Molecular Biology BIOL312

8/12/2019 Molecular Biology BIOL312

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: Molecular Biology

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BIOL 312


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Molecular Biology

BIOL 312

3: BI: G29:

G29: 01:20 11:40: 01:20 11:40: F37: 01:20 11:40: 9:40 8:00:


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Molecular BiologyMolecular biology =

1. The branch of biology that deals with the formation,structure, and function of macromolecules essential to life,

such as nucleic acids and proteins, and especially withtheir role in cell replication and the transmission of geneticinformation.2. The branch of biology that deals with the manipulationof DNA so that it can be sequenced or mutated. Ifmutated, the DNA is often inserted into the genome of anorganism to study the biological effects of the mutation.


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Genetic Engineering1. Genetic Engineering = is the development and

application of scientific procedures and technologiesthat permit direct manipulation of genetic material inorder to alter the hereditary traits of a cell, organism,

or population.

2. Genetic Engineering = is a technique producingunlimited amounts of otherwise unavailable or scarcebiological products by introducing DNA from livingorganisms into bacteria and then harvesting the product,as human insulin produced in bacteria by the humaninsulin gene. Also called biogenetics .


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INTRODUCTION TO ORGANICCOMPOUNDS


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Lifes molecular diversity is based on the properties ofcarbon

Diverse molecules found in cells arecomposed of carbon bonded to other carbons and

atoms of other elements. Carbon-based molecules are called organic

compounds.


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Lifes molecular diversity is based on the properties ofcarbon

Methane and other compounds composed ofonly carbon and hydrogen are calledhydrocarbons.

Carbon, with attached hydrogens, can bondtogether in chains of various lengths.


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Structuralformula

Ball-and-stickmodel

Space-fillingmodel

The four single bonds of carbon point to the corners of a tetrahedron.


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A carbon skeleton is a chain of carbonatoms that can be branched or

unbranched. Compounds with the same formula but

different structural arrangements are call

isomers.

Lifes molecular diversity is based on theproperties of carbon


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Animation: L-DopaRight click on animation / Click play


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Animation: Carbon SkeletonsRight click on animation / Click play


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Animation: IsomersRight click on animation / Click play


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Length. Carbon skeletons vary in length.

Ethane Propane

Butane Isobutane

Branching. Skeletons may be unbranchedor branched.

Double bonds. Skeletons may have double bonds.

1-Butene 2-Butene

Cyclohexane Benzene

Rings. Skeletons may be arranged in rings.


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Length. Carbon skeletons vary in length.

Ethane Propane


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Butane Isobutane

Branching. Skeletons may be unbranchedor branched.


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Double bonds.

1-Butene 2-Butene

Skeletons may have double bonds.


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Cyclohexane Benzene

Rings. Skeletons may be arranged in rings.


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A few chemical groups are key to the functioning ofbiological molecules

An organic compound has unique propertiesthat depend upon the size and shape of the molecule and

groups of atoms (functional groups) attached toit.

A functional group affects a biological

molecules function in a characteristic way. Compounds containing functional groups arehydrophilic (water-loving).


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3.2 A few chemical groups are key to the functioning ofbiological molecules

The functional groups are hydroxyl group consists of a hydrogen bonded

to an oxygen, carbonyl group a carbon linked by a double

bond to an oxygen atom, carboxyl group consists of a carbon double-

bonded to both an oxygen and a hydroxyl group,

amino group composed of a nitrogen bonded totwo hydrogen atoms and the carbon skeleton, and

phosphate group consists of a phosphorus atombonded to four oxygen atoms.


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A few chemical groups are key to the functioning ofbiological molecules

An example of similar compounds that differonly in functional groups is sex hormones. Male and female sex hormones differ only in

functional groups. The differences cause varied molecular actions. The result is distinguishable features of males

and females.


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Testosterone Estradiol


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Testosterone Estradiol


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Cells make a huge number of large molecules from alimited set of small molecules

There are four classes of molecules importantto organisms: carbohydrates,

proteins, lipids, and nucleic acids.


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The four classes of biological molecules containvery large molecules. They are often called macromolecules because of

their large size.

They are also called polymers because they aremade from identical building blocks strung together. The building blocks of polymers are called

monomers.


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Monomers are linked together to formpolymers through dehydration reactions ,which remove water.

Polymers are broken apart by hydrolysis ,the addition of water.

All biological reactions of this sort aremediated by enzymes , which speed up

chemical reactions in cells.


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Animation: Polymers

Right click on animation / Click play

f f


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A cell makes a large number of polymersfrom a small group of monomers. Forexample,

proteins are made from only 20 different aminoacids and DNA is built from just four kinds of nucleotides.

The monomers used to make polymers areuniversal.


Figure 3 3A s1


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Figure 3.3A_s1

Short polymer Unlinkedmonomer

Figure 3.3A s2


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Figure 3.3A_s2

Short polymer Unlinkedmonomer

Dehydration reactionforms a new bond

Longer polymer

Figure 3.3B s1


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g . _

Figure 3.3B s2


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g _

Hydrolysisbreaks a bond


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CARBOHYDRATES


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Monosaccharides are the simplest carbohydrates

Carbohydrates range from small sugarmolecules (monomers) to large polysaccharides. Sugar monomers are monosaccharides , such

as those found in honey, glucose, and fructose.

Monosaccharides can be hooked together toform more complex sugars and polysaccharides.


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The carbon skeletons of monosaccharidesvary in length. Glucose and fructose are six carbons long.

Others have three to seven carbon atoms. Monosaccharides are

the main fuels for cellular work and

used as raw materials to manufacture otherorganic molecules.


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Glucose(an aldose)

Fructose(a ketose)


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Many monosaccharides form rings.

Structuralformula

Abbreviatedstructure

Simplifiedstructure

6

5

4

3 2

1


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Two monosaccharides are linked to form a disaccharide

Two monosaccharides (monomers) canbond to form a disaccharide in adehydration reaction.

The disaccharide sucrose is formed bycombining a glucose monomer and

a fructose monomer. The disaccharide maltose is formed from

two glucose monomers.


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Animation: DisaccharidesRight click on animation / Click play


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Glucose Glucose


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Glucose Glucose

Maltose

Polysaccharides are long chains of sugar units


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Polysaccharides are macromolecules and polymers composed of thousands of

monosaccharides.

Polysaccharides may function as storage molecules or structural compounds.



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Starch is a polysaccharide, composed of glucose monomers, and used by plants for energy storage.

Glycogen is a polysaccharide, composed of glucose monomers, and used by animals for energy storage.



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Cellulose is a polymer of glucose and

forms plant cell walls.

