Chapter 8 Summary

Molecular Cell Biology SCI 231

Restriction nucleases cleave DNA at specific nucleotide sequences.
Restriction enzymes often work as dimers, and the DNA sequence
they recognize and cleave is often symmetrical around a central
point. Here, both strands of the DNA double helix are cut at specific
points within the target sequence (orange). Some enzymes, such as
HaeIII, cut straight across the double helix and leave two bluntended DNA molecules; with others, such as EcoRI and HindIII, the
cuts on each strand are staggered. These staggered cuts generate

sticky endsshort, single- stranded overhangs that help the cut

DNA molecules join back together through complementary basepairing. This rejoining of DNA molecules becomes important for DNA
cloning.

DNA molecules can be separated by size using

gel electrophoresis. (A) Schematic illustration
comparing the results of cutting the same DNA
molecule with two different restriction nucleases,
EcoRI (middle) and HindIII (right). The fragments
are then separated by gel electrophoresis using
a gel matrix of agarose. Because larger
fragments migrate more slowly than smaller
ones, the lowermost bands on the gel contain
the smallest DNA fragments. The sizes of the
fragments can be estimated by comparing them
to a set of DNA fragments of known sizes (left).
Because DNA polymerases synthesize sequences
complementary to an existing DNA molecule,
they are often used in the test tube to create
exact copies of existing DNA molecules. The
copies can include specially modified

nucleotides. To synthesize DNA in this way, the DNA polymerase is

presented with a template and a pool of nucleotide precursors that
contain the modification. As long as the polymerase can use these
precursors, it automatically makes new, modified molecules that
match the sequence of the template. Modified DNA molecules have
many uses. DNA labeled with the radioisotope 32P can be detected
following gel electrophoresis by placing the gel next to a piece of
photographic film.

DNA ligase can join together any two DNA fragments in vitro to
produce recombinant DNA molecules. ATP provides the energy
needed to reseal the sugar-phosphate backbone of DNA.

Human genomic libraries containing DNA fragments that represent

the whole human genome can be constructed using restriction
nucleases and DNA ligase. The DNA is cleaved with restriction
nucleases into millions of genomic DNA fragments, which are then
inserted into plasmids and introduced into bacteria.
An alternative strategy is to begin the cloning process by selecting
only those DNA sequences that are transcribed into mRNA and thus
correspond to protein-encoding genes. This is done by extracting
mRNA from cells and then making a DNA copy of each mRNA
molecule present a complementary DNA (cDNA).

PCR uses repeated rounds of strand separation, hybridization, and

synthesis to amplify DNA. As the procedure outlined in Figure 835 is
repeated, all the newly synthesized fragments serve as templates in
their turn. Because the polymerase and the primers remain in the
sample after the first cycle, PCR involves simply heating and then
cooling the same sample, in the same test tube, again and again.
Each cycle doubles the amount of DNA synthesized in the previous
cycle.

PCR can be used to detect the presence of a viral genome in a

sample of blood. Because of its ability to amplify enormously the

signal from a single molecule of nucleic acid, PCR is an

extraordinarily sensitive method for detecting trace amounts of
virus in a sample of blood or tissue, without the need to purify the
virus.

3 types of matrices used for chromatography:

Proteins usually possess a net positive or

negative charge, depending on the mixture
of charged amino acids they contain. An
electric field applied to a solution containing
a protein molecule causes the protein to
migrate at a rate that depends on its net
charge and on its size and shape. The most
popular application of this property is SDS
polyacrylamide-gel electrophoresis
(SDS-PAGE). It uses a highly cross-linked
gel of polyacrylamide as the inert matrix
through which the proteins migrate. The gel
is prepared by polymerization of monomers;
the pore size of the gel can be adjusted so
that it is small enough to retard the
migration of the protein molecules of
interest. The proteins are dissolved in a
solution that includes a powerful negatively
charged detergent, sodium dodecyl sulfate,
or SDS. Because this detergent binds to
hydrophobic regions of the protein
molecules, causing them to unfold into
extended polypeptide chains, the individual
protein molecules are released from their
associations with other proteins or lipid
molecules and rendered freely soluble in the detergent solution.

