Download Lecture 11 Gene1cs BIOL 335

Lecture11
Gene+cs
BIOL335
Pleasesignana*endancesheet
Classstartsat4pmsharp!
Homework,quizzes,etcreturnedinlabsec>on
Molecularmethods
Bacterialgene>cs
JacobandMonod-lacoperon
Quiz3
Iden>fica>onofpolymorphisms(alleles)by
moleculargenotyping
Wholegenomesequencing:notyetrou>ne
Systema>cpolymorphismdetec>on:rou>nefor
research(GoldenGate)
Examina>onofspecificgenomeregions
Rou>nerightnow
Theregionaroundaknownmuta>oncan
bePCRamplifiedandsequenceddirectly
Wild-type hemoglobin
Sickle-cell hemoglobin
Wild-type hemoglobin DNA
C T T
3ʹ
5ʹ
G A A
5ʹ
3ʹ
Mutant hemoglobin DNA
C A T
3ʹ
G T A
5ʹ
mRNA
5ʹ
5ʹ
3ʹ
mRNA
G A A
3ʹ
A
Normal hemoglobin
5ʹ
G U A
Sickle-cell hemoglobin
Val
Glu
het.AandT
3ʹ
Restric>onFragmentLengthPolymorphism
(RFLP)
Ifyouarelucky,amuta>onorpolymorphism
createsordestroysarestric6onsite
OUenmuchmucheasier,faster,andcheaperthan
sequencing
PCR-amplifiedfragmentsflankingavariable
restric>onsitecanbeusedforgenotyping
Normal β-globin allele
175 bp
DdeI
Normal
allele
Sickle-cell
allele
Large fragment
201 bp
DdeI
DdeI
DdeI
Large
fragment
Sickle-cell mutant β-globin allele
376 bp
DdeI
Large fragment
DdeI
Pro Glu Glu
CCT GAG GAG
DdeI
201 bp
175 bp
TheAàTmuta>on
CCT GTG GAG removesaDdeIsite
Pro Val Glu
376 bp
Old-schoolmethod–SouthernBlotRFLP
DdeI
RecombinantDNAandgenecloning
RecombinantDNA–piecesofDNAsynthe>cally
a*achedtoeachother
Genecloning–molecularisola>onofagene
Transgenic–expressionofaforeigngeneinan
organism
Wormexpressingalight-ac>vated
ionchannel(channelrhodopsin)
fromanalgaeinasingleneuron,
andafluorescentprotein(GFP)
fromajellyfishinintes>nalcells.
Restriction site
5ʹ
3ʹ
GAATTC
CTTAAG
DNA
3ʹ
5ʹ
1 Restriction enzyme
cuts sugar-phosphate
backbones.
Crea>nga
recombinant
DNAmolecule
withrestric>on
enzymes
5ʹ
3ʹ
5ʹ
G
CTTAA
3ʹ
AATTC
G
5ʹ Sticky 3ʹ
3ʹ
end
2 DNA fragment added
from another molecule
cut by same enzyme.
Base pairing occurs.
5ʹ
3ʹ 5ʹ
G AATT C
C TTAA G
3ʹ
5ʹ3ʹ
5ʹ
5ʹ
3ʹ
AATTC
G
G
CTTAA
3ʹ
5ʹ
3ʹ 5ʹ
G AATT C
C TTAA G
5ʹ 3ʹ
3ʹ
5ʹ
3 DNA ligase
seals strands
5ʹ
3ʹ
3ʹ
Recombinant DNA molecule
5ʹ
Plasmidvectors
•  Plasmids-circularDNAof1to200kbthatcontain
thegene>cmachinery(replica>onorigin)necessary
forautonomousreplica>oninbacteriaoryeast...
