Gene transfer mechanisms

Transformation and Conjugation

1. GENE TRANSFER MECHANISMS:
TRANSFORMATION
GRIFFITH’S EXPERIMENT
Dr. M. Sonia Angeline
Assistant Professor
KJC

2. GENE TRANSFER MECHANISMS
•Prokaryotesreproduceasexuallybybinaryfission;theycanalsoexchangegeneticmaterialby
transformation,transduction,andconjugation.
•Binaryfissionisatypeofreproductioninwhichthechromosomeisreplicatedandthe
resultantprokaryoteisanexactcopyoftheparentalprokaryate,thusleavingnoopportunity
forgeneticdiversity.
•TransformationisatypeofprokaryoticreproductioninwhichaprokaryotecantakeupDNA
foundwithintheenvironmentthathasoriginatedfromotherprokaryotes.
•Transductionisatypeofprokaryoticreproductioninwhichaprokaryoteisinfectedbyavirus
whichinjectsshortpiecesofchromosomalDNAfromonebacteriumtoanother.
•ConjugationisatypeofprokaryoticreproductioninwhichDNAistransferredbetween
prokaryotesbymeansofapilus.

4. TRANSFORMATION
•Transformationisaprocessbywhichgeneticinformationistransferredfromonebacterium
toanother.
•ThisphenomenonwasfirstdiscoveredbyFrederickGriffithin1928intheJournalof
Hygiene.
•In1944reportingintheJournalofExperimentalMedicine,OswaldAvery,ColinMacLeod,
andMaclynMcCartyshowedthatDNAwasthemoleculeresponsiblefortransformation.
•Thesetwoexperimentswerepivotalexperimentsshowingthatamaterialsubstancefrom
oneorganismcantransformtheinheritancepatterninanotherorganism(Griffith,1944.)
•Additionally,DNAisamoleculethatstoresandtransmitstheinformationresponsiblefor
transformingbacteria(Avery,MacLeod,andMcCarty,1944.)
•ThesepivotalexperimentsdemonstratedthatthematerialsubstanceDNAisresponsiblefor
storingandtransmittinginheritance.

5. Intransformation,theprokaryotetakesinDNAfoundinitsenvironmentthatisshedby
otherprokaryotes.
IfanonpathogenicbacteriumtakesupDNAforatoxingenefromapathogenand
incorporatesthenewDNAintoitsownchromosome,it,too,maybecomepathogenic.
Transformationisaprocessbywhichforeigngeneticmaterialistakenupbyacell.
Theprocessresultsinastablegeneticchangewithinthetransformedcell.
TRANSFORMATION

6. GRIFFITH EXPERIMENT & TRANSFORMING
PRINCIPLE
•Griffithexperimentwasasteppingstoneforthediscoveryofgeneticmaterial.
•FrederickGriffithexperimentswereconductedwithStreptococcuspneumoniae.
•Duringtheexperiment,GriffithculturedStreptococcuspneumoniaebacteriawhich
showedtwopatternsofgrowth.
•Griffithwasn'ttryingtoidentifythegeneticmaterial,butrather,tryingtodevelopa
vaccineagainstpneumonia.
•Inhisexperiments,Griffithusedtworelatedstrainsofbacteria,knownasRandS.
•Onecultureplateconsistedofsmoothshinycolonies(S)whileotherconsistedofrough
colonies(R).
•ThedifferencewasduetothepresenceofmucouscoatinSstrainbacteria,whereas
theRstrainbacterialackedthem.

7. GRIFFITH EXPERIMENT
R strain:
•When grown in a petri dish, the R bacteria formed colonies, or clumps of related
bacteria, that had well-defined edges and a rough appearance (hence the abbreviation
"R").
•The R bacteria were nonvirulent, meaning that they did not cause sickness when
injected into a mouse.
S strain:
•S bacteria formed colonies that were rounded and smooth (hence the abbreviation
"S").
•The smooth appearance was due to a polysaccharide, or sugar-based, coat produced
by the bacteria.
•This coat protected the S bacteria from the mouse immune system, making them
virulent (capable of causing disease).
•Mice injected with live S bacteria developed pneumonia and died.

