Part Two – Lecture IPart Two – Lecture I

Forms of DNAForms of DNA

A DNAA DNA

Rosalind Franklin focused on this Rosalind Franklin focused on this formform

Prevalent under high salt Prevalent under high salt concentrationsconcentrations

More compactMore compact Modification of major and minor Modification of major and minor

groovesgrooves

Z DNA discovered Z DNA discovered

1979 – Andrew Wang – synthetic 1979 – Andrew Wang – synthetic oligonucleotideoligonucleotide

1.8 nm in diameter1.8 nm in diameter 12 base pairs per turn12 base pairs per turn G-C base pairsG-C base pairs

Ultracentrifugation and the Ultracentrifugation and the Svedburg coefficientSvedburg coefficient

DNA and RNA may be analyzed by DNA and RNA may be analyzed by ultracentrifugationultracentrifugation

RNAs are differentiated according to RNAs are differentiated according to their sedimentation behavior when their sedimentation behavior when centrifuged at high speeds in a centrifuged at high speeds in a concentration gradientconcentration gradient

Sedimentation BehaviorSedimentation Behavior

Sedimentation behavior depends Sedimentation behavior depends upon the molecule’supon the molecule’s

1.1. DensityDensity

2.2. MassMass

3.3. ShapeShape

Sedimentation equilibrium Sedimentation equilibrium centrifugationcentrifugation

A density gradient is created that A density gradient is created that overlaps the densities of the overlaps the densities of the individual components of a mixture individual components of a mixture of molecules.of molecules.

The gradient is usually made of a The gradient is usually made of a heavy metal salt such as CsClheavy metal salt such as CsCl

During centrifugation, the molecules During centrifugation, the molecules migrate until they reach a point of migrate until they reach a point of neutral buoyant densityneutral buoyant density

Sedimentation equilibrium Sedimentation equilibrium centrifugationcentrifugation

Can also be used to study the GC Can also be used to study the GC contentcontent

The number of GC pairs in the DNA The number of GC pairs in the DNA molecule is proportional to the molecule is proportional to the molecule’s buoyant densitymolecule’s buoyant density

Denaturation and Renaturation Denaturation and Renaturation of DNA Moleculesof DNA Molecules

When denaturation of the double When denaturation of the double stranded DNA occurs, the hydrogen stranded DNA occurs, the hydrogen bonds open, the duplex unwinds, bonds open, the duplex unwinds, and the strand separateand the strand separate

No covalent bonds break so that the No covalent bonds break so that the strands stay intactstrands stay intact

Strand separation can be induced Strand separation can be induced by heatby heat

Denaturation and uv Denaturation and uv spectrophotometryspectrophotometry

Nucleic acids absorb ultraviolet light Nucleic acids absorb ultraviolet light most strongly at wavelengths of most strongly at wavelengths of 254-260 nm due to the interaction 254-260 nm due to the interaction of the UV light and the rings of the of the UV light and the rings of the purines and pyrimidinespurines and pyrimidines

UV spectrophotometryUV spectrophotometry

The increase of UV absorption of The increase of UV absorption of heated DNA is referred to as the heated DNA is referred to as the hyperchromic shift and is easiest to hyperchromic shift and is easiest to measuremeasure

RenaturationRenaturation

Denaturation can be reversed – by slowly Denaturation can be reversed – by slowly cooling the DNAcooling the DNA

Single strands of DNA can randomly find Single strands of DNA can randomly find their complementary strands and their complementary strands and reassociatereassociate

The hydrogen bonds will form slowly and The hydrogen bonds will form slowly and then more and more duplexes or double then more and more duplexes or double helixes will formhelixes will form

Molecular HybridizationMolecular Hybridization

This technique is based upon the This technique is based upon the denaturation and renaturation of denaturation and renaturation of DNADNA

In this case DNA from two different In this case DNA from two different sources can be mixedsources can be mixed

DNA and RNA and be mixed DNA and RNA and be mixed together – a transcript can find its together – a transcript can find its complementary sequence in DNAcomplementary sequence in DNA

Molecular Molecular HybridizationHybridization

Used to determine Used to determine the amount of the amount of complementarity complementarity or similarity or similarity between two between two different speciesdifferent species

Proteins are polymersProteins are polymers

ProteinsProteins are polymers of are polymers of amino amino acidsacids. They are molecules with . They are molecules with diverse structures and functions.diverse structures and functions.

Polymers are made up of units Polymers are made up of units called monomerscalled monomers

The monomers in proteins are the The monomers in proteins are the 20 amino acids20 amino acids

Blotting ProceduresBlotting Procedures

AutoradiographAutoradiograph

Fluorescent in situ hybridization - Fluorescent in situ hybridization - FISHFISH

In this procedure mitotic In this procedure mitotic or interphase cells are or interphase cells are fixed to slides and fixed to slides and subjected to hybridization subjected to hybridization

conditionsconditions.. Biotin is complexed with Biotin is complexed with

the DNA and then bound the DNA and then bound to a fluorescent molecule to a fluorescent molecule such as fluoresceinsuch as fluorescein

Examples of fluorescence Examples of fluorescence

Reassociation kinetics - BrittenReassociation kinetics - Britten

Used with small fragments of DNAUsed with small fragments of DNA DNA is then denaturedDNA is then denatured Temperature is lowered and Temperature is lowered and

reassociation monitored reassociation monitored Used to compare different organismsUsed to compare different organisms Originally uncovered repetitive DNA Originally uncovered repetitive DNA

sequences due to a greater than sequences due to a greater than anticipated complmentarityanticipated complmentarity

