Chem 4311 Chapter10 12 Nucleic Acid

7/29/2019 Chem 4311 Chapter10 12 Nucleic Acid

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Chapter 10-12Nucleotides and Nucleic Acids

EXAM - October 19

Ins tructor: Dr. Khairu l I AnsariOffice: 316CPBPhone: 817-272-0616email: [email protected] hours 12 am 1:30 pm Tuesday &.Thursday

CHEM 4311General Biochemistry I

Fall 2012

Chapter 10

Nucleotides and Nucleic Acids

We have discovered thesecret of life.Francis Crick, to patrons ofThe Eagle, a pub inCambridge, England(1953)

Francis Crick (right) andJames Watson (left)point out features of their

model for the structure ofDNA.

Information Transfer in Cells

Figure 10.1 Thefundamental process ofinformation transfer incells.

10.1 What Are the Structure and Chemistry ofNitrogenous Bases?

Know the basic structures

Pyrimidines

Cytosine (DNA, RNA)Uracil (RNA)

Thymine (DNA)

Purines

Adenine (DNA, RNA)

Guanine (DNA, RNA)


Figure 10.2 (a) The pyrimidine ring system; by convention,atoms are numbered as indicated.

(b) The purine ring system; atoms numbered as shown.


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Figure 10.3 The common pyrimidine bases cytosine, uracil, and thymine in the tautomeric formspredominant at pH 7.


Figure 10.4 The common purine bases adenine andguanine in the tautomeric forms predominant at pH 7.

The Properties of Pyrimidines and Purines Can Be Traced to TheirElectron-Rich Nature

The aromaticity and electron-rich nature ofpyrimidines and purines enable them to undergoketo-enol tautomerism

The keto tautomers of uracil, thymine, and guaninepredominate at pH 7

By contrast, the enol form of cytosine predominatesat pH 7

Protonation states of the nitrogens determineswhether they can serve as H-bond donors oracceptors

Aromaticity also accounts for strong absorption of UVlight

The Properties of Pyrimidines and Purines Can Be Traced toTheir Electron-Rich Nature

Figure 10.6 The keto-enol tautomerism of uracil.


Figure 10.7 The tautomerization of the purine guanine.

The Properties of Pyrimidines and Purines Can Be Traced to TheirElectron-Rich Nature

Figure 10.8 The UV absorption spectra of the commonribonucleotides.


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Figure 10.8 The UV absorption spectra of the commonribonucleotides.

10.2 What Are Nucleosides?

Structures to Know

Nucleosides are compounds formed when a base islinked to a sugar via a glycosidic bond

The sugars are pentoses

D-ribose (in RNA)

2-deoxy-D-ribose (in DNA) The difference - 2'-OH vs 2'-H

This difference affects secondary structure andstability


Figure 10.9 The linear and cyclic (furanose) forms ofribose.


Figure 10.9 The linear and cyclic (furanose) forms ofdeoxyribose.


The base is linked to the sugar via a glycosidicbond

The carbon of the glycosidic bond is anomeric

Named by adding -idine to the root name of a

pyrimidine or -osine to the root name of apurine

Conformation can be syn or anti

Sugars make nucleosides more water-solublethan free bases


Figure 10.10 The common ribonucleosides.


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Adenosine: A Nucleoside with Physiological Activity

Adenosine functions as anautacoid, or local hormone, and

neuromodulator. Circulating in the bloodstream, it

influences blood vessel dilation,smooth muscle contraction,neurotransmitter release, and fatmetabolism.

Adenosine is also a sleepregulator. Adenosine rises duringwakefulness, promoting eventualsleepiness.

Caffeine promotes wakefulnessby blocking binding of adenosineto its neuronal receptors.

10.3 What Is the Structure and Chemistry ofNucleotides?

Figure 10.11 Structures of the four common ribonucleotides AMP, GMP, CMP, and UMP. Also shown: 3 -AMP.


Figure 10.13 Formation of ADP and ATP by the succesiveaddition of phosphate groups via phosphoric anhydridelinkages. Note that the reaction is a dehydration synthesis.


Figure 10.13 Formation of ADP and ATP by the succesiveaddition of phosphate groups via phosphoric anhydridelinkages. Note that the reaction is a dehydration synthesis.

Nucleoside 5'-Triphosphates Are Carriers ofChemical Energy

Nucleoside 5'-triphosphates are indispensableagents in metabolism because their phosphoricanhydride bonds are a source of chemical energy

Bases serve as recognition units Cyclic nucleotides are signal molecules and

regulators of cellular metabolism andreproduction ATP is central to energy metabolism GTP drives protein synthesis CTP drives lipid synthesis UTP drives carbohydrate metabolism


Figure 10.14 Phosphoryl, pyrophosphoryl, and nucleotidylgroup transfer, the major biochemical reactions ofnucleotides. Phosphoryl group transfer is shown here.


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Figure 10.14 Phosphoryl, pyrophosphoryl, andnucleotidyl group transfer, the major biochemicalreactions of nucleotides. Pyrophosphoryl grouptransfer is shown here.


Figure 10.14 Phosphoryl, pyrophosphoryl, andnucleotidyl group transfer, the major biochemicalreactions of nucleotides. Nucleotidyl group transfer isshown here.

10.4 What Are Nucleic Acids?

Nucleic acids are linear polymers of nucleotideslinked 3' to 5' by phosphodiester bridges

Ribonucleic acid and deoxyribonucleic acid Know the shorthand notations Sequence is always read 5' to 3' In terms of genetic information, this

corresponds to "N to C" in proteins


Figure 10.15 3',5'-Phosphodiester bridges linknucleotides together to formpolynucleotide chains. The 5'-ends of the chains are at thetop; the 3'-ends are at thebottom. RNA is shown here.


Figure 10.15 3 ,5 -phosphodiester bridges linknucleotides together to formpolynucleotide chains. The

5 -ends of the chains are atthe top; the 3 -ends are atthe bottom. DNA is shownhere.

10.5 What Are the Different Classes of NucleicAcids?

DNA - one type, one purpose

RNA - 3 (or 4) types, 3 (or 4) purposes

ribosomal RNA - the basis of structure and functionof ribosomes

messenger RNA - carries the message for protein

synthesistransfer RNA - carries the amino acids for protein

synthesis

Others: Small nuclear RNA

Small non-coding RNAs


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10.5 What Are the Different Classes of NucleicAcids?

Figure 10.16 Theantiparallel nature of theDNA double helix. The twochains have oppositeorientations.