A specific protein can be identified after its fractionation on a

polyacrylamide gel by exposing all the proteins present on the gel to
a specific antibody that has been labeled with a radioactive isotope
or a fluorescent dye. This procedure is normally carried out after
transferring all of the separated proteins present in the gel onto a
sheet of nitrocellulose paper or nylon membrane. Placing the
membrane over the gel and driving the proteins out of the gel with a
strong electric current transfers the protein onto the membrane. The
membrane is then soaked in a solution of labeled antibody to reveal
the protein of interest. This method

Searching a collection of known sequences for similar genes or

proteins is typically done over the Internet, and it simply involves
selecting a database and entering the desired sequence. A
sequence-alignment programthe most popu- lar is BLASTscans

the database for similar sequences by sliding the submitted

sequence along the archived sequences until a cluster of residues
falls into full or partial alignment.
Fluorescence anisotropy:
This method depends on a fluorescently tagged protein that is
illuminated with polarized light at the appropriate wavelength for
excitation; a fluorimeter is used to measure the intensity and
polarization of the emitted light. If the fluorescent protein is fixed in
position and therefore does not rotate during the brief period
between excitation and emission, then the emitted light will be
polarized at the same angle as the excitation light. This directional
effect is called fluorescence anisotropy. Protein molecules in
solution rotate or tumble rapidly, however, so that there is a
decrease in the amount of anisotropic fluorescence. Larger
molecules tumble at a slower rate and therefore have higher
fluorescence anisotropy. (A) To measure the binding between a small
molecule and a large receptor protein, the smaller molecule is
labeled with a fluorophore. In the absence of its binding partner, the
molecule tumbles rapidly, resulting in low fluorescence anisotropy
(top). When the small molecule binds to its larger partner, however,
it tumbles less rapidly, resulting in an increase in fluorescence
anisotropy (bottom).

< Production of large amounts of a

protein from a protein- coding DNA
sequence cloned into an expression
vector and introduced into cells. A
plasmid vector has been engineered to
contain a highly active promoter, which
causes unusually large amounts of mRNA
to be produced from an adjacent proteincoding gene inserted into the plasmid
vector.
Recombinant DNA techniques make it
possible to move experimentally from
gene to protein and from protein to gene.
If a gene has been identified, its proteincoding sequence can be inserted into an expression vector to
produce large quantities of the protein, which can then be studied
biochemically or structurally. If a protein has been purified based on
its biochemical properties, mass spectrometry can be used to obtain
a partial amino acid sequence, which is used to search a genome
sequence for the corresponding nucleotide sequence. The complete
gene can then be cloned by PCR from a sequenced genome. The
gene can also be manipulated and introduced into cells or
organisms to study its function, a topic covered in the next section
of this chapter (below).

Analternativeapproachtochemicalorradiationmutagenesisiscalled
insertionalmutagenesis.ThismethodreliesonthefactthatexogenousDNA
insertedrandomlyintothegenomecanproducemutationsiftheinserted
fragmentinterruptsageneoritsregulatorysequences.TheinsertedDNA,
whosesequenceisknown,thenservesasamoleculartagthataidsinthe
subsequentidentificationandcloningofthedisruptedgene.
Onceacollectionofmutantsinamodelorganismsuchasyeastorflyhasbeen
produced,onegenerallymustexaminethousandsofindividualstofindthe
alteredphenotypeofinterest.Suchasearchiscalledageneticscreen,andthe
largerthegenome,thelesslikelyitisthatanyparticulargenewillbemutated.
Therefore,thelargerthegenomeofanorganism,thebiggerthescreeningtask
becomes.Thephenotypebeingscreenedforcanbesimpleorcomplex.Simple
phenotypesareeasiesttodetect:onecanscreenmanyorganismsrapidly,for
example,formutationsthatmakeitimpossiblefortheorganismtosurvivein
theabsenceofaparticularaminoacidornutrient.

Example of a conditional mutation:

Genemutationsaffecttheirproteinproductindifferentways.Inthisexample,thewildtype
proteinhasaspecificcellfunctiondenotedbytheredrays.Mutationsthateliminatethis
functionorinactivateitathighertemperaturesareshown.

