Autoradiographic studies of bacterial chromosome replication in amino-acid deficient Escherichia coli 15T-

Abstract

Autoradiographic experiments on amino-acid requiring strains of Escherichia coli T(-) have been performed with fully-labeled cells harvested in log-phase and after periods of amino-acid starvation. The simplest segregation of incorporated label among progeny grown on non-radioactive medium is into two packets. The result corroborates the two-unit model of E. coli DNA inferred from previous studies with partially labeled cells. Following amino-acid starvation, the distribution of label among clones derived from labeled cells indicates cells are grouped into classes having DNA contents in the ratios 1:2:4. The segregation of label among progeny isolated by micromanipulation from such starved cells supports the view that the chromosomes are brought to a state of completed synthesis with different cell classes containing different integral numbers of chromosomes. Infrequent clones interpretable as arising from cells with three chromosomes suggest that the control of replication of chromosomes lying in the same cytoplasm is on an individual basis. The block in DNA synthesis resulting from amino-acid starvation is not perfect. Nevertheless, such starvation permits characterization of the dispersive replication events for more homogeneously labeled and definable DNA units than otherwise possible. The size-frequency distribution of label among progeny following six rounds of chromosome replication is close to that expected from a model of random breakage of linear polymers. The frequency of dispersive events is estimated as 0.5 to 0.7 per chromosome per generation and is only slightly influenced by tritium decays occurring during bacterial growth.