The DNA Double Helix

The double helix is stabilized by hydrogen bonds

"Base pairs" arise from hydrogen bondsA-T; G-C

Erwin Chargaff had the pairing data, but didn'tunderstand its implications

Rosalind Franklin's X-ray fiber diffraction datawas crucial

Francis Crick showed that it was a helix James Watson figured out the H bonds

The Base Pairs Postulated by Watson

Figure 10.17 The Watson-Crick base pairs A:T and G:C.A:T is shown here.

The Base Pairs Postulated by Watson

Figure 10.17 The Watson-Crick base pairs A:T and G:C.

G:C is shown here.

The Structure of DNA

An antiparallel double helix

Diameter of 2 nm Length of 1.6 million nm (E. coli) Compact and folded (E. colicell is only 2000

nm long) Eukaryotic DNA wrapped around histone

proteins to form nucleosomes Base pairs: A-T, G-C

The Structure of DNA

Figure 10.18 Replication of DNAgives identical progeny moleculesbecause base pairing is themechanism that determines thenucleotide sequence of each newlysynthesized strand.


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Digestion of the E. colicell wall releases thebacterial chromosome

Figure 10.19 The chromosome is shown surrounding the cell.

Do the Properties of DNA Invite PracticalApplications?

The molecular recognition between DNA strandscan create a molecule with mechanical propertiesdifferent from single-stranded DNA

DNA double helices are relatively rigid rods DNA chains have been used to construct

nanomachines capable of simple movementssuch as rotation and pincerlike motions

More elaborate DNA-based devices can act asmotors walking along DNA tracks

The construction of DNA tweezers is describedon the following slide

Do the Properties of DNA Invite PracticalApplications?

DNA tweezers a simple

DNA nanomachine.

Messenger RNA Carries the Sequence Informationfor Synthesis of a Protein

Transcription product of DNA

In prokaryotes, a single mRNA contains theinformation for synthesis of many proteins

In eukaryotes, a single mRNA codes for justone protein, but structure is composed ofintrons and exons

Messenger RNA Carries the Sequence Information forSynthesis of a Protein

Figure 10.20 Transcription and translation of mRNAmolecules in prokaryotic versus eukaryotic cells.

In prokaryotes, a single mRNA molecule may contain theinformation for the synthesis of several polypeptidechains within its nucleotide sequence.

Messenger RNA Carries the Sequence Information forSynthesis of a Protein

Figure 10.20 Transcription and translation of mRNA moleculesin prokaryotic versus eukaryotic cells.Eukaryotic mRNAs encode only one polypeptide but are morecomplex.


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Eukaryotic mRNA

DNA is transcribed to produce heterogeneousnuclear RNA (hnRNA)

mixed introns and exons with poly A

intron = intervening sequence

exon = coding sequence

poly A tail - stability?

Splicing produces final mRNA without introns

Ribosomal RNA Provides the Structural andFunctional Foundation for Ribosomes

Ribosomes are about 2/3 RNA, 1/3 protein rRNA serves as a scaffold for ribosomal proteins The different species of rRNA are referred to

according to their sedimentation coefficients rRNAs typically contain certain modified nucleotides,

including pseudouridine and ribothymidylic acid The role of ribosomes in biosynthesis of proteins is

treated in detail in Chapter 30 Briefly: the genetic information in the nucleotide

sequence of mRNA is translated into the amino acidsequence of a polypeptide chain by ribosomes


Figure 10.21 Ribosomal RNAhas a complex secondarystructure due to manyintrastrand H bonds. The grayline here traces apolynucleotide chain consistingof more than 1000 nucleotides.Aligned regions represent H-bonded complementary basesequences.


Figure 10.22 The organization and composition of ribosomes.

Transfer RNAs Carry Amino Acids to Ribosomesfor Use in Protein Synthesis

Small polynucleotide chains - 73 to 94 residues each Several bases usually methylated Each a.a. has at least one unique tRNA which carries

the a.a. to the ribosome 3'-terminal sequence is always CCA-3-OH. The a.a.

is attached in ester linkage to this 3-OH. Aminoacyl tRNA molecules are the substrates of

protein synthesis

Transfer RNAs Carry Amino Acids to Ribosomes for Usein Protein Synthesis

Figure 10.24 Transfer RNA alsohas a complex secondarystructure due to many intrastrandhydrogen bonds. The black linesrepresent base-pairednucleotides in the sequence.


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The RNA World and Early Evolution

Thomas Cech and Sidney Altman showed that RNAmolecules are not only informational they can also becatalytic

This gave evidence to the postulate by Francis Crickand others that prebiotic evolution depended on self-replicating, catalytic RNAs

But what was the origin of the nucleotides? A likely source may have been conversion of

aminoimidazolecarbonitrile to adenine And glycolaldehyde could combine with other

molecules to form ribose Adenine and glycolaldehyde exist in outer space


Aminoimidazolecarbonitrile is a pentamer of HCN andmay be a celestial precursor of adenine.


Glycolaldehyde has been detected at thecenter of the Milky Way and could be aprecursor of ribose and glucose.

The Chemical Differences Between DNA and RNAHave Biological Significance

Two fundamental chemical differencesdistinguish DNA from RNA:

DNA contains 2-deoxyribose instead of ribose

DNA contains thymine instead of uracil

The Chemical Differences Between DNA and RNAHave Biological Significance

Why does DNA contain thymine?

Cytosine spontaneously deaminates to form uracil Repair enzymes recognize these "mutations" and

replace these Us with Cs But how would the repair enzymes distinguish

natural U from mutant U? Nature solves this dilemma by using thymine (5-methyl-U) in place of uracil

DNA & RNA Differences?

Why is DNA 2'-deoxy and RNA is not?

Vicinal -OH groups (2' and 3') in RNA make it moresusceptible to hydrolysis

DNA, lacking 2'-OH is more stable This makes sense - the genetic material must be

more stable RNA is designed to be used and then broken down


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10.6 Are Nucleic Acids Susceptible to Hydrolysis?

RNA is resistant to dilute acid

DNA is depurinated by dilute acid DNA is not susceptible to base

RNA is hydrolyzed by dilute base

See Figure 10.29 for mechanism


Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilicattach by OH- on the P atom leads to 5'-phosphoestercleavage.


Figure 10.27 Alkaline hydrolysis of RNA. Nucleophilic attack byOH- on the P atom leads to 5'-phosphoester cleavage. Random

hydrolysis of the cyclic phosphodiester intermediate gives amixture of 2'- and 3'-nucleoside monophosphate products.


Figure 10.27 Alkaline hydrolysis of RNA. Random hydrolysis ofthe cyclic phosphodiester intermediate gives a mixture of 2'- and

3'-nucleoside monophosphate products.