8. GRIFFITH EXPERIMENT
Griffith's experiment involved the use of two strains of pneumococcus –a deadly virulent
strain (S) or a non-virulent strain (R)
·When Griffith infected mice with the non-virulent bacteria (strain R), the mice survived
·When Griffith infected mice with the virulent bacteria (strain S), the mice died
·When Griffith infected mice with heat-killed virulent bacteria (strain S), the mice survived
as the bacteria had been killed
·When Griffith infected mice with a mix of heat-killed strain S and living strain R, the mice
were found to have died
From this Griffith’s concluded that the living R cells had somehow been transformed into
virulent S cells.
This indicated that there was some form of transferrable genetic material present within the
cells (i.e. DNA).

11. GRIFFITH EXPERIMENT
•Basedontheobservation,GriffithconcludedthatRstrainbacteriahadbeen
transformedbySstrainbacteria.
•TheRstraininheritedsome‘transformingprinciple’fromtheheat-killedSstrain
bacteriawhichmadethemvirulent.
•Andheassumedthistransformingprincipleasgeneticmaterial.

12. DNA AS GENETIC MATERIAL
Griffithexperimentwasaturningpointtowardsthediscoveryofhereditarymaterial.However,it
failedtoexplainthebiochemistryofgeneticmaterial.
Hence,agroupofscientists,OswaldAvery,ColinMacLeodandMaclynMcCartycontinuedthe
Griffithexperimentinsearchofbiochemicalnatureofthehereditarymaterial.
TheirdiscoveryrevisedtheconceptofproteinasgeneticmaterialtoDNAasgeneticmaterial.
Averyandhisteamextractedandpurifiedproteins,DNA,RNAandotherbiomoleculesfromthe
heat-killedSstrainbacteria.
TheydiscoveredthatDNAisthegeneticmaterialanditisaloneresponsibleforthe
transformationoftheRstrainbacteria.
Theyobservedthatprotein-digestingenzymes(proteases)andRNA-digestingenzymes(RNases)
didn’tinhibittransformationbutDNasedid.
Althoughitwasnotacceptedbyall,theyconcludedDNAasgeneticmaterial.

13. CONJUGATION
GeneticrecombinationinwhichthereisatransferofDNAfromalivingdonorbacteriumtoa
livingrecipientbacteriumbycell-to-cellcontact.
InGram-negativebacteriaittypicallyinvolvesaconjugationorsexpilus.
Conjugationisencodedbyplasmidsortransposons.
Itinvolvesadonorbacteriumthatcontainsaconjugativeplasmidandarecipientcellthatdoes
not.
Aconjugativeplasmidisself-transmissible,inthatitpossessesallthenecessarygenesforthat
plasmidtotransmititselftoanotherbacteriumbyconjugation.

14. CONJUGATION
Conjugationistheprocessbywhichonebacteriumtransfersgeneticmaterialtoanotherthrough
directcontact.
Duringconjugation,onebacteriumservesasthedonorofthegeneticmaterial,andtheother
servesastherecipient.
ThedonorbacteriumcarriesaDNAsequencecalledthefertilityfactor,orF-factor.
TheF-factorallowsthedonortoproduceathin,tubelikestructurecalledapilus,whichthedonor
usestocontacttherecipient.
Thepilusthendrawsthetwobacteriatogether,atwhichtimethedonorbacteriumtransfers
geneticmaterialtotherecipientbacterium.

15. CONJUGATION
Typically,thegeneticmaterialisintheformofaplasmid,orasmall,circularpieceofDNA.
Thegeneticmaterialtransferredduringconjugationoftenprovidestherecipientbacteriumwith
somesortofgeneticadvantage.
Forinstance,inmanycases,conjugationservestotransferplasmidsthatcarryantibiotic
resistancegenes.
Conjugationgenesknownastragenesenablethebacteriumtoformamatingpairwithanother
organism,whileoriT(originoftransfer)sequencesdeterminewhereontheplasmidDNAtransfer
isinitiatedbyservingasthereplicationstartsitewhereDNAreplicationenzymeswillnickthe
DNAtoinitiateDNAreplicationandtransfer.

