Section 5 — Mutagenesis 



of a population of competent Bacillus subtilis 

 bacteria was studied. These experiments were 

 performed by means of genetic measurements 

 (double transformation with two unlinked 

 markers) and by means of registration of 

 radioactive DNA uptake by competent bacteria 

 (the track autoradiographs method of Levinthal 

 was applied to study the homogeneity of DNA 

 uptake). The results showed a great inhomogenei- 

 ty of a B. subtilis population in transformation. 

 The knowledge of these details permitted us 

 to choose the conditions for quantitative 

 measurements. 



2. Chemical mutagenesis on purified DNA by 

 means of nitrous acid and u. v. -irradiation was 

 studied. Genetically altered DNA molecules 

 were registered by means of transformation of 

 a recipient strain. Kinetics of mutagenesis and 

 inactivation of DNA were measured and princi- 

 ples and equations of physical chemistry were 

 applied to both processes. Both reactions are one 

 hit processes, requiring one effective collision 

 with the mutagen. 



3. The efficiency of mutagenesis in vitro 

 depends on the ratio of two kinetic constants — 

 rate of mutagenesis versus rate of inactivation. 

 The reaction of inactivation was studied in 

 detail. It was shown that many genetic loci are 

 inactivated simultaneously. 



Possibility for reactivation of chemically 

 modified DNA was studied. Reactivation can 

 be performed by means of fusion and annealing 

 of DNA in solution (Doty — Marmur's method) 

 together with homologous but genetically 

 unmarked DNA, or by a slight fragmentation 

 of damaged DNA with ultrasound. 



These facts give some new points to the 

 mechanism of chemical attack on DNA macro- 

 molecules. 



5.4. The Mutagenic Effect Produced by Radiation 

 and Chemical Agents on Extracellular Phages. 

 A. S. Krivisky (Moscow, U.S.S.R.). 



The phage s (I Escherichia coli SK, isolated by 

 the author, is endowed with a unique capacity to 

 show hereditary variation when irradiated in 

 extracellular state. Below are exposed the results 

 of experiments serving to induce plaque mu- 

 tations of phage Sd acted upon by u. v. -rays, 

 ionizing radiation and nitric acid. The ap- 

 pearance of mutations should not be attributed 

 to selection, multiplicity reactivation or host- 

 reactivation. When the mutagen is applied to 

 the host cell only, no phage mutations arise. 

 The capacity to produce mutations in response 

 to the in vitro action of mutagenes is charac- 



teristic of various strains of phage Sa. The 

 mutants induced appear in equal numbers when 

 plated on different strains of host cells. The 

 initial plaque of the mutant induced contains 

 only phages of this mutant type. It may therefore 

 be concluded that the capacity to produce 

 hereditary variation when exposed to mutagenes 

 in vitro is inherent in phage particles; these 

 mutations arise as a result of a direct action 

 produced by the mutagen on the DNA of a 

 resting phage, and are phenotypically realized 

 directly after the first intracellular replication 

 of the phage DNA. 



The mutations do not revert after the action 

 of the mutagen by which they were induced; such 

 reversions may, however, arise spontaneously. 



The examination of lethal and mutagenic dose 

 response curves shows that the relation between 

 these two types of damages is typical of each 

 mutagen applied. The molecular mechanism of 

 damages seems to be identical in both cases, 

 since radioprotectors are equally efficient in the 

 case of lethal or mutagenic effect. 



The highest mutation ratio per surviving 

 phage (~2-3 per cent) is caused by u. v. -rays. 

 The effect is somewhat smaller in the case of 

 nitric acid, and still smaller in response to various 

 sources of ionizing radiation. The mutagenic 

 effect due to u.v.-rays is characterized by the 

 presence of a maximum corresponding to the 

 viability of phage~10" 2 -10 -3 ; a further increase 

 of the dose leads to a sharp drop of mutation 

 ratio per survivor. No such effect could be 

 observed in our experiments with a temperate 

 phage kappa of Serratia marcescens in which an 

 increase of mutation ratio is observed after u.v. 

 irradiation up to a viability ^10 -6 . The causes 

 responsible for these differences in the behaviour 

 of different phages are being analysed. 



In contrast to other phages, mutations failed 

 to arise after u.v. -irradiation of the vegetative 

 intracellular phage Sa. 



The possible molecular mechanism of muta- 

 genesis of phage Sd and the prospects of its 

 application as a model for radiogenetic investi- 

 gation are discussed. 



5.5. Induced Mutations in Extracellular Actinophages. 



S. I. Alikhanian and N. M. Mkrtumian 

 (Moscow, U.S.S.R.). 



Induced mutations in bacteriophages described 

 in literature include mutations obtained by 

 exposing to various mutagens: (a) phage- 

 infected cells, (b) phages and cells simultaneous- 

 ly, (c) host cells only. Phage mutations obtained 

 after single reproduction of preliminarily mu- 



56 



