Genetic mapping in Bacillus subtilis by 5-bromouracil sensitization to ultraviolet inactivation of transforming...

A new method for chromosome mapping of Bacillus subtilis Marburg is presented which is based on the sensitization to ultraviolet irradiation of transforming deoxyribonucleic acid that has incorporated 5-bromouracil instead of thymine. Deoxyribonucleic acid was extracted at intervals from the outgrowing spores of a thymine-requiring mutant incubated with 5-bromodeoxyuridine and subjected to a definite dose of ultraviolet irradiation. The residual activities of various genetic markers were assayed by transformation. The marker activity of deoxyribonucleic acid that had incorporated 5-bromodeoxyuridine was approximately 10 times as sensitive to ultraviolet irradiation as that of normal deoxyribonucleic acid. The markers proximal to the replication origin were sensitized at earlier times of outgrowth than distal markers. The chromosome replication in outgrowing spores was sufficiently synchronous and allowed the definite determination of when a marker became sensitized by incorporation of 5-bromodeoxyuridine. The time, designated "sensitization time," was estimated by plotting the logarithmic values of relative residual activities versus incubation times. The map constructed with sensitization times as a measurement showed good agreement with those constructed by other methods. The replication of the chromosome under the described conditions appeared to occur in the following marker order: (purA, hisA)-(purB)-(thr, pyrA)-(metC)-(leuA)-(lys, trpC, metB).