Part I. The Transcription of Simple and Complex DNAs by the RNA Polymerase of Escherichia coli. Part II. The Structure and Replication of Intracellular Bacteriophage Lambda DNA

Citation

Kiger, John Andrew (1968) Part I. The Transcription of Simple and Complex DNAs by the RNA Polymerase of Escherichia coli. Part II. The Structure and Replication of Intracellular Bacteriophage Lambda DNA. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/1HYJ-RC42. https://resolver.caltech.edu/CaltechETD:etd-09242002-102646

Abstract

Part I of this thesis is a study of the in vitro transcription of DNA by Escherichia coli RNA polymerase. Experiments on the transcription of phage lambda DNA form Chapter 1. The transcription of lambda DNA is partially asymmetric. When symmetric transcription occurs, RNA so transcribed is found in a double-stranded form which is resistant to digestion by ribonuclease. The temperature of synthesis and the presence of Mn⁺⁺ in the synthetic mixture affect the frequency of symmetric transcription. RNA-DNA hybridization experiments indicate that no more than 50% of the lambda genome (25% of the DNA) is transcribed in vitro.

Experiments on the transcription of some eucaryote DNAs are presented in Chapter 2. Only a small fraction (15-20%) of the RNA transcribed from these DNAs is observed to form hybrid with complementary DNA under a variety of conditions. That RNA which does form hybrid is transcribed asymmetrically from a limited portion (about 10%) of the DNA. Evidence is presented for heterogeneity in the rate at which various RNA and DNA sequences form hybrid. This heterogeneity is believed to be of the same nature as the heterogeneity revealed by renaturation studies of eucaryote DNA.

Part II of this thesis is a study of the structure and replication of intracellular phage lambda DNA. Studies on purified non-replicating intracellular lambda DNA are presented in Chapter 1. Three species of lambda DNA are present following infection of immune bacteria by lambda phage: a closed-circular molecule, component I; an open-circular molecule containing one or more single-strand breaks, component II; and linear phage DNA, component III. The physical and infective properties of these molecules are studied. Component I in the native or denatured state is found to be almost equally infective to spheroplasts. Component II, however, is infective in the native state and shows a large increase in infectivity upon denaturation. This is due to the liberation of single-stranded rings which are more efficient in infecting spheroplasts than are native DNA molecules. Component III decreases greatly in infectivity following denaturation. Evidence is presented from sedimentation studies of components I, II, and III which suggests that the pitch of the Watson-Crick helix is variable in solution.

Experiments on the fractionation of replicating intracellular lambda DNA by chromatography on benzoylated-naphthoylated DEAE cellulose (BNC) are presented in Chapter 2. Intracellular lambda DNA is labeled following induction and mitomycin C treatment (to suppress host DNA synthesis) of lysogens and the purified DNA is adsorbed to BNC. Components II and III along with native phage DNA are eluted from BNC by a gradient of 0.3-1.0M̲ NaCl. A subsequent gradient of 0-2% caffeine elutes a heterogeneous species of intracellular DNA. This species is rapidly labeled by short pulses of ³H-thymidine and is virtually non-infective in the native state to spheroplasts. Denaturation of this species renders it very infective to spheroplasts suggesting that it contains single-strand rings. Analysis of the single-strand composition of this species by alkaline sedimentation reveals material sedimenting up to 1.5 times the rate of single-strand phage DNA. A model for DNA replication, involving initiation of one daughter strand by covalent addition to the 3'-OH of the identical parent strand, is presented based on the single strand composition of the DNA eluted from BNC by caffeine.

Supporting data from pulse and pulse-chase experiments are presented in Chapter 3. Approximately half of the label incorporated in very short pulses into material sedimenting at neutral pH as intracellular lambda DNA sediments in alkali faster than phage DNA single strands.

Very short pulses have revealed the presence of a small, rapidly labeled component in induced cells which appears to be DNA and sediments at about 10S at neutral and alkaline pH. The nature of this component is obscure at present.

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