Figure 10.28 Cleavage in polynucleotide chains. Cleavageon the a side leaves the phosphate attached to the 5'-position of the adjacent nucleotide. b-side hydrolysis yields3'-phosphate products.


Figure 10.28 Cleavage in polynucleotide chains.Cleavage on the a side leaves the phosphate attached tothe 5'-position of the adjacent nucleotide.


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Figure 10.28 Cleavage in polynucleotide chains.b-side hydrolysis yields 3'-phosphate products, amongothers.

Restriction Enzymes

Bacteria have learned to "restrict" the

possibility of attack from foreign DNA bymeans of "restriction enzymes"

Type II and III restriction enzymes cleave DNAchains at selected sites

Enzymes may recognize 4, 6 or more bases inselecting sites for cleavage

An enzyme that recognizes a 6-base sequenceis a "six-cutter"

Type II Restriction Enzymes

No ATP requirement

Recognition sites in dsDNA have a 2-fold axis ofsymmetry

Cleavage can leave staggered or "sticky" ends orcan produce "blunt ends

Type II Restriction Enzymes

Names use 3-letter italicized code:

1st letter - genus; 2nd,3rd - species

Following letter denotes strain

EcoRI is the first restriction enzyme isolated fromthe R strain ofE. coli

Cleavage Sequences of Restriction Endonucleases Cleavage Sequences of Restrict ion Endonucleases


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Restriction Mapping of DNA

Figure 10.29 Restriction mapping analysis.

Chapter 11

Structure of Nucleic Acids

Chapter 11

The Structure of DNA:A melody for the eye of

the intellect, with not a notewasted.Horace Freeland JudsonThe Eighth Day of Creation

11.1 How Do Scientists Determine the PrimaryStructure of Nucleic Acids?

Two simple tools have made nucleic acidsequencing easier than polypeptidesequencing:

The type II restriction endonucleases that cleaveDNA at specific oligonucleotide sites

Gel electrophoresis, which is capable of separatingnucleic acid fragments that differ from oneanother in length by just a single nucleotide


Chain termination method (dideoxy method),developed by Frederick Sanger is the basis for mostDNA sequencing currently.

The method takes advantage of the DNA polymerasereaction, which copies a DNA strand in

complementary fashion to form a new second strand


Figure 11.1 DNA replication yields twodaughter DNA duplexes identical to theparental DNA molecule.


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Figure 11.2 Primed synthesis of a DNA template byDNA polymerase, using the four deoxynucleosidetriphosphates as the substrates.


DNA is a double-helical molecule Each strand of the helix must be copied incomplementary fashion by DNA polymerase

Each strand is a template for copying DNA polymerase requires template and primer Primer: an oligonucleotide that pairs with the

end of the template molecule to form dsDNA DNA polymerases add nucleotides in 5'-3'

direction

Chain Termination Method

Primer extension: A template DNA base-paired witha complementary primer is copied by DNApolymerase in the presence of dATP, dCTP, dGTP,dTTP

Solution contains small amounts of the fourdideoxynucleotide analogs of these substrates, eachof which contains a distinctive fluorescent tag,illustrated here as:

Orange for ddATP

Blue for ddCTP

Green for ddGTP

Red for ddTTP

Occasional incorporation of a dideoxynucleotideterminates further synthesis of that strand

Figure 11.3 The chain

termination method ofDNA sequencing.


Most of the time, the polymerase uses normalnucleotides and DNA molecules grow normally

Occasionally, the polymerase uses adideoxynucleotide, which prevents furtherextension when added to the growing chain

Random insertion of dd-nucleotides leaves(optimally) at least a few chains terminated atevery occurrence of a given nucleotide


Reaction mixtures can be separated by capillaryelectrophoresis

Short fragments go to bottom, long fragments ontop

Read the "sequence" from bottom of gel to top Convert this "sequence" to the complementary

sequence Now read from the other end and you have the

sequence you wanted - read 5' to 3'


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The set of terminated strands can be separatedby capillary electrophoresis

High-Throughput DNA Sequencing by the Light ofFireflies

The importance of DNA sequence information has

motivated development of more rapid and efficientDNA sequencing technologies

454 Technology relies on DNA polymerase but doesnot involve chain termination

Multiple copies of template DNA molecules areimmobilized on microscopic beads

Reagents for primed synthesis are passed over thebeads

Pyrophosphate release is monitored by lightemission via ATP sulfurylase and luciferase reactions

High-Throughput DNA Sequencing by the Light ofFireflies

DNA polymerase action produces PPi:(NMP)n + NTP (NMP)n+1 + PPi

ATP sulfurylase: PPi + APS ATP + SO42-

Luciferase:ATP + luciferin + O2 AMP + PPi + CO2 + oxyluciferin + light

Structures of luciferin and oxyluciferin. Light detectionconfirms that addition of a dNMP by primed synthesis hasoccurred.

High-Throughput DNA Sequencing by the Lightof Fireflies

Figure 11.4

Emerging Technologies to Sequence DNA are Based onSingle-Molecule Sequencing Strategies

Growing demand for sequence information is driving thedevelopment of faster and cheaper methods of DNAsequencing

Most promising are the single-molecule strategies that donot rely on Sanger-based primed synthesis of strandscomplementary to prepared DNA samples

One technique involves passing a single strand of DNAthrough a graphene monolayer pore, measuring thechange in electrical conductance (ion flow) through thepore

Each base alters electrical conductance in a subtle butdifferent way, facilitating the reading of sequence

Figure 11.5 DNA Sequencing through a pore in agraphene monolayer


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11.2 What Sorts of Secondary Structures Can Double-Stranded DNA Molecules Adopt?

Polynucleotide strands are flexible

Each deoxyribose-phosphate segment of thebackbone has six degrees of freedom (Fig 11.4a)

Furanose rings are not planar but instead adoptpuckered conformations, four of which are shown inFigure 11.4b

A seventh degree of freedom per nucleotide arisesbecause of free rotation about the C1'-N glycosidicbond

This freedom allows the plane of the base to rotaterelative to the path of the polynucleotide strand


Figure 11.6 The six degrees of freedom in thedeoxyribose-PO4 units of the polynucleotide chain.


Figure 11.6 Four puckered conformations of the furanoserings.

11.2 What Sorts of Secondary Structures CanDouble-Stranded DNA Molecules Adopt?

Figure 11.6 Free rotation about the C1'-N glycosidic bond.

Figure 11.7

(a) Double-stranded DNA as an imaginaryladderlike structure.(b) A simple right-handed twist converts theladder to a helix.


