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Structure and synthesis of bacteriophage lambda DNA

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  • Biology


The research in this thesis represents an attempt to study the replication of bacteriophage lambda in terms of the structure of the vegetative DNA. A spheroplast assay for lambda DNA is described in Part I and several parameters affecting the efficiency of the assay are investigated. The biological activity of intracellular lambda DNA is then studied using this assay. In contrast to the results obtained with the transformation or helper assay, no eclipse of infectivity of vegetative lambda DNA occurs in the spheroplast assay. The DNA species responsible for most of the infectivity in the spheroplast assay of DNA extracted from infected, immune bacteria, is the fast-sedimenting "super-coiled" form of lambda DNA. In Part II the structure and replication of the fast-sedimenting species of vegetative lambda DNA is examined physically and biologically. Its physical properties are those of a twisted, circular molecule each of whose two strands is closed upon itself. Using a mitomycin C technique to inhibit host DNA synthesis, the accumulation and semi-conservative replication of closed-circular lambda DNA specifically early in the infection is demonstrated with radio-isotopes. Biological analysis using the spheroplast assay verifies this conclusion. Chloramphenicol inhibits the synthesis of viral DNA at a lower-concentration than that needed to inhibit the synthesis of closed-circular DNA. Chloramphenicol does not prevent the conversion from viral to closed-circular DNA. The data presented in Part III suggest that open-circular lambda DNA may act as a precursor to, both closed-circular and viral DNA. The closed-circular species is not a major precursor to viral DNA. Sedimentation analysis also reveals the existence of as yet unidentified intermediates in the replication of viral DNA. Part IV describes several methods for purifying intracellular lambda DNA and examines the biological activity of the purified DNA in the spheroplast assay. The circular DNA is infective before and after denaturation, whereas denaturation inactivates viral DNA. The increased infectivity of open-circular DNA after denaturation (relative to the native state) appears to be due to the appearance of biologicallyactive single-stranded rings of lambda DNA. The closed-circular DNA usually has the same infectivity before and after denaturation.

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