17. CONJUGATION -F+ X F-
•Theprocessofbacterialconjugationisbasedontheprinciplethattheplasmidoranyother
geneticmaterialistransferredfromthedonorcelltotherecipientcellthroughclosephysical
contact.
•Ofalltheconjugativeplasmids,theF(fertility)plasmidofE.coliwasthefirstdiscoveredandis
oneofthebest-studied.
•TheFplasmidispresentinoneortwocopiespercellandisverylarge(about100kilobases).
•E.coliharboringtheFplasmidarereferredtoasdonor(F
+
ormale)cellsandE.colilacking
theFplasmidarereferredtoasrecipient(F
–
orfemale)cells.
•OnlydonorcellsarecapableoftransferringtheFplasmidtorecipientcells.

18. CONJUGATION
•FortransferoftheFplasmidfromdonortorecipient,intimatecontactbetweencells,resultingin
mating-pairformation,isrequired.
•DNAcodesfortheproteinsthatmakeupthesexpilus.ItalsocontainsaspecialsitewhereDNA
transferduringconjugationbegins
•Thetransferofgeneticmaterialisthenbroughtbymembranefusionofthetwocellsbytheactionof
differentenzymes.
•Followingthemembranefusion,thereplicationofdonorDNAoccursandistransferredintothe
recipientcell.
•IftheFfactoristransferredduringconjugation,thereceivingcellturnsintoanF^++start
superscript,plus,endsuperscriptdonorthatcanmakeitsownpilusandtransferDNAtoothercells.

20. HIGH FREQUENCY RECOMBINATION ( HFR)
CELL CONJUGATION
•WhenF-plasmid(sexfactor)integratedwithchromosomalDNAthensuchbacteriaisknownashigh
frequencyrecombination(Hfr)bacteria.
•Inthecross(conjugation)betweenHfrcellandF-cell,frequencyofrecombinationisveryhighbut
frequencyoftransferofwholeF-factorisverylow.
•HfrcellactsasdonorwhileF-cellactsasrecipient.

21. HFR CONJUGATION
•AtfirstF-factormakessexpilusthatjoinsdonorandrecipientcellthenF-factoropensas
replicationoriginthenonestrandiscutdown.
•Nowthe5’endofthisstrandentersintorecipientcellthroughconjugationtube.
•Since,replicationoriginliessomewhereinthemiddleofF-factor,portionofF-factorthatliesat
5’endentersfirstintorecipientcellbuttheportionsituatedat3’endentersonlywhenwhole
chromosomalDNAentersintotherecipientcell.
•TotransferwholechromosomalDNA,ittakes100minutesinE.coli.Inmostofthecases,sex
pilus(conjugationtube)breaksbeforetransferofwholechromosomalDNAtakesplace.So,
frequencyoftransferofwholeF-factorisverylow.
•AfterthecrossbetweenHfrcellandF-cell,recipientcellremainsrecipient.
•Inthisconjugation,chromosomalDNAisalwaysalmosttransferfromdonortorecipientcell
togetherwithportionofF-factor.So,frequencyofrecombinationishigh.

23. F –PRIME (F’) CELL
•BacteriainwhichcontainsF-factorandapartofchromosomalDNAintegratedinitisknownas
F-primebacteria.
•F’cellsareformedfromHfrcellduringinductionofF-factorfromchromosomalDNAinwhichF-
factorcarriesaportionofchromosomalDNAalongwithit.
•Inthecross(conjugation)betweenF-prime(F’)cellandF-cell,frequencyofrecombinationishigh
aswellasfrequencyoftransferofwholeF-factorisalsohigh.
•IftheFplasmidinaccuratelyexcisesfromthechromosomeafterformationofanHfr,itcantakea
portionofthechromosome withit,whichthenbecomespartoftheplasmiditself.Thisformof
theFplasmidiscalledanF'(Fprime).
•F’Cells=DerivativesofHfrcellswhereFplasmidhasdisintegratedfromhostchromosomeand
picksupsomehostgenesnexttoFplasmidintegrationsites