The stability of the DNA double helix is due to:

Hydrogen bonds between base pairs

Electrostatic interactions mutual repulsion ofphosphate groups, which makes them most stable onthe helix exterior

Base-pair stacking interactions

Right-twist closes the gaps between base pairs to 3.4 A(0.34 nm) in B-DNA

See Figure 11.8 for details of DNA secondary structure


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The canonical base pairs

See Figure 11.8 The canonical A:T and G:C base pairs have nearly

identical overall dimensions

A and T share two H bonds

G and C share three H bonds

G:C-rich regions of DNA are more stable

Polar atoms in the sugar-phosphate backbonealso form H bonds


Figure 11.8 Watson-

Crick A:T and G:C basepairs. All H-bonds inboth base pairs arestraight.


Figure 11.8 Watson-Crick A:T and G:Cbase pairs. All H-bonds in both basepairs are straight.

Major and minor grooves

See Figures 11.8, 11.9

The "tops" of the bases (as we draw them) line the"floor" of the major groove

The major groove is large enough to accommodate analpha helix from a protein

Regulatory proteins (transcription factors) canrecognize the pattern of bases and the H-bondingpossibilities in the major groove

Major and minor grooves

Figure 11.9 Themajor and minorgrooves of B-DNA.

Double Helical Structures Can Adopt a Numberof Stable Conformations

The DNA double helix can adopt several stableconformations

Helical twist is the rotation of one base pair relativeto the next, around the axis of the double helix

Successive base pairs in B-DNA show a mean rotation

of 36 with respect to each other

Propellor twist involves rotation around a differentaxis, namely an axis perpendicular to the helix axis

See Figure 11.10


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Figure 11.10 Helical twist and propellor twist in DNA(a) Helical twist: Successive base pairs in B-DNA show arotation with respect to each other.


Figure 11.10 Helical twist and propellor twist in DNA.(b) Propellor twist: Rotation in this dimension allows thehydrophobic surfaces of bases to overlap better

Double Helical Structures Can Adopt a Number ofStable Conformations

Helical twist and propellor twist in DNA. (c) Each of thebases in a base pair shows positive propellor twist as viewed

along the N-glycosidic bond. Note how the hydrogen bondsbetween bases are distorted by this motion, yet remain intact.


Figure 11.11 The B-form of theDNA double helix. In B-form, thepitch (the distance required tocomplete one helical turn) is 3.4nm. Twelve base pairs of DNA areshown.


Figure 11.11 The B-form of theDNA double helix. In B-form, thepitch (the distance required tocomplete one helical turn) is 3.4

nm. Twelve base pairs of DNA areshown.


Figure 11.11 The A-form ofthe DNA double helix. Thepitch of the A-form helix is2.46; thus the A-form is ashorter, wider structure thanthe B-form. One turn in A-form DNA requires 11 bp tocomplete. Twelve base pairsare shown here.


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Figure 11.11 The A-form ofthe DNA double helix. Thepitch of the A-form helix is2.46; thus the A-form is ashorter, wider structure thanthe B-form. One turn in A-form DNA requires 11 bp tocomplete. Twelve base pairsare shown here.

Z-DNA

Discovered by Alex Rich

Found in G:C-rich regions of DNA G goes to syn conformation

C stays anti but whole C nucleoside (base andsugar) flips 180 degrees

Result is that G:C H bonds can be preserved in thetransition from B-form to Z-form!

Z-DNA is a Conformational Variation in theForm of a Left-Handed Double Helix

Figure 11.11 The Z-form of doublehelical DNA.

The N-glycosyl bonds of Gresidues in this alternatingcopolymer are rotated 180 withrespect to their conformation in B-DNA, so now the G nucleoside is inthe syn rather than the anticonformation.

The C residues remain in the antiform.

Because the G ring is flipped, the Cring must also flip to maintainnormal Watson-Crick base pairing.

Z-DNA is a Conformational Variation in the Formof a Left-Handed Double Helix

Figure 11.11 The Z-form ofdouble helical DNA.

The N-glycosyl bonds of Gresidues in this alternatingcopolymer are rotated 180with respect to theirconformation in B-DNA, sonow the purine ring is in thesyn rather than the anticonformation.

The C residues remain in theanti form.

Because the G ring is flipped,the C ring must also flip tomaintain normal Watson-Crickbase pairing.

Comparison of A, B, Z DNA

See Table 11.1

A: right-handed, short and broad, 2.3 , 11 bp perturn

B: right-handed, longer, thinner, 3.32 , 10 bp perturn

Z: left-handed, longest, thinnest, 3.8 , 12 bp perturn

See Figure 11.11

Comparison of A, B, Z DNA


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Comparison of B and Z DNA

Figure 11.12 Comparison of the deoxyguanosine conformationin B- and Z-DNA. It is topologically possible for G to go syn andthe C nucleoside to undergo rotation by 180 without breakingand re-forming the G:C hydrogen bonds.

The Change in Topological Relationships ofBase Pairs from B- to Z-DNA

Figure 11.13

DNA Methylation and Epigenetics

Methylation of cytosine residues (forming 5-methylcytosine) is essential for normal embryonicdevelopment

Cytosine methylation switches genes off, so that theinformation they encode is not expressed

Epigenetics is the study of heritable changes in thegenome that occur without a change in nucleotidesequence (such as cytosine methylation)

Epigenetic changes can influence expression of theinformation encoded by the genome

Intercalating Agents Distort the Double Helix

The double helix is a very dynamic structure

Because it is flexible, aromatic macrocycles flat hydrophobic molecules composed offused, heterocyclic rings, can slip between thestacked pairs of bases

The bases are force apart to accommodatethese intercalating agentsEthidium bromide

Acridine orange

Actinomycin D

Intercalating Agents Distort the Double Helix

Figure 11.14 Thestructures of ethidiumbromide, acridine orange,and actinomycin D, three

intercalating agents, andtheir effects on DNAstructure.

Alternative H-Bonding Interactions Give Riseto Novel DNA Structures

Cruciform structures arise frominverted repeats. In such structures,the normal interstrand base pairing isreplaced by intrastrand pairing.

Figure 11.15 Self-complementary invertedrepeats can rearrange to form H-bondedcruciform stem-loop structures. Cruciformsare not as stable as normal DNA, becausean unpaired segment must exist in the loop.


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Hoogsteen Base Pairs and DNA Multiplexes

Karst Hoogsteen found A:T and G:C base pairs that are different

from the canonical structures. In both A:T and G:C Hoogsteenbase pairs, the purine N-7 atom is an H-bond acceptor.

