J. Mol. Biol. (1971) 69, 65-74 Replication Time of the Chromosome in Thymineless Mutants of Escherichia coli A. ZARITSKYAXD R.H. PRITCHARD Department of Genetic-s University of Leiceder, Leicester, England (Received23 November 1970, and in revised form 14 May 1971) Reducing the concentration of thymine in the growth medium of thy- mutants of Eschetihia coli progressively increases the replication time of their chromo- somes, without affecting significantly the growth rate of the cultures, down to a concentration that is strain-specific. The replication time of the chromosome of thy- mutants of two unrelated strains of E. coli was estimated to be longer than that in E. coli B/r thy+ even in the presence of saturating concentrations of thymine. The replication time could be further reduced by adding deoxygurtnos- ine as well as thymine to the growth medium. 1. Introduction The growth rate of a culture of Escherichia wli and the replication time of the chromo- some can be varied independently of each other. For example, Helmstetter, Cooper, Pierucci & Revelas (1968) have shown that in strain B/r the replication time (C) of the chromosomeis constant in cells growing with mean generation times between 22 and 65 minutes. Conversely, we have shown (Pritchard & Zaritsky, 1970) that in the thy- strain 15T- (555-7)the time taken to complete a round of chromosome replication can be increasedby a factor of at least 25 by reducing the concentration of the thymine in the growth medium without significantly affecting the mean generation time of the culture. The discovery that the replication velocity (reciprocal of the replication time) is affected by the concentration of thymine in the growth medium, and that a substantial change in velocity occurs without any detectable effect on growth rate, introduces a potentially serious source of error in the interpretation of data involving measure- ments of rates of DNA synthesis and determination of the macromolecular composition of cells in thy- mutants, since these parameters are sensitive to changes in the replica- tion time of the chromosome as well as to changesin growth rate (seePritchard & Zaritsky, 1970). In the caseof strain 555-7 this problem is probably not a serious one since only a small increase in the replication velocity occurs on increasing the thymine concentration above 1-O pg/ml. and, in most published work with this strain, concen- trations greater than this have been used. This strain is exceptional, however, in being able to grow normally on very low concentrations of thymine. Concentrations greater than 0.2 pg/ml. will support normal growth (Maalee & Rasmussen, 1963; Lark, Repko & Hoffman, 1963; Pritchard & Zaritsky, 1970), and the major change in replication 5 65