passage of molecules or ions
 Electron carriers in electron transport in
the production of ATP
Functions( continued)i





Clotting proteins
Immune proteins to fight infectious agents
Histones – DNA binding proteins
Toxins to repel or kill other organisms
Bacteriocins – molecules produced by
bacteria against bacteria
Functions of proteins
 Hormones – Growth hormone
 Receptors – to Receive information so that
cell can communicate with other cells
 Neurotransmitters – messenger molecules –
to send information between neurons
 Cytoskeleton – actin, myosin, and collagen –
the structure of connective tissue and
muscles
 Antibodies – Immunoglobulins to fight
disease
Four levels of Protein Structure
 There are four levels of protein structure:
primary, secondary, tertiary, and
quaternary.
 The precise sequence of amino acids is
called its primary structure.
 The peptide backbone consists of repeating
units of atoms: N—C—C—N—C—C.
 Enormous numbers of different proteins are
possible.
The causes of Tertiary
structure