Figure 11.16 Hoogsteen base pairs: A:T (left) and C+:G (right).

Hoogsteen Base Pairs and DNA Multiplexes

Figure 11.17 Base triplets can form when a purine interactswith one pyrimidine by Hoogsteen base pairing and anotherby Watson-Crick base pairing.

H-DNA is Triplex DNA Made of One Purine-Rich Strandand Two Pyrimidine-Rich Strands

Figure 11.18 H-DNA.Pyrimidine-rich strandsare blue; purine-richstrands are green.

DNA Quadruplex Structures

Figure 11.19 G-quadruplexshowing the cyclic array ofguanines linked byHoogsteen hydrogen bonds.

G-quadruplexes are cyclicarrays of four G residuesunited through Hoogsteenbase pairing.


Figure 11.19 Four G-richpolynucleotide strands inparallel alignment with all

bases in anti conformation.


Figure 11.19 Antiparalleldimeric hairpinquadruplex formed byd(G4T4G4)2


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Figure 11.19 Structure ofd(G4T4G4)2K

+ solved by X-ray crystallography. Twod(G4T4G4)2 strands cometogether as hairpins to forma G-quadruplex. Thebackbones of the twostrands are traced in violet.

11.3 Can the Secondary Structure of DNA BeDenatured and Renatured?

See Figure 11.20 When DNA is heated to 80 C or more, its UV

absorbance increases by 30-40% This hyperchromic shift reflects the unwinding of

the DNA double helix Stacked base pairs in native DNA absorb less light

due to , electron interactions When T is lowered, the absorbance drops, reflecting

re-establishment of the double helix and base-pairstacking

11.3 Can the Secondary Structure of DNA BeDenatured and Renatured?

Figure 11.18 Heat denaturation of DNA from various sources.

The Buoyant Density of DNA

Density gradient ultracentrifugation isa useful way to separate and purifynucleic acids.

The net movement of solute particles in an ultracentrifuge isthe result of two processes: diffusion (from regions of higher

concentration to regions of lower concentration) andsedimentation due to centrifugal force.

Single-Stranded DNA Can Renature to FormDNA Duplexes

Denatured DNA will renature to re-form theduplex structure if the denaturing conditionsare removed

Renaturation requires reassociation of the

DNA strands into a double helix, a processtermed reannealing

For this to occur, the strands must realign sothat their complementary bases are onceagain in register and the helix can be

zippered up

Single-Stranded DNA Can Renature to FormDNA Duplexes

Figure 11.21 Steps in thethermal denaturation andrenaturation of DNA.


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If DNA from two different species are mixed,denatured, and allowed to cool slowly, hybridduplexes may form, provided the DNA from onespecies is similar in sequence to the other

The degree of hybridization is a measure of thesequence similarity between the two species

25% of the DNA from a human forms hybrids withmouse DNA, implying some sequence similarity

Hybridization is a common procedure in molecularbiology for identifying specific genes and forrevealing evolutionary relationships

Nucleic Acid Hybridization: Different DNA Strands ofSimilar Sequence Can Form Hybrid Duplexes

Nucleic Acid Hybridization: Different DNA Strands ofSimilar Sequence Can Form Hybrid Duplexes

Figure 11.22 Solutions ofhuman DNA (red) and mouseDNA (blue) are mixed anddenatured; then, the singlestrands are allowed toreanneal.

About 25% of human DNAforms hybrid duplexes withmouse DNA.

11.4 Can DNA Adopt Structures of HigherComplexity?

In duplex DNA, there are ten bp per turn of helix

Circular DNA sometimes has more or less than 10 bpper turn - a supercoiled state

Enzymes called topoisomerases or gyrases canintroduce or remove supercoils

Cruciforms occur in palindromic regions of DNA

Negative supercoiling may promote cruciforms

Supercoils Are One Kind of Structural Complexityin DNA

Double-stranded circular DNA forms supercoils, if thestrands are underwound, or overwound.

Figure 11.23 Toroidal and interwound varieties of supercoiling.

Supercoiled DNA is characterized by a Linking Number(L), Twist (T), and Writhe (W)

Figure 11.24 Linking number (L) is sum of twist (T) and writhe (W)

Supercoiled DNA is characterized by a Linking Number(L), Twist (T), and Writhe (W)

Figure 11.24 Linking number (L) is sum of twist (T) and writhe (W)


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DNA Gyrase is a topoisomerase that introducesnegative supercoils into DNA

Figure 11.25 A model for the action ofbacterial DNA gyrase (topoisomeraseII).

Negative supercoils cause a torsionalstress on the molecule, so themolecule tends to unwind. Negativesupercoiling makes it easier toseparate DNA strands and access theinformation encoded by the sequence.

DNA Gyrase is a topoisomerase that introducesnegative supercoils into DNA

Figure 11.25 Conformationalchanges in the enzyme allow anintact region of the DNA duplexto pass between the cut ends.The cut ends are religated (3),and the covalently completeDNA duplex is released from theenzyme. The circular DNA nowcontains two negativesupercoils (4).

Negative Supercoiling has the Potential to CauseLocalized Unwinding in DNA

Figure 11.26 A 400-bp circular DNA molecule in differenttopological states: (a) relaxed, (b) negative supercoilsdistributed over the entire length, and (c) negative

supercoils creating a localized single-stranded region.

Negatively Supercoiled DNA Can Arrange into aToroidal State

Figure 11.28 The toroidal state is stabilized by wrappingaround proteins that serve as spools for the DNA ribbon .

11.5 What Is the Structure of EukaryoticChromosomes?

Human DNA s total length is ~2 meters!

This must be packaged into a nucleus that is about 5micrometers in diameter

This represents a compression of more than100,000!

It is made possible by wrapping the DNA aroundprotein spools called nucleosomes and thenpacking these in helical filaments

These filaments are thought to arrange in loopsassociated with the nuclear matrix

Nucleosomes Are the Fundamental StructuralUnit in Chromatin

Histones and nonhistone chromosomal proteins are the twoclasses of chromatin proteins. Five distinct histones areknown: H1, H2A, H2B, H3, and H4.

Pairs of histones H2A, H2B, H3, and H4 aggregate toform octameric core structures; the DNA helix is woundaround these core octamers, creating nucleosomes.


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Nucleosome Structure

Chromatin, the nucleoprotein complex,consists of histones and nonhistonechromosomal proteins

Histone octamer structure has been solved(without DNA by Moudrianakis, and with DNAby Richmond)

Nonhistone proteins are regulators of geneexpression

The Structure of the Nucleosome a HistoneOctamer wrapped with DNA

Figure 11.28

Structural Organization of Chromatin Gives Rise toChromosomes

The beads-on-a-string motif is the primarystructure of chromatin.