A large-scale genetic screen can turn up many different mutations

that show the same phenotype. These defects might lie in different
genes that function in the same process, or they might represent
different mutations in the same gene. Alternative forms of the same
gene are known as alleles. The most common difference between
alleles is a substitution of a single nucleotide pair, but different
alleles can also bear deletions, substitutions, and duplications. How
can we tell, then, whether two mutations that produce the same
phenotype occur in the same gene or in different genes? If the
mutations are recessiveif, for example, they represent a loss of
function of a particular genea complementation test can be
used to ascertain whether the mutations fall in the same gene or in
different genes. To test complementation in a diploid organism, an
individual that is homozygous for one mutationthat is, it possesses
two identical alleles of the mutant gene in questionis mated with
an individual that is homozygous for the other mutation. If the two

mutations are in the same gene, the offspring show the mutant
phenotype, because they still will have no normal copies of the gene
in question.

Genesthataffecttheriskof
developingacommondiseasecan
oftenbetrackeddownthrough
linkagetoSNPs.Asegmentofa
typicalchromosomeisshown.For
mostpolymorphicsitesinthis
segment,itisarandommatter
whetheranindividualhasoneSNP
variant(redverticalbars)oranother
(blueverticalbars);thissame
randomnessisseenbothforthe
controlgroupandfortheaffected
individuals.However,inthepartof
thechromosomethatisshadedin
darkergray,abiasisseen:most
normalindividualshavetheblueSNP
variants,whereasmostaffected
individualshavetheredSNP
variants.Thissuggeststhatthis
regioncontains,oriscloseto,agene
thatisgeneticallylinkedtothesered
SNPvariantsandthatpredisposes
individualstothedisease.

Engineeredgenescanbeturnedonandoffwithsmallmolecules.Here,theDNAbinding
portionofabacterialprotein(thetetracycline,Tet,repressor)hasbeenfusedtoaportionofa
mammaliantranscriptionalactivatorandexpressedinculturedmammaliancells.The
engineeredgeneX,presentinplaceofthenormalgene,hasitsusualgenecontrolregion
replacedbycisregulatorysequencesrecognizedbythetetracyclinerepressor.Intheabsence
ofdoxycycline(aparticularlystableversionoftetracycline),theengineeredgeneis
expressed;inthepresenceofdoxycycline,thegeneisturnedoffbecausethedrugcausesthe
tetracyclinerepressortodissociatefromtheDNA.

< Summaryoftheproceduresusedformakinggenereplacementsinmice.Inthefirststep

(A),analteredversionofthegeneisintroducedintoculturedES(embryonicstem)cells.
OnlyafewEScellswillhavetheircorrespondingnormalgenesreplacedbythealteredgene
throughahomologousrecombinationevent.ThesecellscanbeidentifiedbyPCRand
culturedtoproducemanydescendants,eachofwhichcarriesanalteredgeneinplaceofone
ofitstwonormalcorrespondinggenes.Inthenextstepoftheprocedure(B),thesealteredES
cellsareinjectedintoaveryearlymouseembryo;thecellsareincorporatedintothegrowing
embryo,andamouseproducedbysuchanembryowillcontainsomesomaticcells(indicated
byorange)thatcarrythealteredgene.Someofthesemicewillalsocontaingermlinecells
thatcontainthealteredgene;whenbredwithanormalmouse,someoftheprogenyofthese
micewillcontainonecopyofthealteredgeneinalloftheircells.

Themicewiththe
transgeneintheirgerm
linearethenbredto
producebothamaleand
afemaleanimal,each
heterozygousforthe
genereplacement(that
is,theyhaveonenormal
andonemutantcopyof
thegene).Whenthese
twomicearemated(not
shown),onefourthof
theirprogenywillbe
homozygousforthe
alteredgene.