Chitin is a polysaccharide and

used by insects and crustaceans to build anexoskeleton.


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Starch granulesin potato tuber cells

Glycogen granulesin muscletissue Glycogen

Glucosemonomer

Starch

Cellulose

Hydrogen bonds

Cellulosemolecules

Cellulose microfibrilsin a plant cell wall


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LIPIDS


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Fats are lipids that are mostly energy-storage molecules

Lipids are water insoluble ( hydrophobic , or water-

fearing) compounds, are important in long-term energy storage, contain twice as much energy as a

polysaccharide, and consist mainly of carbon and hydrogen atoms

linked by nonpolar covalent bonds.


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Lipids differ from carbohydrates, proteins,and nucleic acids in that they are not huge molecules and

not built from monomers. Lipids vary a great deal in

structure and

function.


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We will consider three types of lipids: fats, phospholipids, and

steroids. A fat is a large lipid made from two kinds of

smaller molecules,

glycerol and fatty acids.


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A fatty acid can link to glycerol by adehydration reaction. A fat contains one glycerol linked to three

fatty acids. Fats are often called triglycerides because of

their structure.


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Fatty acid

Glycerol


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Fatty acids

Glycerol

li id h l l l


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Some fatty acids contain one or more doublebonds, forming unsaturated fatty acids that have one fewer hydrogen atom on each carbon

of the double bond, cause kinks or bends in the carbon chain, and prevent them from packing together tightly and

solidifying at room temperature.

Fats with the maximum number ofhydrogens are called saturated fatty acids.

F li id h l l l


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Unsaturated fats include corn and olive oils. Most animal fats are saturated fats. Hydrogenated vegetable oils are

unsaturated fats that have been converted tosaturated fats by adding hydrogen.

This hydrogenation creates trans fats

associated with health risks.

Phospholipids and steroids are important lipids with a


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p p p pvariety of functions

Phospholipids are structurally similar to fats and the major component of all cells.

Phospholipids are structurally similar to fats. Fats contain three fatty acids attached to

glycerol. Phospholipids contain two fatty acids attached

to glycerol.


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Water

Hydrophobic tails

Water

Hydrophilic heads

Symbol for phospholipid

Phosphategroup

Glycerol


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PROTEINS

Proteins are made from amino acids linked by peptide


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y p pbonds

Proteins are involved in nearly every dynamic function in

your body and very diverse, with tens of thousands of different

proteins, each with a specific structure andfunction, in the human body.

Proteins are composed of differingarrangements of a common set of just 20amino acid monomers.



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y p pbonds

Amino acids have an amino group and a carboxyl group (which makes it an acid).

Also bonded to the central carbon is a hydrogen atom and a chemical group symbolized by R, which

determines the specific properties of each of the20 amino acids used to make proteins.


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Aminogroup

Carboxylgroup

3.11 Proteins are made from amino acids linked by


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peptide bonds

Amino acids are classified as either hydrophobic or

hydrophilic.


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Hydrophobic Hydrophilic

Aspartic acid (Asp)Serine (Ser)Leucine (Leu)



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bonds

Amino acid monomers are linked together in a dehydration reaction, joining carboxyl group of one amino acid to the

amino group of the next amino acid, and creating a peptide bond.

Additional amino acids can be added by thesame process to create a chain of aminoacids called a polypeptide .


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Carboxylgroup

Aminogroup

Amino acidAmino acid


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Carboxylgroup

Aminogroup

Amino acidAmino acid Dipeptide

Peptidebond

Dehydrationreaction

A proteins specific shape determines its function


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A protein s specific shape determines its function

Probably the most important role for proteinsis as enzymes , proteins that serve as metabolic catalysts and

regulate the chemical reactions within cells.



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Other proteins are also important. Structural proteins provide associations between body parts. Contractile proteins are found within muscle. Defensive proteins include antibodies of the immune system. Signal proteins are best exemplified by hormones and other

chemical messengers. Receptor proteins transmit signals into cells. Transport proteins carry oxygen. Storage proteins serve as a source of amino acids for developing

embryos.



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If a proteins shape is altered, it can nolonger function. In the process of denaturation, a

polypeptide chain unravels, loses its shape, and loses its function.

Proteins can be denatured by changes insalt concentration, pH, or by high heat.


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NUCLEIC ACIDS

Avery MacLeod and McCarty


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Avery, MacLeod and McCarty 1944 Avery, MacLeod and McCarty repeated

Griffiths 1928 experiment with modificationsdesigned to discover the transforming factor

Extracts from heat killed cells were digestedwith hydrolytic enzymes specific for differentclasses of macro molecules:

NoNuclease

YesProteaseYesLippase

Transformation?Enzyme

YesSaccharase

Transformation Of Bacteria


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Two Strains Of Streptococcus

Capsules

Smooth Strain(Virulent)

Rough Strain(Harmless)

Transformation Of Bacteria


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Experimental

The Griffith Experiment

- Control

+ Control

- Control

OUCH!

The Hershey-Chase Experiment


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The Hershey Chase Experiment

The Hershey-Chase experiment showeddefinitively that DNA is the genetic material Hershey and Chase took advantage of the fact

that T2 phage is made of only two classes ofmacromolecules: Protein and DNA

HOH

P

O

OHHO O

NH2

Nucleotides containphosphorous, thus DNA containsphosphorous, but not sulfur.

H

OH

OH2N CC

CH2

SH

H

OH

OH2N C

CH3

C

CH2

CH2

S Some amino acidscontain sulfur, thusproteins contain sulfur,

but not phosphorous.

CysteineMethionine

Using S35 Bacteria grown inT2 grown in S 35


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g Bacteria grown innormal non-radioactive media

T2 grown in S containing mediaincorporate S 35 into theirproteins

Blending causes phageprotein coat to fall off

T2 attach to bacteria

and inject geneticmaterial

Is protein the genetic

When centrifuged,

phage protein coatsremain in thesupernatant whilebacteria form apelletThe supernatant is

radioactive, but thepellet is not.

Did protein enter thebacteria?

Using P 32 Bacteria grown inT2 grown in P 32


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g Bacteria grown innormal non-radioactive media

T2 grown in P containing mediaincorporate P 32 into their DNA

Blending causes phageprotein coat to fall off

T2 attach to bacteria

and inject geneticmaterial

Is DNA the genetic material?

When centrifuged,

phage protein coatsremain in thesupernatant whilebacteria form apelletThe pellet is

radioactive, but thesupernatant is not.

Did DNA enter the bacteria?

DNA and RNA are the two types of nucleic acids


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The amino acid sequence of a polypeptide isprogrammed by a discrete unit of inheritanceknown as a gene .