The secondary level of chromatin structure is the30-nm fiber, formed when an array of nucleosomesin a zig-zag pattern adopts a two-start helicalconformation (Figures 11.29a, b, c).

Higher levels of chromatin structural organization areachieved when the 30-nm fiber forms long loops of60-150,000 bp.

Electron microscopic analysis of human chromosome4 suggests that 18 such loops are then arranged

radially about the circumference of a single turn toform a miniband unit of the chromosome.

Higher Order Chromatin Organization

Figure 11.29a


Figure 11.29b


Figure 11.29c


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Higher-Order Structural Organization ofChromatin Gives Rise to Chromosomes

Figure 11.30 A model for chromosome structure, humanchromsome 4, showing nucleosomes in the beads on astring motif.


Figure 11.30 A model for chromosome structure, humanchromsome 4. The 30-nm fiber is created when the arrayof nucleosomes adopts a two-start helical conformation.


Figure 11.30 A model for chromosome structure, humanchromsome 4. The 30 nm filament forms long DNA loops ofvariable length, each containing on average between60,000 and 150,000 bp.


Figure 11.30 A model for chromosome structure, humanchromsome 4. Electron microscopic analysis ofchromosome 4 suggests that 18 loops are arranged

radially about the circumference of a single turn to form aminiband unit of the chromosome.


Figure 11.30 A model for chromosome structure, humanchromsome 4. Approximately a million minibands arearranged along a central axis in each of the chromatids ofchromosome 4 that form at mitosis.

SMC Proteins Aid Chromosome Organization andMediate Chromosome Dynamics

Figure 11.31 SMC protein architecture and func tion. SMCproteins range from 115 to 165 kD in size.


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SMC Proteins Aid Chromosome Organization andMediate Chromosome Dynamics

Figure 11.31 SMC protein architecture and function. Shownhere is the condensation of DNA into a coiled arrangementthrough SMC2/SMC4-mediated interactions.

11.6 Can Nucleic Acids Be SynthesizedChemically?

Laboratory synthesis of nucleic acids requires

orthogonal strategies. Functional groups on the monomeric units are

reactive and must be blocked.

Correct phosphodiester linkages must be made

Recovery at each step must high!

Solid-phase methods are used to satisfy some ofthese constraints.

Solid Phase Oligonucleotide Synthesis

The four-step cycle starts with the first base innucleoside form attached by its 3'-OH.

Then a dimethoxytrityl group blocks the 5'-OH ofthe first nucleoside while it is linked to a solidsupport by the 3'-OH.

Step 1: Detritylation by trichloroacetic acid exposesthe 5'-OH.

Step 2: In coupling reaction, second base is addedas a nucleoside phosphoramidate.


Step 3: Capping with acetic anhydride blocksunreacted 5'-OHs of N-1 from further reaction

Step 4: The phosphite linkage between N-1 andN-2 is reactive and is oxidized by aqueous iodineto form the desired, and more stable, phosphategroup

After the desired oligonucleotide has beenformed, it is freed of blocking groups, hydrolyzedfrom the resin, and purified by gelelectrophoresis

The four-step cycle is shown in Figure 11.32


The structure of the dimethoxytrityl group. (Atriphenylmethyl group is a trityl group.)

Figure 11.32


The DMTr protecting group can be removed bytrichloroacetic acid treatment.

Figure 11.32


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Figure 11.32 Benzoyl chloride can be used to protectNH2 functions


Figure 11.29

Figure 11.32 Isobutyryl chloride can also be used to protect-NH2 functions

Genes Can Be Synthesized Chemically11.7 What Are the Secondary and Tertiary

Structures of RNA?

The double-stranded structure of DNA imposes greatconstraints on its conformational possibilities

RNA molecules are typically single-stranded and thushave six to seven degrees of freedom per nucleotideunit

Thus RNA molecules have a much greater number ofconformational possibilities

Complementary sequences in RNA can join viaintrastrand base pairing

When the base pairing is not complete, a variety ofbulges and loops can form, including hairpin stem-loop structures

11.7 What Are the Secondary and TertiaryStructures of RNA?

Figure 11.33Bulges andloops formed inRNA when

alignedsequences arenot fullycomplementary


A number of defined structural motifs recur withinthe loops of stem-loop structures, such as U-turns,tetraloops, and bulges

Regions where several stem-loop structures meet aretermedjunctions

Stems, loops, bulges, and junctions are the four basicsecondary structural elements in RNA

Other tertiary structural motifs arise from coaxialstacking, pseudoknot formation, and ribose zippers


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Figure 11.34 Junctions and coaxial stacking in RNA.


Figure 11.35 RNApseudoknots are formedwhen a single-strandedregion of RNA base-pairswith a hairpin loop.

Transfer RNA Adopts Higher-Order StructureThrough Intrastrand Base Pairing

In tRNA, with 73-94 nucleotides in a single chain, amajority of the bases are hydrogen- bonded to oneanother

Hairpin turns bring complementary stretches ofbases into contact

Extensive H-bonding creates four double helicaldomains, three capped by loops, one by a stem

Only one tRNA structure (alone) is known

Phenylalanine tRNA is "L-shaped"

Many non-canonical base pairs found in tRNA

Transfer RNA Adopts Higher-Order Structure ThroughIntrastrand Base Pairing

Figure 11.36 Ageneral diagram forthe structure of tRNA.

Transfer RNA Adopts Higher-Order StructureThrough Intrastrand Base Pairing

Figure 11.37 Tertiary structure intRNA arises from base-pairinginteractions between bases in theD loop with bases in the variableand TC loops, as shown here for

yeast phenylalanine tRNA.

Solid lines connect bases that arehydrogen-bonded when thiscloverleaf pattern is folded into thecharacteristic tRNA tertiarystructure (see Figure 11.38).

tRNA Tertiary Structure Arises From InterloopBase Pairing

Figure 11.38


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tRNA Tertiary Structure Arises From InterloopBase Pairing

Figure 11.38 Thethree-dimensionalstructure of yeastphenylalanine tRNA.The anticodon loop isat the bottom and theacceptor end is at thetop right.

