Cosmid Vectors, YAC and BAC Expression Vectors

Info regarding YAC, BAC and Cosmid vectors provided

1. COSMID VECTORS, YAC’S AND
BAC’S EXPRESSION VECTORS
By: Chartha Gaglani

2. COSMID VECTORS

3. INTRODUCTION
•Cosmidvectorsarehybridvectorsderivedfromplasmidswhich
containcossiteofphagelambda.
•Vector-Inmolecularcloning,avectorisaDNAmoleculeusedasa
vehicletoartificiallycarryforeigngeneticmaterialintoanothercell,
whereitcanbereplicatedand/orexpressed.
•Cossite-"Cos"istheabbreviationof"cohesiveendsite".Thisisa
specialityofthelambdaphagewhichhastobelinearizedtofitintothe
phageshead,butcircularizesinthehostcell.
•Cosmid=cossite+plasmid.
•Theyareabout45kb.

4. structure
•Cosmidsareessentiallyplasmidsthatcontainaminimumof250bpof˄DNA,
whichincludesthefollowingsequencesfrom˄genome:
1.Thecossite(thesequenceyieldingcohesiveends)
2.Sequencesneededforbindingofandcleavagebyterminasesothatunder
appropriateconditionstheyarepackagedinvitrointoempty˄phageparticles.
•Atypicalcosmidhas(1)Replicationorigin(2)Uniquerestrictionsites(3)A
selectablemarkersfromaplasmid.

5. •Thecosmidvectorsareopenedbytheappropriaterestrictionenzymeataunique
site,arethenmixedwithDNAinsertspreparedbyusingthesameenzymeand
annealed.
•Amongtheseveraltypesofproducts,longcancatemersarepresent,whicharethe
appropriateprecursorsofpackagingin˄particles.
•ThisprocedureselectsthelongDNAinsertssinceforpackagingthedistance
betweentwocossitesmustbebetween38and52kb.
•TheDNAfragmentsusedforcloningareusuallyproducedbypartialdigestion
witharestrictionenzyme.
•Thisisbecausecompletedigestionalmostalwaysproducesfragmentsthataretoo
smallforcloninginacosmid.
•Cosmidscanaccommodateupto40kblongDNAinserts.

6. •Thetypicalfeaturesofcosmidsareasfollows:
1.TheycanbeusedtocloneDNAinsertsofupto40kb.
2.Theycanbepackagedinto˄particles,whichinfecthostcells;thisismany-fold
moreefficientthanplasmidtransformation.
3.SelectionforrecombinantDNAisbasedontheprocedureapplicabletothe
plasmidmakingupthecosmid.
4.Finally,thesevectorsareamplifiedandmaintainedinthesamemannerasthe
contributingplasmid.

7. DRAWBACKS OF COSMID
•Cosmidsareparticularlyattractiveforconstructionofgenomic
librariesofeukaryotessincetheycambeusedforcloninglarge
fragments.
•ThisapproachisverysensitivetotheexactratioofDNAinsert-to-
vectorDNAbecausevector-to-vectorligationcanoccur.
•Recombinationcosmidshavingduplicatedvectorsequencesare
unstableduringcloning.

8. YEAST ARTIFICIAL
CHROMOSOME (YAC) VECTORS

9. YEAST ARTIFICIAL CHROMOSOME
•Yeastartificialchromosomearegeneticallyengineeredchromosomederivedfrom
DNAoftheyeast.
•Itishuman-engineeredDNAmoleculeusedtocloneDNAsequencesinyeast
cells.
•TheyaretheproductsofarecombinantDNAcloningmethodologytoisolateand
propagateverylargesegmentsofDNAinayeasthost.
•TheamountofDNAthatcanbeclonedintoYACisonaveragefrom200to500
kb.
•Howeverasmuchas1MBcanbeclonedintoaYAC.

10. STRUCTURE OF YAC
•TheYACcloningvectorsconsistoftwocopiesofayeasttelomericsequence,a
yeastcentromere,ayeastars(anautonomouslyreplicatingsequencewhereDNA
replicationbegins)andappropriateselectablemarkers.

11. WORKING PRINCIPLE
•Theprincipleissimilartothatchromosometothatforplasmidsorcosmids.
•Theexperimenterintroducessometypicalelementsthatarenecessaryforcorrect
replication.
•InthecaseofYAC’s,thereplicationoriginsarethecentromeresandtelomeresof
yeastchromosomes,whichmustbeinsertedintotheDNAbeingcloned.

12. ADVANTAGES OF YAC
•YACprovidethelargestinsertcapacityofanycloningsystem.
•Yeastexpressionvectors,suchasYAC’s,YIPs(YeastIntegratingPlasmids),and
YEP’s(YeastEpisomalPlasmids),haveadvantageousoverBAC’S.Theycanbe
usedtoexpresseukaryoticproteinsthatrequirepost-translationalmodification.
•Amajoradvantageofcloninginyeast,aeukaryote,isthatmanysequencesthat
areunable,underrepresentedorabsentwhenclonedintoprokaryoticsystems,
remainstableandintactinYACclones.

13. LIMITATIONS OF YAC
•YACclonesfrequentlycontaindeletions,rearrangementsornoncontiguouspieces
ofclonedDNA.Asaresult,eachYACclonemustbecarefullyanalyzedtobesure
thatnorearrangementsoftheDNAhaveoccurred.
•Theefficiencyofcloningislow.
•YAChavebeenfoundtobelessstablethanBAC.

14. BACTERIAL ARTIFICIAL CHROMOSOME
(BAC) VECTORS

15. BAC
•ABacterialArtificialChromosomeisaDNAconstruct,basedonafunctional
fertilityplasmid,usingfortransformingandcloninginbacteria,usuallyE-coli.
•Theyarecapableofcarryingapproximatelyupto300kbpofinsertedDNA
sequence.
•BACwascreatedbecauseoftheproblemsfacedwithYACe.g.recombination
betweencopiesofinsertsandmoreparticularly,deletionsintheDNAinserts.
•ThefirstBACvectorwaspBAC108L.

16. COMMON GENE COMPONENTS IN BAC
•RepE-forplasmidreplicationandregulationofcopynumber.
•ParAandParB-forpartitioningFplasmidDNAtodaughtercellsduringdivision
andensuresstablemaintenanceofBAC.
•Selectablemarker-forantibioticresistance;someBAC’salsohavelacZatthe
cloningsiteforblue/whiteselection.
•T7&Sp6-phagepromotorsfortranscriptionofinsertedgenes.
•OrlS-originofreplication.

17. APPLICATION OF BAC
•BAC’sarebeinggreatlyusedinmodellinggeneticdiseasesinordertostudytheir
effectsintheexperimentationontransgenicmice.
•BAChavebeenusedtostudytheneurologicaldiseasessuchasAlzheimer’s
diseaseorincaseofDown’ssyndrome.
•ThegenomeofseverallargeDNAvirusesandRNAviruseshavebeenclonedby
BAC’s.Theseconstructsarereferredtoas‘infectiousclones’.

18. DRAWBACKS
•ThechiefdisadvantageofBACvectorsisthesomewhatlaboriousconstructionof
BAClibrariesasinvitromanipulations,suchas,restrictiondigestion,etc.haveto
beperformedinagaroseplugstoavoidshearingofthelargeDNAmolecules.

19. REFERENCES
•BiotechnologyExpandingHorizonsbyB.D.Singh
•www.microbenotes.com