CHARACTERIZATION OF DNAC2 AND DNAC28 MUTANTS BY FLOW-CYTOMETRY

Escherichia coil strains containing thermosensitive dnaC alleles were studied by flow cytometry, Strains containing either the dnaC2 or dnaC 28 allele were shifted between different temperatures, and DNA content distributions were gathered, Inhibition of initiation of chromosome r eplication at nonpermissive temperature, as well as reinitiation of re plication at permissive temperature, were found to be affected by a nu mber of parameters, These included the choice of permissive and nonper missive temperatures, the length of the time of incubation at the nonp ermissive temperature, the growth medium, the type of temperature shif t used for reinitiation of replication (transient or nontransient), th e genetic background of the host cell, and the cell concentration, Rei nitiation of replication required neither transcription nor translatio n, whereas the elongation stage of replication was dependent upon ongo ing protein synthesis in the mutants. Efficient use of dnaC mutants fo r cell cycle studies is discussed.