[Physical mapping of Escherichia coli genome rearrangements using pulsed-field gel electrophoresis and the PCR method].
Physical mapping of Tn10-induced chromosomal rearrangements in Escherichia coli was carried out by means of pulse-field electrophoresis, Southern hybridization, and PCR analysis. Mutants with chromosomal rearrangements were obtained by a method of selection of strains with constitutive udp-gene expression elaborated earlier. Screenings were performed in two E. coli strains that had a udp gene orientation diametrical to oriC and carried in metE :: Tn10- insertion and mutation in the dam gene encoding DNA-methylase. Selection of mutants with constitutive uridine phosphorylase synthesis was aimed at placing the udp-gene under the control of a novel, strong promoter via genome rearrangements. Physical mapping indicated that chromosomal rearrangements, in all cases studied, involved regions of operons encoding ribosomal RNA synthesis and placed the udp gene under the control of an rrn promoter. The majority of rearrangements are inversions of the chromosomal segment between the IS10-element, located in the metE gene, and various sites in different rrn operons. It is shown that the involvement of a particular operon in inversion depends on the direction of rrn operon and udp gene transcription. The rearrangement frequency increases 8-10 times in the presence of dam mutation.
Zaporozhets
D A
DA
Mironov
A S
AS
D001483:Base Sequence; D015139:Blotting, Southern; D002874:Chromosome Mapping / Q000379:methods*; D016521:Electrophoresis, Gel, Pulsed-Field; D004926:Escherichia coli / Q000235:genetics*; D015321:Gene Rearrangement; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction
Fizicheskoe kartirovanie genomykh perestroek Escherichia coli s pomoshch'iu pul's-élektroforeza i PTsR-metod.