Dean Fraser

Synthesis of deoxycytidine-5′-phosphate deaminase in Escherichia coli infected by T2 bacteriophage☆

Abstract Deoxycytidylic deaminase (dCMP deaminase) was found in Escherichia coli after infection with T2 bacteriophage. This enzyme could not be detected in uninfected cells but appears 3–5 min. after infection. Enzyme synthesis could be stopped at any stage by addition of chloramphenicol (20 μg./ml.) to the medium. The enzyme activity reached a maximum approximately 12 min. after infection and declined thereafter. Ultraviolet irradiation of the phage prior to infection resulted in only little loss in the capacity to induce the synthesis of dCMP deaminase under conditions where the replication of the phage was strongly impaired.

Studies in partially resolved bacteriophage-host systems: IV. Some properties of the protoplast-infecting agent derived from T2 bacteriophage

Abstract Properties of the Escherichia coli protoplast-infective agent (π) derived from T2 are compared with those of T2, as well as with the published properties of pneumococcus transforming principle (TP). π is somewhat more resistant to heat inactivation than T2, much less stable than TP. The destruction of π is a complex process characterized by at least three different reactions. Ultraviolet light inactivates π at a rate closely comparable to that observed for T2 and qualitatively similar to that for TP. The data indicate that the π mixture contains many damaged particles incapable of producing intact virus but capable of contributing to multiplicity reactivation. Unlike T2 and TP, π shows a remarkable instability in solutions of high osmolarity and in solutions containing surface-active agents. The pH stability of all three infective agents is roughly similar. Antiserum against T2 inactivates π, but at a slower rate and to a lesser extent than T2. Protein-inactivating reagents (especially those affecting sulfhydryl groups) have a greater effect on T2. On the other hand, agents primarily affecting DNA and presumed mutagens have parallel effects on T2, on π, and, qualitatively, on TP. DNAase and trypsin have no effect on T2 but destroy π rapidly. TP is destroyed more rapidly by DNAase, but is unaffected by trypsin. It is concluded that π is a highly organized structure consisting of other components in addition to DNA. In size and physical properties π is similar to a phage head, but its sensitivity to various reagents indicates that at least some of the physically protecting protein envelope is lacking or altered. More than one component of the π mixture may contribute to the infectivity for protoplasts; these components seem to differ markedly in their stability to various treatments.

The interaction of DNA and internal protein from coliphage T2

The viscosity of DNA from T2 bacteriophage decreased when the nucleic acid complexed with internal protein from the same phage. The viscosity reached a minimum value (60% of that of free DNA) when 0.2 μ g of internal protein had become bound per microgram of DNA; this is considerably below the value for saturation as measured by uptake of radioactive internal protein (25 μ g of protein per microgram of DNA). A similar decrease of viscosity was also observed when calf thymus DNA was substituted for T2 DNA. The effect of internal protein was not specific since the binding of other basic proteins (histone and ribonuclease) to DNA also led to a decrease in viscosity. Bovine serum albumin and T2 coat protein subunits did not bind to, or alter, the viscosity of T2 DNA. The melting point ( T m ) of T2 DNA alone and of DNA complexed with internal protein at a ratio of 0.25 μ g per microgram DNA was the same. Hence this amount of internal protein did not stabilize the DNA helix in a low ionic strength methanolcontaining medium. The maximum decrease in viscosity of DNA is not reached until the concentration of internal protein reaches a value three times larger than is normally found for mature phage. The actual value of the viscosity decrement indicates a very small degree of folding of the DNA under these conditions. No further decrease in viscosity could be obtained by the addition of coat protein subunits to the DNA-internal protein complex. We conclude that internal protein either alone or in conjunction with coat protein is unlikely to be responsible for the folding of DNA during phage maturation.

The effects of nucleases on the reproduction of T3 bacteriophage in protoplasts of Escherichia coli

Abstract 1. 1. Efficiency of conversion of uninfected or T3-infected E. coli to protoplasts and lysates by means of lysozyme and Versene has been studied and found to be essentially unaffected by bacteriophage infection. 2. 2. The stability of uninfected or infected E. coli protoplasts to exposure to RNase and DNase in various media is such as to make studies of the effects of these agents profitable. 3. 3. Brief exposure in appropriate media of T3-infected protoplasts to RNase during early stages of phage development, greatly and consistently inhibits this process. Similar effects, although far less consistent, have also been obtained by DNase pretreatment. 4. 4. The addition of DNase or RNase singly or in combination to the medium throughout growth of bacteriophage T3 leads to almost complete inhibition of this process.

The replication of T2 bacteriophage.

