Uptake and incorporation of 5-bromouracil by cultures of Escherichia coli



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The behavior of the thymine requiring strain, Eschoricia coli 15-TAU, was studied in media containing the analog of thymine, 5-bromouracil. The substitution was capable of permitting continued growth of the organism for two consecutive cell divisions, and two rounds of DNA replication. Colony-forming ability of the cells was reduced as a function of exposure to the analog, and synchronous growth could not be maintained over more -than two generations. The bromouracil content of the cells was measured by two means: elemental chemical analysis, and density gradient ultracentrifugation. Of these, only the density gradient was found usable with the sample volumes available. A technique for preparing up to twelve density gradients at a time is discussed. The incorporation of the 5-bromouracil label, as detected by density gradient analysis of samples from a synchronously growing culture, was found to be in synchrony with the cell division cycle. The first two-thirds of the cell division cycle were found to be involved with incorporation of the label, while the last third of the cycle showed a pause in incorporation. The rate changes observed during incorporation are discussed.