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- W2187994773 abstract "Transfer ofan allele fromadonorDNAtoarecipient DNAmolecule was selected bytheloss ofadominant conditional lethal mutation previously incorporated itothegeneofinterest intherecipient DNA.Boththe Escherichia coli chromosome andplasmids carrying E.coli geneswereusedsuccessfully asdonormolecules. Recipient molecules forthese exchanges were constructed invitro byusing therpsLgene,whichconfers sensitivity tostreptomycin, toreplace segments ofspecific E.coli geneslocated either onmulticopy plasmids or intheE.coli chromosome. Plasmids carrying suchreplacements werecapable ofacquiring chromosomal alleles ofthegene(s) ofinterest, andstrains carrying rpsLreplacements inthechromosome werecapable ofacquiring plasmid-encoded alleles atthesight oftherpsLreplacement. Inbothsituations, these allele transfers resulted inloss oftherpsLgenefromtherecipient DNA molecule. Thedesired transfer eventsconstituted a large percentage ofthese events, which gaverise toviable colonies whenappropriate donor-recipient pairs were subjected tostreptomycin selection. Thus,this isa useful approach fortransferring alleles ofinterest from plasmids totheE.coli chromosome andvice versa. Innumerous bacterial systems, including thechemotaxis systemofEscherichia coli, cloning a geneofinterest into a multicopy plasmid canbeamajor steptoward understanding thefunction ofthis gene or theassociated gene product because itplaces thegene ina formthat can bereadily isolated, characterized, andselectively mutagenized. However,whenone assessestheability ofwild-type andmutant versions ofthegenetocomplement chromosomal lesions of thegene,complications may arisefromexpression ofthe cloned geneatinappropriate levels because of(i) theloss of cis-acting regulatory elements or (ii) theincreased copy numberoftheplasmid-borne gene.Forthis reason,itis sometimes desirable todetermine theeffects ofmutations generated invitro (with theplasmid-borne gene) whenthey are presentinsingle copy (inthebacterial chromosome) rather thaninmultiple copies. Inother instances, interesting chromosomal alleles may already exist, andcloning each allele wouldbeatedious expenditure oftimeandresources. Inbothsituations, theinvestigator wouldbenefit froma methodofselecting forallele transfer, either fromthe chromosome toa plasmid carrying a copy ofthegene of interest orfromsucha plasmid tothechromosome. Inthis communication, we describe howselection forlossofa dominant antibiotic sensitivity marker canbeusedtoisolate recombinant plasmidsresulting from chromosometo-plasmid allele transfer andtocreatestrains produced by plasmid-to-chromosome allele transfer. We demonstrate the utility ofthisapproach intransferring alleles ofseveral chemotaxis genesinE.coli. Ourapproach provides some advantages overpreviously reported techniques fortransferring chromosomal alleles of cloned genesontoplasmids. Theseother techniques involve an intermediate stepinwhichtheplasmid istemporarily integrated into a segmentofthechromosomal DNA (4,7, 13). Whentheplasmid issubsequently recovered fromthe chromosome, theallele istransferred totheplasmid ifthe locations oftheexcision eventsareappropriately oriented withrespect tothelocation oftheinsertion event.Ifthe" @default.
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- W2187994773 date "1989-01-01" @default.
- W2187994773 modified "2023-09-27" @default.
- W2187994773 title "Exchange ofChromosomal andPlasmid Alleles inEscherichia coli bySelection forLossofa Dominant Antibiotic Sensitivity Marker" @default.
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