Reassociation kinetics and Reassociation kinetics and repetitive DNArepetitive DNA

ElectrophoresisElectrophoresis

Separates molecules ina mixture by Separates molecules ina mixture by causing them to migrate under the causing them to migrate under the influence of an electric fieldinfluence of an electric field

A sample is placed in a porous media A sample is placed in a porous media such as agarose or polyacrylamide gelsuch as agarose or polyacrylamide gel

They are then placed in a solution They are then placed in a solution (buffer) which conducts an electric (buffer) which conducts an electric currentcurrent

Separation of DNASeparation of DNA

DNA has a strong negative charge DNA has a strong negative charge due to the phosphate groupsdue to the phosphate groups

When the DNA is placed in the gel, When the DNA is placed in the gel, it will migrate toward the positive it will migrate toward the positive electrodeelectrode

Agarose Gel Agarose Gel ElectrophoresisElectrophoresis

StainingStaining

SDS Polyacrylamide GelsSDS Polyacrylamide Gels

Vertical gelVertical gel SDS used to SDS used to

denature proteinsdenature proteins Proteins run or Proteins run or

separate separate according to their according to their molecular massmolecular mass

Native Protein GelsNative Protein Gels

Native GelsNative Gels

In native gels, the proteins migrate In native gels, the proteins migrate according to a mass/charge ratioaccording to a mass/charge ratio

In the case of hemoglobin the In the case of hemoglobin the variant forms are able to be variant forms are able to be separated based upon a difference separated based upon a difference of charge due to the substitution of of charge due to the substitution of amino acids from the Beta globin amino acids from the Beta globin chainchain

Protein FactsProtein Facts

Proteins: Polymers of Amino AcidsProteins: Polymers of Amino Acids ProteinsProteins are polymers of are polymers of amino acidsamino acids. They . They

are molecules with diverse structures and are molecules with diverse structures and functions.functions.

Each different type of protein has a Each different type of protein has a characteristic amino acid composition and characteristic amino acid composition and order.order.

Proteins range in size from a few amino acids to Proteins range in size from a few amino acids to thousands of them. thousands of them.

Folding is crucial to the function of a protein and Folding is crucial to the function of a protein and is influenced largely by the sequence of amino is influenced largely by the sequence of amino acids.acids.

Proteins: Polymers of Proteins: Polymers of Amino AcidsAmino Acids

Each different type of protein has a Each different type of protein has a characteristic amino acid characteristic amino acid composition and order.composition and order.

Proteins range in size from a few Proteins range in size from a few amino acids to thousands of them. amino acids to thousands of them.

Folding is crucial to the function of a Folding is crucial to the function of a protein and is influenced largely by protein and is influenced largely by the sequence of amino acids.the sequence of amino acids.

Proteins are complex Proteins are complex moleculesmolecules

They have levels of structureThey have levels of structure Structure based upon the sequence Structure based upon the sequence

of the amino acidsof the amino acids

Polar side chainsPolar side chains

Non Polar Hydrophobic Non Polar Hydrophobic side chainsside chains

Electrical charged Electrical charged hydrophilichydrophilic

Function of Proteins - Function of Proteins - continuedcontinued

Enzymes – Biological catalystsEnzymes – Biological catalysts Transport of small molecules – Albumin Transport of small molecules – Albumin

and haptoglobinand haptoglobin Transport of oxygen – hemoglobin and Transport of oxygen – hemoglobin and

myoglobinmyoglobin Membrane proteins – to assist in supportMembrane proteins – to assist in support Channels in membranes – to allow the Channels in membranes – to allow the

passage of molecules or ionspassage of molecules or ions Electron carriers in electron transport in Electron carriers in electron transport in

the production of ATPthe production of ATP

Functions( continued)iFunctions( continued)i

Clotting proteinsClotting proteins Immune proteins to fight infectious Immune proteins to fight infectious

agentsagents Histones – DNA binding proteinsHistones – DNA binding proteins Toxins to repel or kill other organismsToxins to repel or kill other organisms Bacteriocins – molecules produced by Bacteriocins – molecules produced by

bacteria against bacteriabacteria against bacteria

Functions of proteinsFunctions of proteins

Hormones – Growth hormoneHormones – Growth hormone Receptors – to Receive information so that Receptors – to Receive information so that

cell can communicate with other cellscell can communicate with other cells Neurotransmitters – messenger molecules Neurotransmitters – messenger molecules

– to send information between neurons– to send information between neurons Cytoskeleton – actin, myosin, and collagen Cytoskeleton – actin, myosin, and collagen

– the structure of connective tissue and – the structure of connective tissue and musclesmuscles

Antibodies – Immunoglobulins to fight Antibodies – Immunoglobulins to fight diseasedisease

Four levels of Protein Four levels of Protein StructureStructure

There are four levels of protein structure: There are four levels of protein structure: primary, secondary, tertiary, and primary, secondary, tertiary, and quaternary. quaternary.

The precise sequence of amino acids is The precise sequence of amino acids is called its called its primary structureprimary structure. .

The peptide backbone consists of The peptide backbone consists of repeating units of atoms: N—C—C—N—Crepeating units of atoms: N—C—C—N—C—C.—C.

Enormous numbers of different proteins Enormous numbers of different proteins are possible.are possible.

The causes of Tertiary The causes of Tertiary structurestructure