Genes consist of DNA (deoxyribonucleicacid ), a type of nucleic acid .

DNA is inherited from an organisms parents. DNA provides directions for its own

replication. DNA programs a cells activities by directing

the synthesis of proteins.



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DNA does not build proteins directly. DNA works through an intermediary,

ribonucleic acid (RNA) .

DNA is transcribed into RNA.

RNA is translated into proteins.


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Gene

DNA


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Gene

DNA

Transcription

RNA

Nucleic acids


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Gene

DNA

Transcription

RNA

ProteinTranslation

Aminoacid

Nucleic acids

Nucleic acids are polymers of nucleotides


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p y

DNA (deoxyribonucleic acid ) and RNA (ribonucleic acid ) are composed ofmonomers called nucleotides.

Nucleotides have three parts: a five-carbon sugar called ribose in RNA and

deoxyribose in DNA, a phosphate group, and

a nitrogenous base.


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Phosphate

groupSugar

Nitrogenousbase

(adenine)



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p y

DNA nitrogenous bases are adenine (A), thymine (T), cytosine (C), and guanine (G).

RNA also has A, C, and G, but instead of T, it has uracil (U).

Pyrimidines

Purines


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Pyrimidines

NH2

O

N

N NH

N

Guanine

N

N

Adenine

N

N

NH2

N O

NH2

N O

NH2

NCytosine

Uracil(RNA)CH3

N ON

O

NH

N ON

O

NH

Thymine(DNA)

Purines



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p y

A nucleic acid polymer, a polynucleotide,forms from the nucleotide monomers, when the phosphate of one nucleotide bonds to

the sugar of the next nucleotide, by dehydration reactions, and by producing a repeating sugar-phosphate

backbone with protruding nitrogenous bases.


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A

T

C

G

T

Nucleotide

Sugar-phosphatebackbone



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p y

Two polynucleotide strands wrap aroundeach other to form a DNA double helix. The two strands are associated because

particular bases always hydrogen bond to oneanother.

A pairs with T, and C pairs with G, producingbase pairs.

RNA is usually a single polynucleotidestrand.

The Watson - Crick A T

- -


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The Watson - CrickModel Of DNA

3.4 nm1 nm

0.34 nm

Majorgroove

Minorgroove

A T

T A G C

C G

C G G C

T A

A T

G C T A

A T C G

- -

-

-

- - -

- -

- -

- -

-

---

-

--- -

--

---

-

Forms of the Double Helix


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0.26 nm

2.8 nmMinorgroove

Majorgroove

1.2 nm

A DNA

1 nm

Majorgroove

Minorgroove

A T

T A G C

C G

C G

G C T A

A T

G C T A

A T C G

0.34 nm

3.9 nm

B DNA

+34.7 o Rotation/Bp11 Bp/turn

-30.0 o Rotation/Bp12 Bp/turn

+34.6 o Rotation/Bp10.4 Bp/turn

0.57 nm

6.8 nm

0.9 nm

Z DNA


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Question:

If you had 30% Adenine in a pieceof DNA, how much Guanine isthere?

Distribution Of Negative Charge PreventsDNA Annealing


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H

PO

-O

O

O

CH2

HOH

PO

O-

O

O

O

CH2

H

P

O

O-

-O

O

O

CH2

NH2

NN

N

N

O

O

NH2 NNH

NN

N O

NH2

N

O H

P

O

O

O

CH2

H

P O-

O

O

CH2

O

O

H OH

P

OO-

O

O

CH2

O-

O-

g

Salts Allow DNA Annealing


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NaCl

H

PO

-O

O

O

CH2

HOH

PO

O-

O

O

O

CH2

H

P

O

O-

-O

O

O

CH2

NH2

NN

N

N

O

O

NH2 NNH

NN

N O

NH2

N

O H

P

O

O

O

CH2

H

P O-

O

O

CH2

O

O

H OH

P

OO-

O

O

CH2

O-

O-

Cl-

Na+

Cat ions can cancelout the negativecharge carried onthe sugarphosphatebackbone.

Na+

Na+

O-

O NH

H OH



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Na +

Na +

Na+

Na+

Na +

Na+ H

PO

-O

O

O

CH2

HOH

P

O

O-O

O

O

CH2

H

P

O

-O

O

O

CH2

NH2

NN

N

N

O

O

NH2 N

NH

N

N

N O

NH2

N

O H

P

O

O

O

CH2

H

P O-

O

O

CH2

O

O

H OH

P

OO-

O

O

CH2

O-

O-

Na +

Na+


O-

Na +


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Na+

Na+

Na +

Na +

O H

P

O

O

O

CH2

H

P O-

O

O

CH2

O

O

H OH

P

OO-

O

O

CH2

O-

O-

H

PO

-O

O

O

CH2

HOH

P

O

O-O

O

O

CH2

H

P

O

-O

O

O

CH2

NH2

N

NN

N

O

O

NH2 NNH

N

N

N O

NH2

N

Na+

Na +

Na +


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Central Dogma of Molecular

Biology

DNA mRNA Protein

replicationtranscription translation

DNA Replication


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3` end

Sugar-phosphate

backbone

Base pair (joined byhydrogen bonding)

Old strands

Nucleotide about to be

added to a new strand

A

3` end

3` end

5` end

New strands

3` end

5` end

5` endC G

C G

ATC G

A T A T

G C A T

A TT A

5` end

DNA Replication

Is DNA Replication Dispersive,


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Conservative or Semiconservative?

All models fitthe Watson-Crick model

of DNAprediction:exactcopying of

geneticinformation.

E ilib i


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EquilibriumDensity

Centrifugation inDNA Analysis

M. Meselson

W. F. Stahl

Semiconservative Re

plication of Density-Labeled DNA


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January 2008

Is DNA Replication UnidirectionalBidi i l?


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or Bidirectional?

(a). Linear DNA virus (b). Some bacterial

plasmid

(c). Most organisms


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Evidence forBidirectional

Replication

DNA Replication


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Replication begins at specific siteswhere the two parental strandsseparate and form replicationbubbles.

The bubbles expand laterally, asDNA replication proceeds in bothdirections.

Eventually, the replicationbubbles fuse, and synthesis ofthe daughter strands iscomplete.

1

2

3

Origin of replication

Bubble

Parental (template) strand

Daughter (new) strand

Replication fork

Two daughter DNA molecules

In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of eachchromosome.

(a)

DNA Replication

DNA replicates, following the process of semi-conservativereplication.

