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Part I: The abortive infection of bacteriophage øx174 at low temperatures. Part II: The early stages in the process of infection by bacteriophage øx174

Citation

Newbold, John Edward (1970) Part I: The abortive infection of bacteriophage øx174 at low temperatures. Part II: The early stages in the process of infection by bacteriophage øx174. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:08122015-154248368

Abstract

Part I

The infection of E. coli by ΦX174 at 15°C is abortive; the cells are killed by the infection but neither mature phage nor SS (single-stranded) DNA are synthesized. Parental RF (replicative form) is formed and subsequently replicated at 15°C. The RF made at 15°C shows normal infectivity and full competence to act as precursor to progeny SS DNA after an increase in temperature to 37°C. The investigations suggest that all of the proteins required for SS DNA synthesis and phage maturation are present in the abortive infection at 15°C.

Three possible causes are suggested for the abortive infection at 15°C: (a) A virus-coded protein whose role is essential to the infection is made at 15°C and assumes its native conformation, but its rate of activity is too low at this temperature to sustain the infection process. (b) Virus maturation may involve the formation of a DNA-protein complex and conformational changes which have an energy threshold infrequently reached at 15°C. (c) A host-coded protein present in uninfected cells, and whose activity is essential to the infection at all temperatures, but not to the host at 15°C, is inactive at 15°C. An hypothesis of this type is offered which proposes that the temperature-limiting factor in SS DNA synthesis in vivo may reflect a temperature-dependent property of the host DNA polymerase.

Part II

Three distinct stages are demonstrated in the process whereby ΦX174 invades its host: (1) Attachment: The phage attach to the cell in a manner that does not irreversibly alter the phage particle and which exhibits "single-hit" kinetics. The total charge on the phage particle is demonstrated to be important in determining the rate at which stable attachment is effected. The proteins specified by ΦX cistrons II, III and VII play roles, which may be indirect, in the attachment reaction. (2) Eclipse: 'The attached phage undergo a conformational change. Some of the altered phage particles spontaneously detach from the cell (in a non-infective form) while the remainder are more tightly bound to the cell. The altered phage particles detached (spontaneously or chemically) from such complexes have at least 40% of their DNA extruded from the phage coat. It is proposed that this particle is, or derives from, a direct intermediate in the penetration of the viral DNA.

The kinetics for the eclipse of attached phage particles are first-order with respect to phage concentration and biphasic; about 85% of the phage eclipse at one rate (k = 0.86 min-1) and the remainder do so at a distinctly lesser rate (k = 0.21 min-1).

The eclipse event is very temperature-dependent and has the relatively high Arrhenius activation energy of 36.6 kcal/mole, indicating the cooperative nature of the process. The temperature threshold for eclipse is 17 to 18°C.

At present no specific ΦX cistron is identified as affecting the eclipse process. (3) DNA penetration: A fraction of the attached, eclipsed phage particles corresponding in number to the plaque-forming units complete DNA penetration. The penetrated DNA is found in the cell as RF, and the empty phage protein coat remains firmly attached to the exterior of the cell. This step is inhibited by prior irradiation of the phage with relatively high doses of UV light and is insensitive to the presence of KCN and NaN3. Temporally excluded superinfecting phages do not achieve DNA penetration.

Both eclipsed phage particles and empty phage protein coats may be dissociated from infected cells; some of their properties are described.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biophysics
Degree Grantor:California Institute of Technology
Division:Biology
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Sinsheimer, Robert L.
Thesis Committee:
  • Unknown, Unknown
Defense Date:16 October 1969
Record Number:CaltechTHESIS:08122015-154248368
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:08122015-154248368
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9100
Collection:CaltechTHESIS
Deposited By: Bianca Rios
Deposited On:13 Aug 2015 15:22
Last Modified:13 Aug 2015 15:47

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