Althoughknockingout(orconditionallyexpressing)ageneinanorganismand
studyingtheconsequencesisthemostpowerfulapproachforunderstandingthe
functionsofthegene,RNAinterference(RNAi,forshort),isanalternative,
particularlyconvenientapproach.AsdiscussedinChapter7,thismethod
exploitsanaturalmechanismusedinmanyplants,animals,andfungitoprotect
themselvesagainstvirusesandtransposableelements.Thetechnique
introducesintoacellororganismadoublestrandedRNAmoleculewhose
nucleotidesequencematchesthatofpartofthegenetobeinactivated.Afterthe
RNAisprocessed,ithybridizeswiththetargetgeneRNA(eithermRNAor
noncodingRNA)andreducesitsexpression.

RNAlevelscanbemeasuredbyquantitativeRTPCR.Thefluorescencemeasuredis
generatedbyadyethatfluorescesonlywhenboundtothedoublestrandedDNAproductsof
theRTPCR(seeFigure836).TheredsamplehasahigherconcentrationofthemRNA
beingmeasuredthandoesthebluesample,sinceitrequiresfewerPCRcyclestoreachthe
samehalfmaximalconcentrationofdoublestrandedDNA.Basedonthisdifference,the
relativeamountsofthemRNAinthetwosamplescanbepreciselydetermined.

DNAmicroarraysareusedtoanalyzetheproductionofthousandsofdifferentmRNAsina
singleexperiment.Inthisexample,mRNAiscollectedfromtwodifferentcellsamplesfor
example,cellstreatedwithahormoneanduntreatedcellsofthesametypetoallowfora

directcomparisonofthespecificgenesexpressed
underbothconditions.ThemRNAsareconverted
tocDNAsthatarelabeledwitharedfluorescent
dyeforonesampleandagreenfluorescentdyefor
theother.Thelabeledsamplesaremixedandthen
allowedtohybridizetothemicroarray.Each
microscopicspotonthemicroarrayisa50
nucleotideDNAmoleculeofdefinedsequence
madebychemicalsynthesisandspottedonthe
array.TheDNAsequencerepresentedbyeachspot
isdifferent,andthehundredsofthousandsofsuch
spotsaredesignedtospanthesequenceofthe
genome.TheDNAsequenceofeachspotiskept
trackofbycomputer.Afterincubation,thearray
iswashedandthefluorescencescanned.Onlya
smallproportionofthemicroarray,representing
676genes,isshown.Redspotsindicatethatthe
geneinsample1isexpressedatahigherlevel
thanthecorrespondinggeneinsample2,andthe
greenspotsindicatetheopposite.Yellowspots
revealgenesthatareexpressedataboutequal
levelsinbothcellsamples.Theintensityofthe
fluorescenceprovidesanestimateofhowmuch
RNAispresentfromagene.Darkspotsindicate
littleornoexpressionofthegenewhoseprobeis
locatedatthatpositioninthearray.

< Chromatin immunoprecipitation. This

method allows the identification of all the
sites in a genome that a transcription
regulator occupies in vivo. The identities of
the precipitated, amplified DNA fragments are
determined by DNA sequencing.
In this approach, proteins are covalently
cross-linked to DNA in living cells, the cells are
broken open, and the DNA is mechanically
sheared into small fragments. Anti- bodies
directed against a given transcription
regulator are then used to purify the DNA that
became covalently cross-linked to that protein
in the cell. This DNA is then sequenced using
the rapid methods discussed earlier; the
precise location of each precipitated DNA
fragment along the genome is determined by
comparing its DNA sequence to that of the
whole genome sequence. In this way, all of

the sites occupied by

the transcription
regulator in the cell
sample can be
mapped across the
cells genome

Ribosomeprofiling>.RNAis
purifiedfromcellsanddigestedwithanRNAsetoleaveonlythoseportionsofthemRNAs
thatareprotectedbyaboundribosome.TheseshortpiecesofprotectedRNA(approximately
20nucleotidesinlength)areconvertedtoDNAandsequenced.Theresultinginformationis
displayedasthenumberofsequencereadsalongeachpositionofthegenome.Inthediagram

here,thedataforonlyonegene,whosemRNAisbeingefficientlytranslated,areshown.
RibosomeprofilingprovidesthistypeofinformationforeverymRNAproducedbythecell.