Publisher Summary This chapter discusses the life cycle of T2 bacteriophage, which is one of a somewhat an arbitrary family of phages capable of attacking Escherichia coli , strain B. The physical properties, morphology, biochemical make-up, and life cycles of these phages are well known. T2 is the archetype for virulence among viruses because of its ability to commandeer the metabolism of the host cell in the interests of its own reproduction. Hence, it is, in many ways, an ideal model for a study of the processes of cell control. Chemically and metabolically inert by itself, T2 consists externally of a complex of some eight proteins. Its head seems composed of a single protein whose function is to protect and stabilize, both chemically and mechanically, the contained genetic apparatus of the virus. The proteins of T2 in their native state, such as those of viruses in general, are remarkably stable to proteolytic enzymes. When the virus and host cell are mixed in liquid suspension at adequate concentrations, the virus collides with the cell as the result of random Brownian motion. The structural information of the invading phage particle must be transmitted not only heterocatalytically or functionally––that is, into directions for the synthesis of specific proteins––but also autocatalytically––that is, into the replication to form new phage particles.

Physical effects of the deoxyribonucleic acid inhibitor β-phenethyl alcohol

Abstract Several aspects of β-phenethyl alcohol inhibition of DNA synthesis in Escherichia coli suggested a possible interaction of the inhibitor and DNA. To test this assumption effects of β-phenethyl alcohol on thermal denaturation, renaturation and viscosity of DNA were examined. These parameters were found not to be affected by β-phenethyl alcohol. Turning to a study of the effects of β-phenethyl alcohol on intact virus, differences in sedimentation were found which indicated that β-phenethyl alcohol caused shrinkage of the phage head. The results of dye permeability studies are compatible with this interpretation. β-Phenethyl alcohol-induced head shrinkage is completely reversible and therefore does not involve any damage to the head subunits. The DNA core appears necessary for β-phenethyl alcohol to induce the head form change. It is not at present clear what the role of DNA is in this reaction. A variety of bacteriophages was found to be inactivated by β-phenethyl alcohol and phenylacetaldehyde under conditions normally used to inhibit DNA synthesis with β-phenethyl alcohol. An effect of these inhibitors on protein is indicated.

Studies in partially resolved bacteriophage-host systems: VI. The involvement of ribonucleic acid in various aspects of the reproduction of bacteriophage T2

Abstract A variety of inhibitors has been shown to have pronounced effects on the replication of T2 bacteriophage in protoplasts of E. coli B. Ribonuclease applied in 5 or 10 min pulses exerts a maximum effect (40% reduction of burst size) when applied between 5 and 10 min after infection. p -chloromercury benzoate shows a sharp maximum of effectiveness and inhibits the burst completely when added 15 to 20 min after infection. Of a variety of purine and pyrimidine antagonists, 5-fluorouridine and 2′-deoxy-5-fluorouridine showed the most striking effects. The latter compound appears to have a specific inhibitory action on DNA synthesis and the inhibition (some 98%) is essentially completely reversed by thymidine. The action of 5-fluorouridine appears to be considerably more complex. In part its effect is also directly on DNA synthesis and is reversible by thymidine. During the first 10 min of infection, however, this inhibitor, especially when tested in the presence of thymidine, appears to exert its primary effect of RNA synthesis with resulting effects on the synthesis of “early protein” and hence DNA. These effects can be overcome by uridine. A combination of thymidine and uridine completely abolishes the inhibition by 5-fluorouridine even at quite high concentrations. These conclusions have been reached on the basis of measurements of phage yield and also of incorporation of 35 SO 4 and 32 PO 4 into the appropriate macromolecules.

Studies in partially resolved bacteriophage-host systems: V. A possible role of a polyribonucleotide in the infection of E. coli B with bacteriophage T2

1. 1. Brief treatment of protoplasts of E. coli with RNase virtually abolishes their ability to be infected by the protoplast-infecting agent (π) from T2. 2. 2. This effect can be reversed completely by brief incubation of the RNase-treated protoplasts either in nutrient broth or in a synthetic medium containing the 4 ribonucleosides and ATP. 3. 3. The optimal concn. of all 5 components is about 0.001 M. All are required simultaneously. 4. 4. Ribonucleosides can be replaced by ribonucleoside-5′-mono; di; or tri phosphates. Of these only the monophosphates require ATP in addition. 5. 5. The restoration reaction is inhibited by 5-hydroxyuridine, 5-fluorouridine and 5,6-dichloro-ribosylbenzimidazole. These inhibitions are reversed by uridine, uridine, and adenosine, respectively. 6. 6. The result suggest the involvement of a polyribonucleotide in the infection process. This polymer is, then, destroyed by RNase and rebuilt in the restoration reaction. It may again be destroyed by a second RNase treatment. 7. 7. Evidence is presented to indicate the location of this essential polyribonucleotide receptor on the surface of the proplast. 8. 8. Protein synthesis is not necessary for the re-synthesis of this polyribonucleotide receptor. 9. 9. A tentative scheme is presented describing the involvement of RNA in the initial stages of phage replication.