Sensitivity of phage precursor nucleic acid, synthesized in the presence of chloramphenicol, to ultraviolet irradiation

Abstract

Infected bacteria, in which phage precursor nucleic acid (DNA) has been allowed to accumulate in the presence of chloramphenicol, can be irradiated with multiple phage-lethal doses of ultraviolet light (UV) without preventing formation of phage particles after subsequent removal of chloramphenicol. Considerable numbers of such particles prove to be noninfective. With a small dose of UV, phage particles and deoxyribonucleic acid (DNA) are produced more or less normally after resumption of protein synthesis, but more than half of the particles lack infectivity. The number of noninfective particles produced is roughly proportional to the amount of precursor DNA present at the time of irradiation. Irradiation of bacteria before infection does not cause the production of noninfective particles. Large doses of irradiation permit synthesis of protein and maturation of phage particles with little concomitant DNA synthesis, after removal of chloramphenicol. About 99% of the particles produced under these conditions are noninfective. With such preparations, photoreactivation, cross reactivation, and multiplicity reactivation can be demonstrated with an efficiency appropriate to the dose of UV actually used. These results mean that phage precursor DNA formed in the presence of chloramphenicol presents a characteristic UV-sensitive target equivalent in all respects to that contained in finished phage particles. Since this target is presumed to be the phage chromosome, its formation in the presence of chloramphenicol seems to show that multiplying phage chromosomes are composed of DNA to the exclusion of all other known phage constituents. Irradiation of such chromosomes under the conditions described produces persistent lesions which do not prevent the subsequent formation of normal chromosomes; the lesions themselves are neither repaired nor replicated. Other experiments show that DNA synthesized at different times is incorporated into single phage particles and therefore that the phage precursor DNA destined for a given particle does not consist of a unitary structure finished in advance. Moreover, both irradiated and unirradiated DNA is found in both live and dead particles after maturation. Preliminary analysis of the manner of mixing shows that the bulk of the phage precursor DNA, like that of parental origin, consists of roughly equal parts of DNA that is (in a formal sense) resistant or sensitive, respectively, to UV damage.