essen>allyanar>ficialchromosome
•  Containanan>bio>cresistancemarkerforselec>ng
cellsthatcontaintheplasmid
•  Op>onal,dependingontheplasmid:
Controlregions(promoter)forregula>ng
expressionofthetransgeneinatargetorganism
Plasmidvectors
Recombinant
DNA
Gene of
interest
1 Plasmid put into
bacterial cell - Transformation
Recombinant
bacterium
2 Host cell grown in culture to
form a clone of cells containing
the cloned gene of interest
Gene of
interest
Protein expressed from
gene of interest
Copies of gene
Do stuff
with the
gene
3 Basic research
and various
applications
Protein harvested
Do stuff with
recombinant
protein
StoringclonedgenesinDNAlibraries
Genomiclibrary-collec>onofrecombinantvector
clonesproducedbycloningDNAfragmentsfroman
en>regenome
ComplementaryDNA(cDNA)library–collec>onof
DNAmadeinvitrobyreversetranscrip>onofallthe
mRNAproducedbyapar>cularcell
-onlythesubsetofgenestranscribedintomRNA
Crea>ng GenomicDNA
digestedwith
genomic restric>onenzyme
Ligategenomic
DNAlibraries fragmentsinto
plasmidvectors
Bacterialgenome
Transforminto
Ecolibacteria
Plasmidcontaininggenomicinsert
amplifiedbybacteria
BacteriareplicateplasmidDNA
whentheydivide
Clone1
Clone2
ReverseTranscrip6on:
RNAàDNA
DNA in
nucleus
mRNAs in
cytoplasm
5ʹ
mRNA
Reverse
transcriptase
DNA
strand
A A A A A A 3ʹ
T T T T T 5ʹ
5ʹ
3ʹ
5ʹ
3ʹ
3ʹ
5ʹ
Primer
AAAAAA
TTTTT
3ʹ
5ʹ
3ʹ
DNA
polymerase
Poly-A tail
3ʹ
5ʹ
3ʹ
5ʹ
reversetranscriptaseenzymefrom
retrovirusesusedtomake
complementaryDNA(cDNA)
frommRNA
RNaseHofreversetranscriptase
removesRNAstrand
DNApolymerasegenerates
secondDNAstrand
cDNA
Screeninglibrariesbyhybridiza6on–oldschool
Aprobecanbesynthesizedthatiscomplementarytothe
geneofinterest
Forexample,ifthedesiredgeneis:
5ʹ
⋅⋅⋅ CTCAT CACCGGC⋅⋅⋅
3ʹ
Synthesizethisradiolabelledprobe:
3ʹ G A G T A G T G G C C G
5ʹ
TheDNAprobecanbeusedtoscreenalargenumberof
clonessimultaneouslyforthegeneofinterest
Onceiden>fied,theclonecarryingthegeneofinterestcan
beisolated
Screeninglibrariesbyhybridiza6on–oldschool
Radioactively
labeled probe
molecules
Gene of
interest
Probe
DNA
Multiwell plates
holding library
clones
Nylon membrane
5ʹ
3ʹ
⋅⋅⋅ CTCATCACCGGC⋅⋅⋅
GAGTAGTGGCCG
5ʹ
3ʹ
Singlestranded
DNA from
cell
Location of
DNA with the
complementary
sequence
Film
Nylon
membrane
Today
Easy:ifyourorganismhasasequencedgenomeand
organized,sequencedlibraries,simplyselectthe
cloneofinterest,digest,andcloneintovectorofyour
choice
Li*lebitharder:DesignprimerstoPCR-amplifythe
DNAofinterest,clonethePCRproductintothevector
ofyourchoice
PCR
GenomicDNA
Greenfluorescentprotein(GFP)fromjellyfish:
apowerfultoolformodernbiology
Usefulforlabelingspecificcellsorproteinsinanorganismto
examinewheretheygoandwhen
Canbeengineeredintogene>cally-encodedbiosensorsfor
detec>ngspa>otemporalregula>onofspecificcellular
molecules
Brainsliceofmousedesigned
toexpressadis>nctcolorin
eachneuron(Brainbow)
Bacteriaexpressingdis>nct
fluorescentproteinsusedto
paintaseascape
PCR
GFP
GFP
A.victoriacDNA
GFP
Gibson
Assembly
transformintoE.coli
GFP
transcrip>on
RNApolymerase
mRNA transla>on
ribosome
Gibsonassembly–restric>on-enzyme-less,
one-potDNAassembly
iden>cal
sequences
~25bp
chewsback,
crea>nglong
single-stranded
s>ckyends
Heatto50˚C1hr
Inac>vateexonuclease
Anneals>ckyends
Thermostable
polymerasefillsingaps
Thermostableligase
sealsthegaps
Bacteriacan
becultured
onsolidagar
plates
Avisiblebacterialcolonyisacloneofa
singlefoundercell
Dependingonwhatgenesithasandwhat
enzymesitproduces,apar>cularbacterialstrain
mayormaynotbeabletogrowonpar>cular
typesofmedia
•  Escherichiacoli–wild-typecangrowonminimal
mediacontainingoneofseveraltypesofsugarsasa
carbonsourceandinorganicsalts(prototroph)
•  Auxotrophs–mutantsthatcannotgrowon
minimalmediaunlessitissupplementedwith
par>cularcompounds
Example–BeadleandTatum'sNeurosporaarginine