Bacterial Replication and Cell division


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p

No apparent chromosomecondensation

Origin used to separate chromosomes

Rate of DNA replication


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Rate of DNA replication

E. coli 42 minutes 4,639,221 bp 1.4 mm

1000 bp/second/fork Human

8 hours 3 x 10 9 bp

2 m 100 bp/second/fork 10,000 to 100,000 replicons

DNA Replication Machinery


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More than a dozen enzymes and other proteins participate in DNA replication

Much more is known about replication in bacteria than in eukaryotes

Each strand of the original molecule acts as a template for the synthesisof a new complementary DNA molecule

p y

DNA Replication Machinery


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In E. coli , two different DNA polymerases are involved in replication: DNApolymerase III and DNA polymerase I.

In eukaryotes, at least 11 different DNA polymerases have been identifiedso far

p y

DNA P l


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DNA Polymerase

Unable to separate the two strands of DNA Only elongate a pre-existing DNA or RNA

(Primer) Only add nucleotides to the 3 -hyforxyl

group, i.e., only 5 -3 synthesis

Genes and Proteins in DNA


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Genes and Proteins in DNAReplication

Gene Protein dnaA DnaA

dnaB DnaB or Helicase dnaC DnaC dnaG Primase

Initiation of DNA Replication


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Initiation of DNA Replication DnaA Protein Initiates Replication in E. coli

Initiation complex Prepriming complex

I iti ti f DNA R li ti


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Initiation of DNA Replication

DnaB is a helicase. An enzyme moves

along DNA duplexutilizing the energy of

ATP hydrolysis toseparate the strands. 5-3 Processive

SSB: single strand binding protein.

The Role of RNA Primer


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in DNA Replication

E. coli primasecatalyze the RNAPrimer for DNASynthesis dnaG

Primosome:

primases+DnaB Primer:


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600 kDa Holoemzyme: 10

peptides

Core polymerase: a: active site : 3-5 exonuclease : unkown

b -subunit dimer tethers the core of


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E. coli DNA polymerase III to DNA

Rest of Pol III

distributive toprocessive (5x10 5 nts)

b : clamp

b subunit dimer tethers the core of


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b -subunit dimer tethers the core ofE. coli DNA polymerase III to DNA

Growing Fork


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Growing Fork

Leadingstrand:continuous

Laggingstrand:discontinuous


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Fidelity of DNA Replication


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a subunit of Pol III error rate: 1 in 10 4 Observed mutation rate: 10 -9

3-5 proofreading exonuclease activity of DNA

polymerase E. coli Pol I E. coli Pol III subunit.

Mismatch repair system that distinguish newlysynthesized DNA from the old one.

Proofreading by 3 to 5


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Exonuclease



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Exonuclease activity of DNA Pol

DNA Pol I Structure


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DNA Pol I Structure

A Summary of


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A Summary ofDNA Replication

in Bacteria

A Summary of


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A Summary ofDNA Replication

in Bacteria

E. col i replication proteins at a


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growing fork subunit

Holds two core polymerase

Contacts DnaB protein.

Properties of DNA Polymerase


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E. coli PolymeraseI II III

Polymerization + + +

Exonuclease3-5 + + +5-3 + - -

Synthesis fromIntact DNA - - -Primed Single Strand + - -Primed Single Strand + - + +SSB

Overwinding that occurs when


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Overwinding that occurs when

duplicating circular DNA.

Topoisomerase I


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Topoisomerase I

Nicking and then closing one strand of dsDNA

Topoisomerase II


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Breaking andrejoining double-strand DNA


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Topoisomerase IIseparates chromosomes

at the end of DNAreplication.

Eukaryotic


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yReplicationMachinery


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PCNA and b Clamp


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PCNA and b Clamp



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Mammalian Polymerase a

b g d

Replication of Circular DNA


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January 2008

Replication of Circular DNA

Th E d


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The End-

ReplicationProblem

The


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The

Extension ofTelomeres byTelomerase

Summary of DNA Replication


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General Features Semiconservative Bidirectional Replication origin

DNA replication machinery Initiation proteins Helicase Primase Polymerases

Leading and lagging strands Telomerase

Topoisomerases in DNA replication

Gene Expression


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Overview

The information content of DNA is in the form of specificsequences of nucleotides along the DNA strands.

The DNA inherited by an organism leads to specific traitsby dictating the synthesis of proteins.

Gene expression, the process by which DNA directsprotein synthesis, includes two stages called transcriptionand translation.

Proteins are the links between genotype and phenotype.

Transcription


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Transcr ip t ion : synthesis of complementary RNA from

coding strand of DNA.this RNA molecule is called a messenger RNA(mRNA)

mRNA is synthesized by RNA polymerase .

mRNA and DNA have same language (i e code)


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Question:If you have the following DNA sequence on atemplate strand

3` ATG GGC CAC GTC AGA ACG GAA TAA CAT 5`

What would be the mRNA transcript?

3` ATG GGC CAC GTC AGA ACG GAA TAA CAT 5`Template

5` TAC CCG GTG CAG TCT TGC CTT ATT GTA 3`Coding

mRNA 5` UAC CCG GUG CAG UCU UGC CUU AUU GUA 3`

Transcription


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Transcription occurs in 3 stages: 1. RNA Pol binding and in i t ia t ion of transcription

Promoter

Transcription factors

2. Elongat ion of RNA strand

3. Terminat ion of transcription


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Regulation of GeneExpression

Control of Enzyme Synthesis


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Cells can turn the synthesis of enzymes onand off- usually based on environmentalclues.

Control is at the level of the DNA May be slow to react- maybe hour to make

new one or longer to remove one.

Substrate or product of reaction usually theregulatory compound- lactose most studied.

Operons Are Groups Of GenesExpressed By Prokaryotes


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Expressed By Prokaryotes

Bacterial genes grouped in an operon are allneeded to complete a given task

Each operon is controlled by a single controlsequence in the DNA Because the genes are grouped together, they

can be transcribed together then translatedtogether

The Lac Operon


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Genes in the lac operon allow E. coli bacteria to

metabolize lactose E. coli is unlikely to encounter lactose, so it would

be wasteful to produce the proteins needed to

metabolize it unless necessary Metabolizing lactose for energy only makes sense

when two criteria are met:

Other more readily metabolized sugar (glucose) isunavailable

Lactose is available

The Lac Operon - Parts


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The lac operon is made up of a control region and

four genes:1 LacZ - b-galactosidase - An enzyme that hydrolizes

the bond between galactose and glucose

2LacY

- Codes for a permease that lets lactose acrossthe cell membrane3 LacA - Transacetylase - An enzyme whose function

in lactose metabolism is uncertain4 Repressor - A constitutively expressed protein that

works with the control region to modulateexpression

Lactose and b -Galactosidease


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b -GalactosideaseLac Z

gene product

OH

O

OH

H2COH

HO

GlucoseO

O

OH

HOCH 2

HO

HO Galactose

LactoseO-b-D-galactopyranosyl-(1->4)- b -D-glucopyranose

H2O

Lactose and b -Galactosidease


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-GalactosideaseLac Z

gene product

GlucoseGalactose

The Lac Operon - ControlTh l i i d f


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The control region is made up of two parts:

1 Promoter Promoters are specific DNA sequences to which RNA

Polymerase binds so that transcription can occur

The lac operon promoter also has a binding site for protein called Catabolite Activator Protein (CAP)

2 Operator

The binding site of the repressor protein The operator is located down stream (in the 3 direction) from the promoter so that if repressor is

bound RNA Polymerase cant transcribe

The Lac Operon:When Glucose Is Present But Not Lactose


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Repressor Promoter L acY LacALacZOperatorCAP Binding

RNAPol.

