Nuclear Segregation and the Growth of Clones of Bacterial Mutants induced by Ultraviolet Light

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Section: Discussi~nmentioning

confidence: 99%

Spontaneous Mutation in Spheroplasts of Escherichia colid

Ryan

Okada

Nagata

1963

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“…1 would already have taken place during the initiation of the colony from which the mutant clone was first isolated. The activity of growth-factorindependent mutants in heterozygotes (Lederberg, 1949) and the fact that they probably occur as zero-point mutants (Ryan, 1954; Ryan, Fried & Schwartz, 1954) would indicate that the h+ condition is dominant. If this were true and the development of the h+ phenotype required less than a generation, then the lag in the increase of the mutant clone, imposed by the necessity for the segregation of mutant from non-mutant nuclei, would have the following effect on the rise of mutant number.…”

Section: -2mentioning

confidence: 99%

Nuclear Segregation and the Growth of Clones of Spontaneous Mutants of Bacteria

Ryan

Wainwright

1954

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SUMMARY: An investigation of the rate of increase of spontaneous mutants and the subsequent increase in mutant proportion in a bacterial culture revealed discrepancies between the observed results and those expected on the assumption that mutant and parent grew with equal rates. These discrepancies could not be accounted for in terms of a selective difference between established mutants and their parents since, when the two were mixed together in reconstruction experiments, they fared equally well for hundreds of generations. Rather the discrepancies indicated a difference between parents and new mutants. The data were consistent with the hypothesis that the mutation occurred independently among one of four mutable units (nuclei) and that the mutant nucleus was 'dominant' over its sister non-mutant nuclei in the heterocaryon so formed. As a consequence, a delay of two generations ensued before the mutant unit segregated into the homocaryotic ancestor of a mutant clone. This process delayed the onset of an increase in mutant numbers after mutation. The accurate prediction of the pseudo-equilibrium level of mutants, based on verifiable assumptions of periodic selection, mutation and segregation lag, is added evidence for the occurrence of a two-generation delay before the increase of the mutant clone. This phenomenon, called segregation lag, is a source of error in the calculation of mutation rate by methods involving the numbers of mutants found in bacterial cultures. Furthermore, because bacteria may be multinucleate, the rate of mutation/bacterium/generation is not the same as the rate of mutation/mutable unit (nucleus)/generation.That a growing bacterium may contain more than one body consisting of deoxyribonucleic acid is well known. Witkin (1951) was the first, however, to offer direct evidence indicating that these bodies contain genetic material and are the counterparts of nuclei. Her evidence was threefold. First, the ratio of wholly mutant colonies to those containing mutant sectors was proportional to the ratio of uninucleate bacteria in the irradiated culture which gave rise to the colonies. This finding is consistent with the hypothesis that sectored colonies originate through segregation of the nuclear bodies during growth. Secondly, there was a proportionality between the frequency of induced mutants, whole and sectored, and the frequency of bacteria with more than one nuclear body. Finally, there was some correlation between the types of sectored colonies and the most frequent number of nuclear bodies/multinucleate cell; cultures with mostly binucleate bacteria gave rise predominantly to half sectors while cultures consisting for the most part of cells with four nuclei gave rise predominantly to quarter sectors. A more extensive correlation was not attempted because of the difficulty in determining nuclear numbers.This difficulty is even greater with bacteria that have been grown 0.n chemically defined media. Yet it is sometimes necessary to know the average number

“…If the effect of pre-treatment is solely on the very early generations in the treatment media containing the analogue, it would not be easily detected in the present experiments because the majority of the mutants are induced in the terminal generations of growth. In view of the findings of Witkin (1956), Ryan (1954Ryan ( , 1955a and Ryan, Fried & Schwartz, (1954) the number of generations in post-treatment media may not be as critical as the rate of growth in the first generation after treatment. If post-treatment effects can only be expressed when they are applied before a bacterial division occurs as is the case with photo-reactivation and some chemical post-treatment effects on ultra-violet light, their detection in the present system would be improbable since the reversal would affect only one generation (the last) of induced mutants.…”

Section: Antimutagenesismentioning

confidence: 92%

Growth Inhibitors and their Antagonists as Mutagens and Antimutagens in Escherichia coli

Greer

1958

Thirty-five substituted benzimidazoles, benzotriazoles and quinoxalines were tested as growth inhibitors on three mutants of Escherichia coli, strain W, having different purine requirements and on the ciliate Tetrahymena geleii. A number of the compounds, especially those with a nitro group in the benzene ring, were inhibitory. Both organisms were affected in a similar way by the compounds. Several of these analogues were tested for mutagenic activity a t the purine and streptomycin loci in a purine-requiring mutant of E. coli. Only inhibitory compounds were found to be mutagenic ; non-inhibitory concentrations of one analogue, 4-nitrO-6-hydroxybenzimidazole (NHB), were, however, also mutagenic. No specific mutagenesis was demonstrated. NHB was found to increase the rate of mutation as determined by the papilla method. Evidence is presented which eliminates the hypothesis that selection rather than mutagenesis was involved. This analogue did not increase the frequency of mutants in non-dividing cells, and was not incorporated into bacterial DNA. The mechanism of this mutagenesis is thought to include the inhibition of enzymes concerned with DNA synthesis. DNA and RNA and their constituents, purine and pyrimidine precursors and analogues, vitamin B,, and other vitamins, were found to have no effect on the inhibition or mutagenesis produced by NHB. Several amino acid combinations and one non-mutagenic analogue, 4-hydroxy-6-nitrobenzotriazole, were, however, found to interfere with mutagenesis by NHB. These compounds did not affect the frequency of spontaneous mutants. The amino acid combination was effective only when present simultaneously with the mutagen and did not act by a selection mechanism. The mechanism of antimutagenesis by amino acids may involve their ability to increase the growth rate and also to modify the inhibited nucleic acid metabolism. The mutagenesis by 5-nitroquinoxaline, a pterin analogue, was also found to be depressed by amino acids in the same purine-requiring mutant. The annulment of mutagenic activity by amino acids was strain and mutagen specific. Twenty-five known inhibitors of nucleic acid synthesis and antagonists to inhibition were tested for mutagenic activity in five strains of Eschmichia coZi. Several, including 6-mercaptopurine, were found to be mutagenic ; in addition, NHB, 5-nitroquinoxaline and 5-aminouracil were mutagenic in most of the strains tested. The mutagenic activity of 5-aminouracil was not annulled by thymine, thymidine or other nucleic acid components, but the mutagenesis of 6-mercaptopurine was annulled by purine bases and by ribosides.