auxotrophscannotgrowwithoutarginine
Unlikeeukaryotes,
bacteriahaveasingle
copyofacircular
chromosome
(a) Origin of replication in an E. coli cell
Origin of
replication
(
Parental (template) strand
Daughter (new)
strand
Doublestranded
DNA molecule
Replication
fork
Replication
bubble
Theyarehaploid,andonly
haveasinglealleleofeach
Two daughter
DNA molecules
gene
0.5 µm
Thesefactsareonly
mostlytrue
Discoveryofbacterialsex(conjuga6on)
Foundbya21yearoldJoshuaLederberg
workingwithEdwardTatum(1946)
StrainA=requiredmethionine(met)andbio>n(bio)
forgrowth(metandbioauxotroph)
StrainB=requiredthreonine(thr),leucine(leu),and
thiamine(thi)forgrowth(thr,leu,thiauxotroph)
Genotype/phenotypenota>on:
StrainA:met-bio-thr+leu+thi+
StrainB:met+bio+thr-leu-thi-
Discoveryofbacterialsex
(conjuga6on)
StrainA:
met bio-thr+leu+thi+
StrainB:
met+bio+thr-leu-thi-
mixture
Washandplate Washandplate Washandplate
108cells
108cells
108cells
minimalmedia
No
colonies
minimalmedia minimalmedia
No
Colonies!!! colonies
BernardDavisshowedthatthisisnotdueto
transforma?onbysomefactor(DNA);
thecellsneedtocontacteachother
Result–nocolonieson
minimalmedia
DNAistransferred
betweenbacteriaviapili
WilliamHayes(1953):bacterialgenetransferis
unidirec6onal
StrainA:met-bio-thr+leu+thi+
x
StrainB:met+bio+thr-leu-thi-
recombinantstrain=strainB+
thr,leu,andthifromstrainA
NOT
strainA+metandbiofromstrainB
strainA=donorstrain;
strainBisrecipientstrain
WilliamHayes(1953):abilitytodonategenes
canbetransmi*ed
donorStrainA:met-bio-thr+leu+thi+
x
recipientStrainB:met+bio+thr-leu-thi-
recombinantstrain=strainB+
thr,leu,andthifromstrainA
AND
newstrainnowcandonatetootherstrains
Saidthedonorstrainshaveatransmi*ablegene
calledthefer6lity(F)factor
TransferofF+plasmidfromdonortorecipient
bacterial
chromosome
Fplasmid
F+donorcell
F-recipientcell
Plasmid–smallcircularDNAseparatefromthemain
bacterialchromosome(doesnotneedtohaveFfactor)
TransferofF+plasmidfromdonortorecipient
F+cellextendspilustoF-cell
F-plasmidisnicked,andasingle
strandispassedthroughthepilus
totherecipientcell
TransferofF+plasmidfromdonortorecipient
strandsarereplicated
inbothcell
F-plasmidtransfercomplete
F-cellisnowF+!!!
Highfrequencyrecombina>on(Hfr)strains
DiscoveredbyLucaCavalli-Sforza(1953)
1.1000-foldmorerecombinantsthanusualF+strains
(hencethename....)
2.Therecipientcellsareonlyrarelyconverted
todonors
Highfrequencyrecombina>on(Hfr)strains
HfrstrainshavetheF-factor
integratedontotheir
mainchromosome
Conjuga>onofHfrstrainsissimilartoF+strains,
viaapilus
F-plasmid
integra>on
Hfrcell
HfrcellextendspilustoF-cell
Conjuga>onofHfrstrainsissimilartoF+strains,
viaapilus
F-containingchromosomeis
nickedattheFlocus,andasingle
strandispassedthroughthepilus
totherecipientcell
DNAistransferredun>lcomplete,
orun>lthepilusfallsapart.
Strandsarereplicatedinbothcells
Thetransferredfragment(exogenote)must
recombineintotherecipientchromosome)
X
exogenote
double-
recombina+on
endogenote
exogenoteislost
recombined
into
chromosome
ElieWollmanandFrancoisJacob(1957)–use
Hfrcrossestomakemapsofbacteria
chromosomes
HfraziRgal+tonRstrS
x
Resistanttoaziandtondrugs,
cangrowongalactose,
killed(sensi>ve)bystreptomycin
F-aziSgal-tonSstrR
killedbyaziandtondrugs,
cannotgrowongalactose,
resistanttostreptomycin
letcrossfor2hrs
getcellsthatareresistanttoallthreedrugs,can
growongalactose,andcanactasHfrdonors
ElieWollmanandFrancoisJacob(1957)–use
Hfrcrossestomakemapsofbacteria
chromosomes
Puongamixtureofconjuga>ngbacteriathrougha
blenderbreaksthepili,stoppingDNAtransfer
Calledthe"coitusinterruptus"assayaUerthe
Va>can-approvedbirthcontrolmethod
ElieWollmanandFrancoisJacob–useHfr
crossestomakemapsofbacteriachromosomes
HfraziRgal+tonRstrS
x
F-aziSgal-tonSstrR
Systema>callyinterruptconjuga>onatvarious>mes
andassayforrecombinants.
