[Isolation and properties of recombinant DNA from a plasmid and filamentous phage].
In the course of studying extrachromosomal DNA with composite replicons, a hybrid has been constructed by the in vitro recombination of the filamentous phage M13mp2 DNA (RF) and plasmid pUR222 (ApR). Both parental DNAs contain a fragment of lac-operon (ca. 800 bp), which includes the distal end of lacI gene, lacPO segments, and the lacZ gene proximal region coding for 145 N-terminal amino acid residues of beta-galactosidase and thus providing for alpha-complementation, the effect being cancelled with a polynucleotide insertion at the unique EcoRI site in the lacZ gene segment. E. coli BMH71-18 cells were transformed with the ligated mixture of EcoRI restricts of both DNAs. A phage-like nucleoprotein was isolated from colourless plaques (on the Xgal- and IPTG-supplemented medium); its deproteinization yielded a DNA which contains the ApR-determinant and, according to PAGE, structurally specific staining, restriction analysis, sequencing by the Sanger procedure, and electron microscopy data, is a linear double-stranded molecule comprising the phage and plasmid genomes in an equimolar ratio. Since the hybrid DNA does not display the alpha-complementation effect, both bacterial inserts are in the opposite orientation. Transformation of both phage (F+) and plasmid (F-) hosts with the hybrid DNA led to cultures which, after precipitation of the nucleoprotein from the extracellular medium and deproteinization, afforded the same composite DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
D002874:Chromosome Mapping; D003090:Coliphages / Q000032:analysis* / Q000235:genetics; D004262:DNA Restriction Enzymes; D004270:DNA, Circular / Q000032:analysis / Q000235:genetics; D004274:DNA, Recombinant / Q000032:analysis / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D009693:Nucleic Acid Hybridization; D010957:Plasmids
D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral; D004262:DNA Restriction Enzymes
Poluchenie i svoĭstva rekombinantnoĭ DNK na osnove plazmidy i nitchatogo bakteriofaga.