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IIIegitimate integration of non‐replicative vectors in the genome of Rhodococcus fascians upon electro‐transformation as an insertional mutagenesis system
Author(s) -
Desomer J.,
Crespi M.,
Montagu M.
Publication year - 1991
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.1991.tb02141.x
Subject(s) - replicon , plasmid , biology , genetics , insertional mutagenesis , transformation (genetics) , genome , gene , t dna binary system , recombination , homology (biology) , bacterial conjugation , site specific recombination , vector (molecular biology) , recombinant dna , recombinase
Summary Electrotransformation of Rhodococcus fascians by non‐replicating plasmids containing a suitable resistance marker resulted in stable transformants by integration of these constructs at various sites in the genome, thereby generating different mutations. Tagged genes could be isolated in Escherichia coli owing to the presence of a CoIE1 replicon and an ampicillin resistance gene in the inserted sequences. Southern analysis and nucleotide sequencing revealed that recombination can occur at defined locations in the plasmid, while no site preference for target sequences could be detected. Low homology between the recombining sequences indicates illegitimate recombination. The specificity of the plasmid sites could be explained by assuming a linear recombination intermediate, generated by cleavage of the transformed plasmid.