PlateonstreptomycintokillHfrdonorcells
ElieWollmanandFrancoisJacob–useHfr
crossestomakemapsofbacteriachromosomes
HfraziRgal+tonRstrS
x
F-aziSgal-tonSstrR
ElieWollmanandFrancoisJacob–useHfr
crossestomakemapsofbacteriachromosomes
HfraziRgal+tonRstrS
azi
5min 10min
x
F-aziSgal-tonSstrR
gal
ton
F
12min
25min
2hrs
Blendt=5min–norecombinantsseen
Blendt=10min–Findaziresistantcells,butnotothers
Blendt=12min–Findaziandtonresistantcells
Blendt=25min–azi,tonresistanceandgalactose
growthseen
Blendt=2hrs–Seestr-resistant(recipient),azi,ton
resistant,galactosegrowingcellsthatcanbeFdonors
WollmanandJacobusedthiswithotherstrains
O=origin,F=fer>lity
Eachstrainhadadifferentorderofgenes!?!?
Intwostrains,thehisgeneistransferredbeforetheglygene(hisis
closertoO),butinthreestrains,theglygeneistransferredbeforethe
hisgene.
AllanCampbellhypothesizedthatbacteriahave
circularchromosomes,andthatthedifferentHfr
strainshaveF
integratedatdis>nct
loca>ons
Aswitheukaryotes,recombina>on-frequencybased
mappingispossiblewithbacteria
exogenote
fromHfr
strain
Amajorcaveatisthatrecombina>onfreqscanbemeasuredonlyfor
genesontheexogenotefragmentthathasbeentransferredover
Aswitheukaryotes,recombina>on-frequencybased
mappingispossiblewithbacteria
Onlyrecombinantswith
evennumbersof
cross-overscanbe
observed;single
recombinantsarenot
resolved
singlecrossover
doublecrossover
Aswitheukaryotes,recombina>on-frequencybased
mappingispossiblewithbacteria
Result
leu+arg-met-
leu+arg+met-
leu+arg+met+
leu+arg-met+
Gene>cmapofEcoli
InsteadofcM,units
are"minutes"–
conjuga>ontransfer>me
Some>mestheF-factorcanpopoutofthe
chromosome
F-plasmid
excision
Hfrcell
F+cell
Some>mestheF-factorcanpopoutofthe
chromosomeandtakesomeDNAalong–messy
orimpreciseexcision
Messyexcision;
Result:F'plasmidthat
hasadonatablelacgene,
alacepisome
Diploidjustforthelacgene
F'episomesareveryveryusefulforbacterial
gene>cs
-Canbeusedtotransferallelesfromonestrainto
another
-Canbeusedtoseewhetheragivenalleleis
dominantorrecessive
ThesetoolswereusedbyFrancoisJacoband
JacquesMonodtodiscoverhowbacterialgenes
areregulated
"WhatistrueforE.coliistruefortheelephant”
-JacquesMonod
Ecoliproducestheenzymeβ-galactosidase
(beta-gal)tometabolizelactosesugar
Ecolialsomakesatransporterprotein,lacpermease,
totransportthesugarfromoutsidetothecytoplasm
Ecoliproducestheenzymeβ-galactosidase
(beta-gal)tometabolizelactosesugarONLY
whenlactoseisaround
-savesmaterialandenergy....wastefultomakean
enzymeifitssubstrateisnotaround
-ifglucoseANDlactoseareavailable,Ecolimakes
theenzymeonlyaUeralltheglucose,itspreferred
foodisusedup
Non-ediblelactoseanalogscalledinducerswere
knowntotrickEcolitoincreasebetagallevels
1000-fold
isopropyl-b-D-thiogalactoside
(IPTG)
Usingthebacterialgene>csbeingdeveloped,
JacobandMonodiden>fiedandmappeda
numberofgenesandmutantswithabnormal
betagalregula>on
structuralgenes
lacI
lacZ
lacY
lacA
beta-gal
permease
transacetylase
"theinducer"
lacI-cellsproducethe
enzymesallthe+me
(cons6tu6vely)
theseenzymesare
co-regulated,so
lacZexpressionis
agoodproxy
lacIwastheverymutanteverfoundtohaveadefect
notintheac>vityofanenzyme,butinthecontrolof
enzymeproduc>on.
structuralgenes
lacI
lacO
lacZ
lacY
lacA
"theoperator"
Theyalsodiscoveredasetofmuta>onsinagene,lacO
thatmappedveryclosetolacZ
Muta>onsonlacOwerealsocons>tu>veorfailed
toproduceanyenzyme,dependingontheallele
Next>me
HowtheJacobandMonodworkedouthow
thelacoperonworks
Otherexamplesofgeneregula>oninbacteria
Thingsonecandowithregulatedbacterial
genes
Now:Quiz