A biophysical study of bacteriophage [phi]X-174 replication

by Denhardt, David T.

Abstract (Summary)
NOTE: Text or symbols not renderable in plain ASCII are indicated by [...]. Abstract is included in .pdf document. Techniques for synchronizing, by cyanide and starvation, the development of [phi]X-174 in all of the infected cells of a culture of E. coli C were developed. The infection of starved cells in buffer, followed by the addition of broth, was found most satisfactory. One-step growth curves of [phi]X and [...] (a plaque morphology mutant of [phi]X that has a longer mean latent period and will not lyse cells in concentrated cultures) were determined. The rate of phage maturation is constant. A probit plot of the percentage of phage released vs. the logarithm of the time after infection is linear. The replication of [phi]X DNA was investigated using E. coli CR (a thymine requiring strain), 5-bromodeoxyuridine, the technique of equilibrium CsCl density gradient centrifugation, and the protoplast assay technique for [phi]X DNA. The DNA combining with the infecting single strand to form the first replicative form (RF) is synthesized after infection from low molecular weigh precursors from the medium. The rate of increase in the amount of infective RF in the culture during the eclipse period is constant, and the RF molecule containing the parental single strand replicates semiconservatively. The conserved subunit is a single strand of DNA. The material constituting the progeny single strands is derived directly from the medium, not from RF. [phi]X carrying 2-4 P(32) atoms per particle were used to infect cells in cold medium. The phage and early complexes were killed with an efficiency of 1; mature complexes were inactivated with an efficiency of about 0.2, even though unlabeled and infective RF had been synthesized in them. At about the same time as progeny single strands were synthesized the complexes became completely refractory to suicide. Chloramphenicol did not prevent the P32 suicide efficiency of the complexes from decreasing to about 0.2; complexes formed in chloramphenicol from cells which had not been starved became fully resistant to inactivation, whereas complexes formed from starved cells did not. The ultraviolet radiation sensitivity of [phi]X complexes formed with E. coli C, E. coli Cs (a non host cell reactivating strain), and E. coli CR were determined at various wavelengths, at various stages of development, and under various conditions. Free [phi]X is not host cell reactivable; the target that is observed in the Luria-Latarjet curves of the complex is host cell reactivable. The most uv sensitive structure in the complex is the single strand of DNA synthesized after infection and associated with the infecting single strand. Budr present in this newly synthesized strand will sensitize the complex to uv. Experiments entailing a switch between Budr and thymidine media indicated that the uv sensitive target in the complex could be identified with the single strand of DNA associated with the infecting parental strand. The DNA constituting this strand "turns over" each time the parental RF replicates. The uv sensitivity of the bacterial capacity to support [phi]X growth was also studied. Although the colony forming ability of Cs is more sensitive to uv than C, the capacity is not. There was evidence that a single nucleic acid like structure and a large number of protein like structures had to be inactivated in order to destroy the capacity. Starvation of CR for thymine, or growth in Budr, increased the uv sensitivity of the capacity; as the cells in a thymine starved culture underwent thymineless death, they also lost their capacity to support [phi]X growth. Action spectra for the complexes and for capacity are presented. The uv sensitivities of MS-2, Bu-[phi]X, and [...] (in the presence and absence of cysteamine) are described.
Bibliographical Information:

Advisor:Robert Sinsheimer; Max Delbruck

School:California Institute of Technology

School Location:USA - California

Source Type:Master's Thesis



Date of Publication:09/01/1964

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