Repressor

Repressor

RepressormRNA

Hey man, Imconstitutive

Come on,

let me through

The Lac Operon:When Glucose And Lactose Are Present


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Repressor Promoter LacY LacALacZOperatorCAP Binding

Repressor

RepressormRNA

Hey man, Im

constitutive

Repressor

Repressor

X

RNAPol.

RNAPol.

Great, I can

transcribe!

This lactose hasbent me

out of shape

We need CAP binding to thepromoter

But I need

cAMP/CAP

The Lac Operon:When Lactose Is Present But Not Glucose


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CAPcAMPRepressor

RepressormRNA

Hey man, Im

constitutive

Repressor

Repressor

XThis lactose has

bent meout of shape

CAPcAMP

Bind to mePolymerase

RNAPol.

Yipee!

CAPcAMP

RNAPol.

At last we meetCAP my love

Genes in the operon are efficiently transcribed

Hey CAP,lets get together

The Lac Operon:When Neither Lactose Nor Glucose Is Present

Al i h I ff


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CAPcAMP

CAPcAMP

CAPcAMP

Bind to mePolymerase

RNAPol.

Repressor

RepressormRNA

Hey man, Im

constitutive

Repressor

STOPRight therePolymerase

Alright, Im off tothe races . . .

Come on, letme through!

Translation


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Transla t ion : synthesis of polypeptide under direction of

mRNA.- involves a change from DNA language into proteinlanguage.- involves mRNA, ribosomes, transfer RNA (tRNA)

TRANSLATION

TRANSCRIPTION DNA

mRNA

Ribosome

Polypeptide

Prokaryotic cell. In a cell lacking a nucleus, mRNAproduced by transcription is immediately translatedwithout additional processing.

(a)

TRANSCRIPTION

RNA PROCESSING

TRANSLATION

mRNA

DNA

Pre-mRNA

Polypeptide

Ribosome

Nuclear

envelope Eukaryotic cell. The nucleusprovides a separatecompartment for transcription.The original RNAtranscript, called pre-mRNA, isprocessed in various

ways before leaving the

nucleus as mRNA.

(b)

replication

Translation


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Synthesis of polypeptide under the direction of mRNA

The message in mRNA is translated (interpreted) into proteinTranslation process involves:- Change from DNA language into protein language.

- mRNA, ribosomes, transfer RNA (tRNA) Ability to extract intended message from written languagedepends on reading the symbols in the correct sequence ofgroupings. This ordering is called the read ing f ram e .

DNA mRNA Protein

replication

transcription translation

The Genetic Code

l d


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Triplet code

Codon = 3 nucleotides (bases)

Three consecutive bases specify an amino acid, creating43 (64) possible code words.

Since there are 64 possible code words, and only 20 aminoacids, some amino acids are encoded by more than one codeword. Thus the genetic code is redundant

The genetic instructions for a polypeptide chain are writtenin the DNA as a series of three-nucleotide words.

The Genetic Code


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P

OH

HO

ONH 2

A Codon


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H

PO

O

HO

O

O

CH 2 NH 2 NNH

N

N

HOH

P

O

O

HO

O

O

CH 2

NH 2

N

N

N

N

H

O

OCH 2 N

N

N

N

O Guanine

Adenine

Adenine

Arginine

How Codons Work:tRNA the Translators


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tRNA - Transfer RNA Relatively small RNA molecules that

fold in a complex way to produce a 3dimensional shape with a specificamino acid on one end and an

anticodon on another part Associate a given amino acid with the

d h RNA h d f i


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Translation - Initiation


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AE

Largesubunit

P

Smallsubunit

fMet

UACGAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5 mRNA

3

Translation - Elongation


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AE

Ribosome P

Arg

Aminoacyl tRNAPhe

Leu

Met

SerGly

Polypeptide

CCAGAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5 mRNA

3



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AE

Ribosome P

PheLeu

Met

SerGly

Polypeptide

Arg

Aminoacyl tRNA

UCUCCAGAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5 mRNA

3

ACIDAMINE

Protein SynthesisOH


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ANYTHING

ACID

C

O

OHCN

H

HH

C

HO HC

H

O

CN

H

HH

C

H H

C

H

O

OHCN

H

HH

C

HO H

Serine

C

H

O

OHCN

H

HH

C

H H

Alanine

HC

OHC

R

N

H

Amino Acid

H 2O



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AE

Ribosome P

CCA

Arg

UCU

PheLeu

Met

SerGly

Polypeptide

GAG...CU -AUG--UUC--CUU--AGU--GGU--AGA--GCU--GUA--UGA- AT GCA...T AAAAAA 5 mRNA

3



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AE

Ribosome P

Aminoacyl tRNA

Ala

CCA

Arg

UCU

PheLeu

Met

SerGly

Polypeptide


3



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AE

Ribosome P

Arg

UCU

PheLeu

Met

SerGly

Polypeptide

CGA

Ala


3


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Restriction EnzymeDigestion

C i DNA l l ifi l i


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Cutting DNA molecules at specific locations.

Enzymes are the Tools of DNATechnology

The tools of DNA technology are the same enzymes used


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The tools of DNA technology are the same enzymes used

by cells to modify their own DNA: DNA Polymerases DNA Ligase

One special class of enzyme is pivotal to the cloning ofDNA and many other techniques used in DNATechnology

These enzymes are the restriction endonucleases

Restriction - Because for the way they work, they restrict bacteriophages to only one host bacterial strain. They are alsorestricted to acting on only specific DNA sequences

Endonuclease - They cut nucleic acids in the middle not just

th d

What is a Palindrome?