Ribosomal RNA

Ribosomes synthesize proteins All ribosomes contain large and small subunits

rRNA molecules make up about 2/3 of ribosome

High intrastrand sequence complementarity leadsto extensive base-pairing

Secondary structure features seem to beconserved, whereas sequence is not

There must be common designs and functionsthat must be conserved

Ribosomal RNA also Adopts Higher-OrderStructure Through Intrastrand Base Pairing

These secondary structures of several 16S rRNAs are based on computeralignment of rRNAnucleotide sequences into optimal H-bonding segments.

Figure 11.39 Comparison of secondary structures of 16S-likerRNAs from several organisms.

rRNA Tertiary Structure

Figure 11.40 Detailedstructures of ribosomes havebeen revealed by X-raycrystallography andcryoelectron microscopy.

These images reveal details ofboth tertiary and quaternaryinteractions that occur whenribosomal proteins combinewith rRNAs to form thecomplete ribosome.

Riboswitches Act as Regulators of GeneExpression

Riboswitches, naturallyoccurring aptamers, areconserved regions of mRNAsthat reversibly bind specificmetabolites and coenzymes

and act as gene expressionregulators.

Figure 11.41

Chapter 12Recombinant DNA: Cloning and

Creation of Chimeric Genes


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12.1 What Does It Mean To Clone ?

Clone: a collection of molecules or cells, all

identical to an original molecule or cell

To "clone a gene" is to make many copies of it -for example, in a population of bacteria

Gene can be an exact copy of a natural gene

Gene can be an altered version of a natural gene

Recombinant DNA technology makes it possible

Plasmids Are Very Useful in Cloning Genes

Plamids are naturally-occurring

extrachromosomal DNA Plasmids are circular dsDNA

Plasmids can be cleaved by restriction enzymes,leaving sticky ends

Artificial plasmids can be constructed by linkingnew DNA fragments to the sticky ends of plasmid

These recombinant molecules can beautonomously replicated, and hence propagated

Cloning Vectors

Cloning vectors are plasmids that can be modified to

carry new genes

Plasmids useful as cloning vectors must have

a replicator (origin of replication)

a selectable marker (antibiotic resistance gene)

a cloning site (site where insertion of foreignDNA will not disrupt replication or inactivateessential markers)

Plasmids as Cloning Vectors

Figure 12.1 One ofthe first widely usedcloning vectors wasthe plasmid pBR322.

Note the antibioticresistance genes(ampr and tetr).

Virtually Any DNA Sequence Can Be Cloned

Nuclease cleavage at a restriction sitelinearizes the circular plasmid so that aforeign DNA fragment can be inserted.

Recombinant plasmids are hybridDNA molecules consisting of plasmidDNA sequences plus inserted DNAelements (pink here).

Such hybrid molecules are calledchimeric plasmids.

Figure 12.2 An EcoRI restrictionfragment of foreign DNA can beinserted into a plasmid.

Chimeric Plasmids

Named for mythological beasts with bodyparts from several creatures

After cleavage of a plasmid with a restriction enzyme, aforeign DNA fragment can be inserted

Ends of the plasmid/fragment are joined to form a"recombinant plasmid"

Recombinant plasmid can replicate when placed in asuitable bacterial host

See Figure 12.2


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Short DNA Duplexes With Restriction Sites Can BeUsed as Linkers

Figure 12.3 The use oflinkers to create tailor-madeends on cloning fragments.

Directional Cloning

Often one desires to insert foreign DNA in a particular

orientation This can be done by cleaving the plasmid with twodifferent restriction enzymes

Cleave the foreign DNA with same two restrictionenzymes

Foreign DNA can only be inserted in one direction

See Figure 12.4

Directional Cloning

Figure 12.4 Directional cloning.DNA molecules whose endshave different overhangs canbe used to form chimericconstructs in which the foreignDNA can enter the plasmid inonly one orientation.

Biologically Functional Chimeric Plasmids

Plasmids can be used to transform recipient E. colicells

( Transformation means the uptake and replicationof exogenous DNA by a recipient cell.)

To facilitate transformation, the bacterial cells arerendered somewhat permeable to DNA by Ca2+

treatment and a brief 42 C heat shock

The useful upper limit on cloned inserts in plasmidsis about 10 kbp. Many eukaryotic genes exceed thissize.

Biologically Functional Chimeric Plasmids

Figure 12.5 A typicalbacterial transformationexperiment. HerepBR322 is the cloningvector.

Shuttle Vectors Are Plasmids That Can Propagatein Two Different Organisms

Figure 12.6 A typical shuttle vector. LEU2+ is a gene in theyeast pathway for leucine biosynthesis.

Shuttle vectors are plasmids capable of propagating andtransferring ( shuttling ) genes between two different organisms.


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12.2 What Is a DNA Library?

A DNA library is a set of cloned DNA

fragments that together represent thegenes of a particular organism

Any particular gene may represent a tiny, tinyfraction of the DNA in a given cell

Can't isolate it directly

Trick is to find the fragment or fragments in thelibrary that contain the desired gene

12.2 What is a DNA Library?

The probabilities are daunting

Consider the formula on page 386 for probability of finding aparticular fragment in N clones

Suppose you want a 99% probability of finding a givenfragment in N clones of 10 kbp fragments

If your library is from the human genome, you would need1,400,000 clones to reach 99% probability of finding thefragment of interest!

Colony HybridizationA way to screen plasmid-based genome libraries for a DNA

fragment of interest

Host bacteria containing a plasmid-based library of DNAfragments are plated on a petri dish and allowed to growovernight to form colonies

Replica of colonies on the dish made with a nitrocellulose disc

Disc is treated with base or heated to convert dsDNA tossDNA and incubated with a labeled probe

Colonies that bind probe (labeled with 32P or other tag) holdthe fragment of interest

What is a DNA Library?

Figure 12.7 Screening a genomiclibrary by colony hybridization. Hostbacteria transformed with a plasmid-based genomic library are plated on apetri plate and incubated overnight toallow bacterial colonies to form.

A replica of the colonies is obtainedby overlaying the plate with a flexibledisc composed of absorbent material(such as nitrocellulose or nylon).

Probes for Southern Hybridization Can BePrepared in a Variety of Ways

Figure 12.8 Cloning genes usingoligonuceotide probes from a known aminoacid sequence. A radioactively labeled setof DNA (degenerate) oligonucleotidesrepresenting all possible mRNA codingsequences is synthesized and is used toprobe the genomic library by colonyhybridization (see Figure 12.7).

Labeling methodologies other thanradioactivity are also available.

Identifying Specific DNA Sequences by SouthernBlotting

Finding one particular DNA segment among a vast populationof different DNA fragments (e.g., in a genomic DNApreparation) is to exploit its sequence specificity to identify it.

