Replicon rescue: a novel strategy to clone the genomic DNA flanking insertions of integrating shuttle vector DNA.
A novel cloning strategy, replicon rescue, was developed for cloning genes disrupted by plasmid insertions. After ligation to a tetracycline resistance cassette, fragments containing a bacterial origin of replication from the insertion are recovered in Escherichia coli because they replicate autonomously. Restriction enzymes for cloning are so chosen that the only legitimate two fragment ligation yielding TetR clones involves a fragment spanning the boundary of the insertion. Replicon rescue was used successfully firstly in a test system to clone the chromosomal orl from a Klebsiella aerogenes strain, and secondly to recover a disrupted gene from a phototaxis-deficient mutant of Dictyostelium.
McMahon
T L
TL
School of Microbiology, La Trobe University, Melbourne, Victoria, Australia.
Wilczynska
Z
Z
Barth
C
C
Fraser
D J
DJ
Pontes
L
L
Fisher
P R
PR
D000818:Animals; D003001:Cloning, Molecular / Q000379:methods*; D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism; D004023:Dictyostelium / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000235:genetics; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D018741:Replication Origin / Q000235:genetics; D012093:Replicon / Q000235:genetics*
D004262:DNA Restriction Enzymes