A palindrome is anything that reads the same


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A palindrome is anything that reads the same

forwards and backwards: English palindromes: Mom

Dad Tarzan raised Desi Arnaz rat. Able was I ere I saw Elba (supposedly said by

Napoleon) Doc note I dissent, a fast never prevents a fatness,

I diet on cod.

DNA Palindromes Because DNA is double stranded and the strands

ti ll l li d d fi d


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run antiparallel, palindromes are defined as anydouble stranded DNA in which reading 5 to 3

both are the same Some examples:

The EcoRI cutting site: 5'-GAATTC-3' 3'-CTTAAG-5'

The HindIII cutting site: 5'-AAGCTT-3'

3' TTCGAA 5'

Type II restriction enzymenomenclature


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Eco RI Escherichia coli strain R, 1 st enzyme Bam HI Bacillus amyloliquefaciens strain H, 1 st enzyme Dpn I Diplococcus pneumoniae , 1 st enzyme HindIII Haemophilus influenzae , strain D, 3 rd enzyme Bgl II Bacillus globigii , 2 nd enzyme Pst I Providencia stuartii 164, 1 st enzyme Sau 3AI Staphylococcus aureus strain 3A, 1 st enzyme Kpn I Klebsiella pneumoniae , 1 st enzyme

Joining linear DNA fragments together with covalent bonds

Ligation


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is called ligation. More specifically, DNA ligation involvescreating a phosphodiester bond between the 3' hydroxyl ofone nucleotide and the 5' phosphate of another.

The enzyme used to ligate DNA fragments is T4 DNAligase , which originates from the T4 bacteriophage.

DNA cloning requires restrictionenzymes and DNA ligase


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Consider a plasmid with a unique Eco RIsite:

5' NNNN GAATTC NNNN 3'3 NNNNCTTAAG NNNN 5'

An Eco RI restriction fragment of foreign DNA can beinserted into a plasmid having an Eco RI cloning site by:a) cutting the plasmid at this site with Eco RI,b) annealing the linearized plasmid with the Eco RI foreignDNA fragment, and,

c) sealing the nicks with DNA ligase.

5' NNNN GAATTCNNN N 3'3' NNNN CTTAA GNNNN 5

This results in a recombinant DNA molecule.

Gel Electrophoresis


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Wells

Gel Electrophoresis


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Wells

Gel Electrophoresis-


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+

Directionof

DNATravel

Wells

Small

Large

gelelectrophoresis.exe

Restriction fragment length polymorphism(RFLP)


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Restriction fragment analysis detects DNA differences thataffect restriction sites.

Does a particular gene differ from person to person? Are certain alleles associated with a hereditary disorder?

Where in the body and when during development is a geneexpressed?What is the location of a gene in the genome?Is expression of a particular gene related to expression of

other genes?Restriction fragment analysis is sensitive enough to distinguishbetween two alleles of a gene that differ by only one base pairin a restriction site.


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Genetic EngineeringCloning Vectors


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g

Plasmids are circular pieces of DNA found naturally inbacteria.

Plasmids can carry antibiotic resistance genes, genes forreceptors, toxins or other proteins.

Ori

pUC18

Amp r

MCS

LacZ

Plasmids replicate separately fromthe genome of the organism.

Plasmids can be engineered to beuseful cloning vectors .

pUC 18A Typical Plasmid
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.1587http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.1585http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.1585http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.figgrp.1587


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2,686 bp

Lac ZGene

Multiple CloningSite

aagcttgcatgcctgcaggtcgactctagaggatccccgggtaccgagctcgaattc HindIII SphI PstI SalI XbaI BamHI XmaI KpnI SstI EcoRI

AccI SmaI BanIIHincII

BspMI

Originof Replication

Amp r Gene



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g



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Plasmid vectors can be designed with a varietyof features:

Antibiotic resistance Colorimetric markers Strong or weak promoters for driving expression of

a protein

g



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AntibioticResistance

Gene

g

MultipleCloningRegion

The cloning marker for this plasmid is the lac Z gene.



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Selecting for Transformants

The transformed bacteria cells are grown on

selective media (containing antibiotic) to selectfor cells that took up plasmid.

For blue/white selection to determine if the

plasmid contains an insert, the transformantsare grown on plates containing X-Gal and IPTG.

g

So How Do You Know IfYou Cloned Something?


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IPTG - Inducesexpression of lacZ

X-Gal - A lactose analogwhich turns blue whensplit by b-galactosidase

Ampicillin - Kills all

bacteria that lack the plasmid

X-Gal5-Bromo-4-chloro-3-indolyl b -D-galactopyranoside


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OH

O

OH

HOCH 2

HO

GlucoseO

O

OH

HOCH 2

HO

HO Galactose

LactoseO-b-D-galactopyranosyl-(1->4)- b -D-glucopyranose



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-GalactosideaseLac Z

gene product

N

H

Br

Cl

O

O

OH

HOCH 2

HO

HO Galactose

X-Gal(Colorless)

H2O



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-Galactosidease

OH

O

OH

HOCH 2

HO

HO Galactose

Blue

N

H

Br

Cl

HO

So How Do You Know IfYou Cloned Something?

Blue colonies - Express b-galatosidase


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which metabolizes colorles X-gal to blueand turn blue thus lacZ is not disruptedand there is no foreign DNA cloned

Cloned fragments disrupt lacZ thus makeno b-galactosidase andcolonies remain white

IPTG - Inducesexpression of lacZ

X-Gal - A lactose analogwhich turns blue whensplit by b-galactosidase

Ampicillin - Kills all

bacteria that lack the plasmid

StrategyExtract DNA


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Fragment DNA

Insert into vector

DNA Library in bacteria

Screen libraryand grow up bacteria with clone of interest

A Library

The clone of interest


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The clone of interest


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Polymerase ChainReaction

History

The Polymerase Chain Reaction (PCR) was not a


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y ( )

discovery, but rather an invention PCR uses a special DNA polymerase to make

many copies of a short length of DNA (100 -

10,000 bp) that is defined by primers Kary Mullis was the inventor of PCR PCR is so important that Mullis was awarded the

1993 Nobel Prize in Chemistry

What PCR Can Do PCR can be used to make many copies of any

DNA that is supplied as a template


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It can start with only one original and make analmost infinite number of copies

Amplified fragments of DNA can be sequenced

to discover the code for a given gene Defective genes can be amplified to diagnose any

number of illnesses

Genes from pathogens can be amplified to identifythem (ie. HIV)

Amplified fragments can act as genetic fingerprints

How PCR Works PCR is an artificial way of doing DNA

replication


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replication Instead of replicating all the DNA present, only a small segment is

replicated, but this small segment isreplicated many times As in replication, PCR involves:

Melting DNA = Denaturation Priming = Annealing Polymerization = Extension

Components of a PCR Reaction

++


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Buffer (containing Mg++

) Template DNA 2 Primers that flank the fragment of

DNA to be amplified dNTPs

Taq DNA Polymerase (or anotherthermally stable DNA polymerase)

A thermophilic (heat-loving) bacteria called Thermus aquaticus isthe source of Taq DNA polymerase used in PCR reactions.


