Fragmentary transfer of P 32-labeled parental DNA to progeny phage

Abstract

The fate and mode of transfer of parental deoxyribonucleic acid (DNA) in T4 phage have been examined. “Heavy” 5-bromouracil (5-BU) bacteria were infected with P 32-labeled “light” bacteriophage. The fate of parental phage P 32 was analyzed in a number of ways: 1. 1. Parental and progeny phage were centrifuged in a cesium chloride (CsCl) gradient. The viable phage and P 32 contents were determined in aliquots collected from the gradient. The results showed a coincidence in density of P 32 and viable parental “heavy” phage, and a similar coincidence of P 32 and viable “light” progeny phage. 2. 2. DNA was extracted from “heavy” cells at different times after infection with P 32-labeled phage and was centrifuged in a CsCl gradient. The density of the P 32-containing DNA was estimated in fractions derived from the gradient and was compared with DNA extracted from purified progeny and from parental phage. In progeny phage no hybrid DNA of intermediate density, containing parental P 32, was found. The greater part of the parental P 32 overlapped with the distribution of the ultraviolet (UV)-absorbing material, at a density which indicated that the DNA molecules containing the P 32 were constituted predominantly of newly synthesized DNA. In prematurely opened infected bacteria, the P 32 distribution shifted progressively from the parental density (light) to intermediate positions nearer the final location of dense progeny DNA. 3. 3. Progeny DNA was fragmented by sonication and subsequently analyzed as in (2). After sonication most of the P 32-containing material was separated from the greater part of the UV-absorbing material. The UV-absorbing DNA banded at a density corresponding to the density found before sonication, whereas the P 32 banded as a symmetrical peak at a position corresponding to a density calculated for hybrid DNA. The mechanism of DNA replication—crossing-over—in phage, and the molecular organization of the highly polymerized phage DNA preparations are discussed.