Southern blots (invented by E.M. Southern) do this

DNA fragments (the library ) are fractionated by size withagarosegel electrophoresis

Separated molecules are blotted to an absorbent support andthen incubated with labeled (radioactive or otherwise)oligonucleotideprobes

Detection of the label shows the location of DNA fragmentsthat hybridized with the probe


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Identifying Specific DNA Sequences by SouthernBlotting

The Southern blottingtechnique involves thetransfer ofelectrophoreticallyseparated DNAfragments to anabsorbent sheet andsubsequent detection ofthe specific DNAsequences.

cDNA Libraries Are DNA Libraries Preparedfrom mRNA

cDNAs are DNAs copied from mRNA templates. cDNA libraries are constructed by synthesizing cDNA from

purified cellular mRNA.

Because most eukaryotic mRNAs carry 3'-poly(A) tails, mRNA canbe selectively isolated from preparations of total cellular RNA byoligo(dT)-cellulose chromatography (Figure 12.9)

DNA copies of the purified mRNAs are synthesized by firstannealing short oligo(dT) chains to the poly(A) tails.

These serve as primers for reverse transcriptase-driven synthesisof DNA (Figure 12.10)


Figure 12.9 Isolation of eukaryotic mRNA viaoligo(dT)-cellulose chromatography.


Reverse transcriptase is an enzyme that synthesizes a DNAstrand, copying RNA as the template

DNA polymerase is then used to copy the DNA strand andform a double-stranded duplex DNA

Linkers are then added to the DNA duplexes rendered fromthe mRNA templates

The cDNA is then cloned into a suitable vector

Once a cDNA derived from a particular gene has beenidentified, the cDNA becomes an effective probe for screeninggenomic libraries for isolation of the gene itself


Figure 12.10 Reversetranscriptase-drivensynthesis of cDNA from

oligo(dT) primers annealedto the poly(A) tails ofpurified eukaryotic mRNA.

DNA Microarrays Are Arrays of DifferentOligonucleotides Immobilized on a Chip

Robotic methods can be used to synthesize combinatoriallibraries of DNA oligonucleotidesdirectly on a solid support.

The completed library is a 2-D array of differentoligonucleotides

The final products of such procedures are referred to as genechips because the sequences synthesized upon the chip

represent the sequences of chosen genes The oligonucleotideson such gene chips are used as probes in

hybridization experiments to reveal gene expression patterns


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DNA Microarrays Are Arrays of DifferentOligonucleotides Immobilized on a Chip

Figure 12.11 Gene chips (DNAmicroarrays) in the analysis ofgene expression.

The Human Genome Project

A working draft of thehuman genome wascompleted in June, 2000and published inFebruary 2001.

The genomes of manyother organisms havenow been sequenced aswell.

Information about wholegenome sequences hascreated a new branch ofscience calledbioinformatics.

12.3 Can the Cloned Genes in Libraries BeExpressed?

Figure 12.12 Expression vectorscarrying the promoter recognized bythe RNA polymerase of bacteriophageSP6 are useful for the production of

multiple RNA copies of any DNAinserted at the polylinker.

Expression vectors are engineeredso that the RNA or protein productsof cloned genes can be expressed.


Figure 12.13 A typical expression-cloning vector.

To express a eukaryotic protein in E. coli, the eukaryoticcDNA must be cloned in an expression vector that containsregulatory signals for transcription and translation.


Figure 12.14 A ptacprotein expressionvector contains thehybrid promoter ptacderived from fusionof the lac and trppromoters.

Strong promotershave beenconstructed to drivesynthesis of foreignproteins to levels of30% of total E. coliprotein.


Figure 12.15 A typicalexpression vector forthe synthesis of ahybrid protein.

Some expressionvectors carry cDNAinserts cloned directlyinto the codingsequence of aprotein-coding gene.


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12.3 Can the Cloned Genes in Libraries BeExpressed? Reporter Gene Constructs

Figure 12.16 Greenfluorescent protein(GFP) as a reportergene.

Reporter gene

constructs arechimeric DNAmoleculescomposed of generegulatorysequences next toan easily

expressible geneproduct.

Specific Protein-Protein Interactions Can BeIdentified Using the Two-Hybrid System

Figure 12.17 The yeast two-hybrid system for identifyingprotein-protein interactions.If proteins X and Y interact,the lacZ reporter gene isexpressed. Cellsexpressing lacZ exhibit -galactosidase activity.

12.4 What Is the Polymerase Chain Reaction(PCR)?

What if you don't have enough DNA forcolony hybridization or Southern blots?

The small sample of DNA can serve as template for DNApolymerase

Make complementary primers; add DNA polymerase Add primers in more than 1000-fold excess Heat to separate dsDNA stran ds, then cool Run DNA polymerase (usually Taq) reaction again Repeat heating, cooling, polymerase cycle

12.4 What Is the Polymerase Chain Reaction(PCR)?

Figure 12.18 Polymerase chainreaction (PCR).

In Vitro MutagenesisFigure 12.19 One method of PCR-based site-directed mutagenesis.

(1) Template DNA strands areseparated and amplified byPCR.

(2) Following many cycles of PCR,the DNA product can be used totransform E. colicells.

(3) The plasmid DNA can beisolated and screened for thepresence of the uniquerestriction site (by restrictionendonuclease cleavage.


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12.5 How Is RNA Interference Used to Reveal theFunction of Genes?

RNA interference (RNAi) has emerged as amethod of choice in eukaryotic geneinactivation

RNAi leads to targeted destruction of aselected gene s transcript

The consequences following loss of genefunction reveal the role of the gene product incell metabolism

12.6 Is It Possible to Make Directed Changes inthe Heredity of an Organism?

Figure 12.20 Gene knockdown byRNAi.

The dsRNA is processed byDICER. Following unwinding byDICER, the guide strand isdelivered to the RISC complex.The guide strand and acomplementary mRNA arebrought together by Ago. RNaseon Ago cleaves the gene transcript(mRNA), rendering it incapable oftranslation by ribosomes.

Human Gene Therapy Can Repair GeneticDeficiencies

Figure 12.21 Retrovirus-mediatedgene delivery ex vivo using MMLV.

A basic strategy of human gene therapyinvolves incorporation of a functionalgene into target cells.Retroviruses (RNA viruses that makeDNA from RNA) provide a route forpermanent modification of host cells exvivo.

Human Gene Therapy Can Repair GeneticDeficiencies

Figure 12.22 Adenovirus-mediatedgene delivery in vivo.Adenoviruses are DNA viruses.

Adenovirus vectors are a possible invivo approach to human gene therapy.

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