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The first round of PCR


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94C 37-65C 70-75C

PCR increases the yield of DNAexponentially


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PCRMelting94 oC

p e r a

t u r e

100


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T e m

p

0

50

T i m e

5 3

3 5

PCRMelting94 oC

p e r a

t u r e

100


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T e m

p

0

50

T i m e

3 5

5 3

Heat

PCRMelting94 oC

AnnealingPrimers

Extension72 oC p e

r a t u r e

100 Melting94 oC


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50 oC T e m

0

50

T i m e

3 5

5 3 5

5

PCRMelting94 oC

Melting94 oC

AnnealingPrimers

Ext

ension72 oC p e

r a t u r e

10030x


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50oC T e m

0

50

T i m e

3 5

5 3

Heat

Heat

5

5

5

PCRMelting94 oC

Melting94 oC

AnnealingPrimers

Ext

ension72 oC p e

r a t u r e

10030x


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50oC T e m

0

50

T i m e

3 5

5 3 5

5

5

5

5

5

PCRMelting94 oC

Melting94 oC

AnnealingPrimers

Ext

ension72 oC p e

r a t u r e

10030x


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50oC T e m

0

50

T i m e3 5

5 3 5

5

5

5

5

5

Heat

Heat

PCRMelting94 oC

Melting94 oC

AnnealingPrimers

Ext

ension72 oC p e

r a t u r e

10030x


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50oC T e m

0

50

T i m e3 5

5 3 5

5

5

5

5

5

5

5

5

5

PCRMelting94 oC

Melting94 oC

AnnealingPrimers

Ext

ension72 oC p e

r a t u r e

10030x


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Fragments of

defined length

50oC T e m

0

50

T i m e3 5

5 3 5

5

5

5

5

5

5

5

5

5

DNA Between The Primers Doubles WithEach Thermal Cycle

Number


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0Cycles

1

3

8

2

4

1

2

4

16

5

32

6

64

Gel electrophoresis


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Indirect method to analyze and compare genomes

Separation of nucleic acids or proteins on the basis of theirrate of movement through a gel in an electrical field.


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A method of separating large molecules (such as DNAfragments or Proteins ) from a mixture of similarmolecules.

An electric current is passed through a mediumcontaining the mixture, and each kind of molecule travelsthrough the medium at a different rate, depending on itselectrical charge and size .

Agarose and acrylamide gels are the media commonlyused for electrophoresis of proteins and nucleic acids

The pH and other buffer conditions are arranged so


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that the molecules being separated carry a net(negative ) charge so that they will me moved by theelectric field toward the positive pole .

As they move through the gel, the larger molecules

will be held up as they try to pass through the poresof the gel, while the smaller molecules will beimpeded less and move faster.

This results in a separation by size, with the larger

molecules nearer the well and the smaller moleculesfarther away.


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The Phosphate groups on the backbone ofthe DNA molecule readily give up their H +ions, therefore nucleic acids are negativelycharged in most buffer systems.

DNA molecules will migrate away from thenegative electrode (cathode) , and migrate

towards the positive electrode (anode) .

DNA molecules will migrate away from the negative


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electrode (cathode) , and migrate towards the positiveelectrode (anode) .


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Rate of movement depends on :

1- Size


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2- Electrical charge3- Other physical properties of the macromolecules

Agarose (a polysaccharide isolatedfrom seaweed). Ethidium bromide (a stain which

fluoresces under ultraviolet light whenbound to DNA).

Materials required

Agarose Gel Electrophoresis


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Analysis of isolated DNA Separation of DNA restricted with Hae III

(RFLP analysis) followed by a Southern Blotand Hybridization with a labeled probe

Post Amplification confirmation andqualitative assessment of PCR product


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DNA sequencing Reading the genetic code

Review of DNA replication(synthesis) requirements DNA synthesis occurs in the 5 to 3 direction.


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y

DNA synthesis requires a template and a primer.

DNA replication is semi-conservative (one strand copied).

DNA replication is carried out by an enzyme called DNApolymerase.

DNA synthesis requires a 3 -OH to make thenext phosphodiester bond during DNA

synthesis


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normaldNTP

Dideoxy NTPs block DNAsynthesis


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H

A mixture of dNTPs and ddNTPsare used in DNA sequencing


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Polyacrylamide gel electrophoresis isused to visualize the results of the

sequencing reaction


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Automated DNA sequencingwith fluorescent dyescoupled to each reaction


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Fluorescent dye coupled toreaction allows visualizationof di-deoxy terminationevents by means of a laserthat detects the coloredproduct.

This shows four differentreactions as done with theold manual sequencing.

Automated DNA sequencingwith fluorescent dyes coupledto each reaction


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Automated DNA sequencing output-4 reactions carried out in one tube


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Mutations Mutation = Change

IntroductionThe Central Dogma

of Molecular Biology


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DNAmRNA

Transcription

Cell

Polypeptide(protein)

TranslationRibosome

Macromutations Four major types of Macromutations are

recognized:


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1 Deletions - Loss of chromosome sections2 Duplications - Duplication of chromosome

sections3 Inversions - Flipping of parts of chromosomes4 Translocations - Movement of one part of a

chromosome to another part

Macromutation - Deletion

Chromosome


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Centromere

A B C D E F G H

Genes

E F

A B C D G H

Macromutation - DuplicationChromosome

Centromere


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A B C D E F G HGenes

A B C D E F E F G HE F

Duplication

Macromutation - InversionChromosome

Centromere


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A B C D F E G H

GenesA B C D E F G H

Inversion

Macromutation - TranslocationChromosome

Centromere


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A B E F C D G H

GenesA B C D E F G H

Micro or Point Mutations Two major types of Macromutations are recognized:1 Frame Shift - Loss or addition of one or two nucleotides


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2 Substitutions - Replacement of one nucleotide byanother one. There are a number of different types: Transition - Substitution of one purine for another purine,

or one pyrimidine for another pyrimidine.

Transversion - Replacement of a purine with a pyrimidineor vice versa.

