Part Two Lecture I

choppedspleenMechanics

Feb 21, 2014 (3 years and 1 month ago)

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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 254
-
260 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