Biomedical Sciences ETDs

Publication Date



The experiments reported in this dissertation were aimed at studying two problems. The first of these was the occurrence and role of DNA rings in cells of Micrococcus lysodeikticus infected by any of several bacteriophages, particularly bacteriophage N1. Difficulties in labeling vegetative N1 DNA with radioactive pre­cursors led to the second problem, utilization of nucleosides by bacteriophage N1-infected N1-lysodeikticus.

It had been shown previously that N1-infected M. lysodeikticus contains during the latent period a DNA ring of the same molecular weight as the linear DNA isolated from the intact phage.

This result was confirmed and extended to include M. lysodeikticus cells infected with any of six phages representing three different sero­logical groups. For these experiments, DNA rings were isolated from phage-infected cells by CsCl-ethidium bromide equilibrium density gradient centrifugation and prepared for electron-microscopic examination by a modification of the Kleinschmidt technique. DNA rings were obtained from M. lysodeikticus cells infected with phages N1, N2, N3, N4, NS, and N6. The molecular weights of these DNA' s corresponded to the reported molecular weights of the linear DNA' s extracted from these phages.

The Campbell model for lysogenization predicts the formation of intracellular DNA rings after infection of bacteria by temperate phages. However, of the phages used in this study, only NS is known

to be temperate for any strain of M. lysodeikticus. Therefore, a search for lysogens of N1 on two commonly used host strains was made, using the method suggested by Bradley. No surviving clones were found to be lysogens.

Studies in the role of DNA rings were hampered by the finding that N1 DNA cannot be labeled with methyl-3H-thymidine experiments on nucleoside utilization by N1-infected cells gave the following results:

  1. DNA and infected cells can be labeled by methyl-3H-thymidine

  2. labeled thymidine is not degraded by thymidine phosphorylase in infected cells

  3. infected cells are permeable to thymidine

  4. identical levels of thymidine kinase are also found in infected and control cells

  5. N1 DNA can be labeled by a number of other nucleosides and deoxynucleosides including the uridine- 6-3H and deoxyuridine-6-3H.

These results are consistent with the hypothesis that N1 DNA does not contain thymine, and thymine is replaced by a similar base with a novel substitution on the 5-carbon of the pyrimidine ring. This hypothesis will be tested by mass spectroscopy of the nucleosides isolated from N1 DNA.

The results presented in this dissertation extend the catalogue of bacterial viruses known to cause the formation of intracellular DNA rings. Since five of the six phages for M. lysodeikticus are apparently virulent, these DNA rings may have some role in phage physiology other than establishment of lysogeny. The studies of nucleoside utilization by N1-infected cells suggest that N1 DNA may contain an unusual base in place of thymine.

Document Type




Degree Name

Biomedical Sciences

Level of Degree


Department Name

Biomedical Sciences Graduate Program

First Committee Member (Chair)

Joseph Victor Scaletti

Second Committee Member

Leslie Frank Smith

Third Committee Member

Ulrich Hollstein