Frame Shift Mutations3 AGTTCAG-TAC-TGA-A C A-CCA-TCA-ACT-GATCATC 5

5 AGUC-AUG-ACU-U GU-GGU-AGU-UGA-CUAGAA


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5 AGUC-AUG-ACU-U UG-GUA-GUU-GAC-UAG-AAA3

AGTTCAG-TAC-TGA-A AC-CAT-CAA-CTG-ATCATC5

Met Thr Cys Gly Ser

Met Thr ValVal ValLeu

Frame shift mutations tend to have a dramatic effect on proteins as

all codons down stream from the mutation are changed and thuscode for different amino acids. As a result of the frame shift, thelength of the polypeptide may also be changed as a stop codon willprobably come at a different spot than the original stop codon.

Substitution Mutations3 AGTTCAG-TAC-TGA-A C A-CCA-TCA-ACT-GATCATC 5

5 AGUC-AUG-ACU-U GU-GGU-AGU-UGA-CUAGAA


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Purine toPyrimidine

Transversion

Pyrimidine to

Pyrimidine

Transition

3 AGTTCAG-TAC-TGA-A T A-CCA-TCA-ACT-GATCATC 5

Met Thr Cys Gly Ser

3 AGTTCAG-TAC-TGA-A A A-CCA-TCA-ACT-GATCATC 5

3 AGTTCAG-TAC-TGA-A C A-CCA-TCA-ACT-GATCATC 5 5 AGUC-AUG-ACU-U GU-GGU-AGU-UGA-CUAGAA

Met Thr Cys Gly Ser

5 AGUC-AUG-ACU-U AU-GGU-AGU-UGA-CUAGAA

Met Thr Gly SerTyr

5 AGUC-AUG-ACU-U UU-GGU-AGU-UGA-CUAGAAMet Thr Gly SerPhe

TC TNormal -globin DNA

TC AMutant -globin DNA

The Sickle Cell Anemia Mutation


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ValMutant -globin

H 2NOH

OH

CO

H 2CH

C

CH 2 C

O Acid

GluNormal -globin

H 2NOH

CO

H 3CH

C

CH CH 3

Neutral Non-polar

AG AmRNA

AG UmRNA

Thymine Dimers

OH


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Thymine

Thymine

H

P

O

HO

O

O

CH 2

OH

H

P

O

HO

O

O

CH 2

O

OH

H

P OH

O

O

CH 2

O

O

H

H OH

P

O

OH

O

O

CH 2

NH 2

N

N

N

N

NH 2

N

N

N

N

Thymine Dimers

OH


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Thymine

Thymine

H

P

O

HO

O

O

CH 2

OH

H

P

O

HO

O

O

CH 2

O

OH

H

P OH

O

O

CH 2

O

O

H

H OH

P

O

OH

O

O

CH 2

NH 2

N

N

N

N

NH 2

N

N

N

N

Thymine Dimers

OH


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Thymine

Thymine

OHH

P

O

HO

O

O

CH 2

OH

H

P

O

HO

O

O

CH 2

O

H OH

O

OCH 2

NH 2

N

N

N

N

NH 2

N

N

N

N

OH

H

P

O

O

CH 2

O

O

HP OHO

P h

o t ol y

a s e

Thymine Dimers

OH

H OH

NH


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Thymine

Thymine

H

P

O

HO

O

O

CH 2

OH

H

P

O

HO

O

O

CH 2

O

OH

H

P OH

O

O

CH 2

O

O

H

H OH

P

O

OH

O

O

CH 2

NH 2

N

N

N

N

NH 2

N

N

N

N

P h

o t ol y

a s e

Thymine Dimers

OH

H OH

NH


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Thymine

Thymine

H

P

O

HO

O

O

CH 2

OH

H

P

O

HO

O

O

CH 2

O

OH

H

P OH

O

O

CH 2

O

O

H

H OH

P

O

OH

O

O

CH 2

NH 2

N

N

N

N

NH 2

N

N

N

N


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Recombination

Ways Bacteria Exchange GeneticMaterial


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1 Transformation - Bacteria take up DNA fromtheir environment and incorporate it into theirgenome (i.e. the Griffith experiment)

2 Conjugation - The direct transfer of DNA bybacteria usually via plasmids

3 Transduction - Movement of DNA between

bacteria by viruses

Transformation Of BacteriaTwo Strains Of Streptococcus


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CapsulesSmooth Strain

(Virulent)

Rough Strain(Harmless)

Transformation Of BacteriaThe Griffith Experiment

+ Control

OUCH!


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Experimental

- Control

- Control

Avery, MacLeod and McCarty 1944 Avery, MacLeod and McCarty decided to

repeat Griffiths 1928 experiment and try to


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discover the transforming factor

They did this by using extracts from the heatkilled cells and digesting specific classes ofmolecules with enzymes

No Nuclease

YesProtease

YesLipase

Transformation?Enzyme

YesSaccharase

1 Transformation


255/273

Insertion

Crossingover

1 Transformation


256/273

1 Transformation


257/273

1 Transformation


258/273

1 Transformation


259/273

1 Transformation


260/273

2 ConjugationF plasmid

Mating Bridge

F+ bacteria


261/273

F- bacteria

Mating Bridge

F plasmid

2 ConjugationF plasmidF+ bacteria

Mating Bridge


262/273

F- bacteria

F plasmid

Mating Bridge


Mating Bridge


263/273

F- bacteria

F plasmid

Mating Bridge


Mating Bridge


264/273

F- bacteria

F plasmid

Mating Bridge


Mating Bridge


265/273

F- bacteria

F plasmid

F plasmid

Mating Bridge

Hfr Recombination


266/273

Transfer ofgenetic material

F+ bacteria

F- bacteria

F plasmidIntegration

Hfr cell

Hfr Recombination


267/273

F+ bacteria Hfr cellF plasmidIntegration

F- bacteria

RecombinantBacteria


268/273

Phage Strategies

Bacteriophage AttackDestruction ofthe bacterias

DNA

Infection


269/273

Lysis

DNA

Replication of

the viralgenomeProduction ofviral parts

Packaging

Phage Reproduction:The Lytic CycleDestruction ofthe bacterias

DNA

Infection


270/273

DNA

Replication of


PackagingLysis

Phage Reproduction:The Lysogenic CycleCircularizationof phage DNA

Infection


271/273

Many generationsof bacteria

Temperate phage

Integration of

phage DNA intothe bacterialgenome

On to thelytic cycle

Exit of phage

3 TransductionGeneralized

Destruction ofthe bacterias

DNA

Infection


272/273

DNA

Lysis

Replication of


Packaging

3 TransductionSpecialized

TemperatePhage

Part of the bacterias

DNA


273/273

DNA

Date post:	03-Jun-2018
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Molecular Biology BIOL312

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