J.F. Bleichrodt

Contribution of various types of damage to inactivation of a biologically-active double-stranded circular DNA by gamma-radiation.

SummaryThe double-stranded circular DNA of the bacteriophage PM2 has been irradiated in oxygenated solution by 60Co gamma rays. The following quantities have been determined as a function of dose: The average number of single-strand breaks and that of double-strand breaks per molecule, the biological activity of the irradiated sample as such, the biological activity after mild denaturation (to denature molecules containing a break) and the biological activity after denaturation and membrane filtration (i.e. the biological activity of DNA without single- and double-strand breaks.)From the collected data it can be deduced that 4·5 ± 0·5 per cent of the inactivation is a consequence of double-strand breaks, 8·5 ± 4·2 per cent of single-strand breaks and 87·0 ± 4·2 per cent of nucleotide damage. Only about 2 per cent of the single-strand breaks is lethal, whereas the efficiency of inactivation of nucleotide damage is about 30 per cent of the nucleotide damage which is lethal if present in single-stranded DNA....

Infection of spheroplasts of Pseudomonas with DNA of bacteriophage PM2

SummarySpheroplasts of Pseudomonas BAL-31/PM2, obtained by treatment of the bacteria with lysozyme, can be infected with purified DNA from bacteriophage PM2. After 4 h of incubation the yield of progeny phage reaches a value of 107-6×107 plaque forming units/μg PM2 DNA. The yield increases linearly with the concentration of DNA over at least 3 orders of magnitude.The biological activity of double-stranded circular PM2 DNA containing one or more single-strand breaks per molecule (component II), does not differ significantly from that of intact PM2 DNA (component I). Single-stranded PM2 DNA obtained by denaturation of component II, and the irreversible alkali-denatured form of component I are also infective.

Binding of radiation-induced phenylalanine radicals to DNA: influence on the biological activity of the DNA and on its sensitivity to the induction of breaks by gamma rays

When an aqueous solution of double-stranded DNA of bacteriophage PM2 containing phenylalanine and saturated with N2O is irradiated with gamma-rays, radiation-induced phenylalanine radicals are bound covalently. Under the conditions used, about 25 phenylalanine molecules may be bound per lethal hit. For single-stranded PM2 DNA, most of the phenylalanine radicals bound are non-lethal. Evidence is presented that, in double-stranded DNA, an appreciable fraction of the single-strand breaks is induced by phenylalanine radicals. Radiation products of phenylalanine and the phenylalanine bound to the DNA decrease the sensitivity of the DNA to the induction of single-strand breaks. There are indications that the high efficiency of protection by radiation products of phenylalanine is due to their positive charge, which will result in a relatively high concentrations fo these compounds in the vicinity of the negatively-charged DNA molecules.

Contribution of various types of damage to inactivation of bacteriophage PM2 DNA in frozen solution by gamma-radiation

SummaryWhen biologically-active double-stranded circular DNA of the bacteriophage PM2 in a solution of nutrient broth is irradiated at −196°C by 60Co gamma-rays, about 85 per cent of the inactivation is due to nucleotide damage, i.e. damage in the nucleotides not associated with a chain break. Double-strand breaks contribute some 10 per cent to lethality and single-strand breaks only a minor fraction, if at all. About 10 per cent of the nucleotide damage that is lethal if present in single-stranded DNA is lethal in the double-stranded PM2 DNA. These results do not show large differences from those obtained previously for irradiation of PM2 DNA under conditions of indirect effect.

The transition between two forms of bacteriophage φX17A differing in heat sensitivity and adsorption characteristics

If cells of the bacterium Escherichia coliC infected with bacteriophage φX174 are lysed artificially in the cold, the phage obtained is relatively heat resistant at temperatures around 65° and unable to adsorb to host cells at 4°. At 37° the phage particles become more thermolabile with respect to inactivation at 65° and, concomitantly, obtain the ability to adsorb to E. coli C in the cold. A minor fraction remaining thermoresistant at 37° does not adsorb to host cells even at this temperature. Optimal conditions for the transition of the resistant into the sensitive form in phosphate buffer are 37°, pH ≤ 7 and a concentration of phosphate ≤= 25 mM. At lower pH and high titers of phage the thermosensitive form changes partly back into the thermoresistant one.

Effect of oxygen on inactivation of biologically active DNA by. gamma. rays in vitro: influence of metalloporphyrins and enzymatic DNA repair

Biologically active DNA dissolved in a bacterial extract shows a higher sensitivity to ..gamma.. rays under oxygen than under anoxic conditions. This oxygen effect depends on the presence of dialyzable, probably organometallic, compounds in the extract. Metalloporphyrins mimic these cellular components with regard to the effect of oxygen on DNA irradiated in vitro. Anoxic irradiation leads to less double-strand breaks in the DNA than irradiation under oxygen, but the oxygen effect in vitro is mainly due to nucleotide damage. No oxygen effect is observed when the biological activity of the irradiated DNA is assayed on spheroplasts of a bacterial strain carrying a uvrA mutation, i.e., a deficiency in the excision repair system, and the sensitivity of the DNA is almost equal to that found for irradiation under oxygen and assay on a repair-proficient strain. It may be concluded, therefore, that the oxygen effect observed with DNA in cellular extracts or in the presence of metalloporphyrins results from more efficient cellular repair of the otherwise lethal nucleotide damage inflicted under anoxic conditions. Comparison of the oxygen effect on DNA in vitro with the radiosensitization of bacterial cells by oxygen shows that in bacteria part of the radiation damage may be similarmorexa0» to that induced in DNA in vitro, but, in addition, the cells sustain another type of damage which is subjected to an oxygen effect but not to excision repair.«xa0less

Thermal inactivation of bacteriophage ∅X174 and two of its mutants

Abstract The thermal inactivation of the two forms (∅∗ and ∅) of bacteriophage ∅X174 with different adsorption properties was investigated under various conditions of pH, ionic strength, and temperature. A reaction mechanism is proposed that is similar for both forms. At high temperatures inactivation occurs mainly as a result of protein destruction and is dependent on the structure of the adsorption site(s). At lower temperatures the contribution of DNA destruction is higher. In addition to a difference in heat sensitivity, the two forms show a different dependence of their inactivation rate on pH and ionic strength. Application of the theory of absolute reaction rates, using the estimates of Stearn concerning enthalpy and entropy of activation for bonds broken during denaturation of proteins, suggests that the structural difference between ∅∗ and ∅ may be determined by only one weak noncovalent bond. The Arrhenius curves of the host-range mutant ∅X174 h 1 h 2 and of the thermoresistant mutant ∅X174 st are similar to that of the wild type. Comparison of the Arrhenius curves of these three phages and of their rate of transition from the ∅∗ into the ∅ form suggests that the bonds primarily involved in this transition are not located in the immediate vicinity of the adsorption site(s).