The effect of extracellular metabolites on the frequency of thy+ revertants in Salmonella typhimurium populations

There is convincing evidence that adaptation and survival processes in bacterial populations depend on cell-to-cell interactions. Our studies showed that the frequency of stress-induced His⁺ reversions in an amino-acid-starved Salmonella typhimurium culture is inversely proportional to cell density in this culture. The effects of cell density and of different culture liquids prepared from cultures starved for histidine on the frequency of Thy⁺ revertants were also studied. It was found that the frequency of Thy⁺ revertants is inversely proportional (r = ?0.74) to the density of the bacterial culture starved of thymine. The culture liquid prepared from the culture starved of histidine exerted an inhibitory effect on the frequency of Thy⁺ reversions, indicating that mutations induced by different types of stress have a common mechanism. The study of the effect of the culture liquid prepared from a histidine-starved culture on the frequency of ethyl-methanesulfonate-induced His⁺ revertants showed that this liquid prevented the induction of His⁺ reversions.     

UV-mutagenesis in Bacillus subtilis. IV. Analysis of revertants to methionine prototrophy]

UV light induces in Bacillus subtilis met5 ade6 two classes of revertants to prototrophy to methionine which can be easily distinguished by their phenotype: double (Met+Ade+) and solitary (Met+) revertants. Crosses of revertants with the wild type, carried out in transformational experiments, showed that original (direct) mutation met5 is presented in chromosome of double revertants. Consequently they are extragenic suppressor revertants. In the chromosome of solitary revertants Met+ an extragenic suppressor was not detected; reversions Met+ seem to be of an intragenic nature. It is possible to use reversions to prototrophy to methionine as a model to study UV-mutagenesis in suppressor and non-suppressor genes.     

Escherichia coli K-12 mutants with increased resistance to ionizing radiation. V. The effect of mutations on spontaneous and radiation mutagenesis

The frequencies of spontaneous mutations (reversions his-4----His+ and forward mutations to rifampicin-, nalidixic acid- or valine-resistance) in radiation-resistant mutants Gamr444 and Gamr445 are much lower than in the wild-type strain AB1157. His+ revertants and rifampicin-resistant mutants Rifr are induced by low doses of gamma-rays more efficiently than in the wild-type. Low doses of UV light only enhanced mutagenic activity in Gamr strains for induction of His+ reversions but not for Rifr mutations. For the wild-type strain the frequencies of His+ and Rifr mutations increase proportionally to the square of dose both of UV light and gamma-rays. For the most radioresistant Gamr444 mutant the frequencies of UV- and gamma-rays-induced Rifr mutations and of gamma-rays-induced reversions increase linearly with the dose. Possible reasons for these anomalies of radiation-induced mutagenesis in Gamr mutants are discussed.     

Dynamics of x-ray-induced reversion in heterogeneous S. cerevisiae populations.

The survival and frequency of adenine and homoserine revertants after X-irradiation have been studied in starved and growing populations of haploid S. cerevisiae (strain 5483/1b). A growing population is heterogeneous to cell killing, and a mathematical model can be used to determine possible correlation between sensitivity to killing and sensitivity to mutation induction. The results indicate correlation between sensitivity to ade2-1 reversion and sensitivity to cell killing, whereas no such correlation was found between sensitivity to hom3-10 reversion and sensitivity to killing. The difference in the dynamics of homoserine and adenine reversions was reduced by adding caffeine to the post-irradiation media.     

Reversal of malignant transformation induced by the oncogenic virus SV40. I. Induction of reversal to normal type for the contact inhibition trait

The possibility of induction by the oncogenic DNA-containing virus SV40 of reversions to normal phenotype as regards contact inhibition ("flat" revertants), was studied in spontaneously transformed chinese hamster fibroblasts. Negative selection was used for detection of revertants. The method adopted allowed to study the mutagenic activity of the virus, while excluding its transforming effect. In all experiments the frequency of revertants after infection exceeded that in control series. The value of induction varied from 1.2 to 28.4 X 10(-6). The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) known to increase the frequency of mutations induced by carcinogens in vitro, displayed no enhancing effect on the frequency of revertants induced by SV40. The lack of enhancement of virus-induced reversions after TPA treatment might be explained by the lack of the transforming effect of SV40 in the system studied. Some of the normal "flat" colonies were T-antigen positive, i. e. the viral oncogene was expressed. The role of mutations induced by SV40 in cellular genes controlling malignancy is discussed.     

Mechanism of reversion of the gal3 mutation of Escherichia coli

The gal3 mutation is an insertion of a DNA sequence in the operator-promoter region of the galactose operon of E. coli. It reverts spontaneously to produce three kinds of gal+ revertants, which are: (i) stable and inducible, (ii) stable and constitutive, and (iii) unstable and constitutive. The constitutive revertants also show drastically reduced frequencies of transduction with lambda. The mechanism by which these reversions occur has remained unknown. It is proposed that the stable and inducible revertants arise by accurate excision of the insertion sequence. The unstable and constitutive revertants arise by tandem duplications of the gal operon in such a way that the structural genes of the extra copy of gal operon become connected to a different promoter. The resulting tandem configuration (gal3 ETK...P'E'T'K') permits constitutive expression and gal3 segregation (by internal recombination) simultaneously. The proposal was tested by comparison of the buoyant densities in CsCl of derivatives of a lambdagal phage carrying gal+, gal3, and the inducible and constitutive revertants. The densities of the inducible revertants were identical to the wild type, and the slight increase in density found to be associated with the gal3 insertion was missing. It was concluded that inducible revertants arise by excision of the inserted sequence. In contrast, lysates of a constitutive revertant exhibited several anomalous properties. The lysates contained a small quantity of phage whose density was identical to lambdagal3, produced few gal+ transductants (10(-3)-10(-4) of a normal HFT lysate), and the transductants were stable and constitutive. In turn, these abnormal transductants produced lysates which showed no lambdagal particles on centrifugation, and no transducing activity whatsoever. These anomalous properties of the constitutive revertant were attributed to the failure of lambda to package the DNA duplication efficiently. Transduction experiments with P1 (which can package more DNA than lambda) show that the unstable, constitutive reversions were located adjacent to prophage lambda. Segregation of the gal and lambda markers among the gal+ transductants was in accordance with the pattern expected for a duplication. Introduction of a recA marker resulted in stabilization of the reversion without affecting its constitutive expression. It was concluded that the unstable, constitutive reversion was a tandem duplication. It is further proposed that the stable, constitutive class of revertants might represent inverted (gal3 ETK...K'T'E'P') or partial tandem (gal3 ET...E'T'K') duplications of the gal operon.     

Effects of the antimutagen cinnamaldehyde on reversion and survival of selected Salmonella tester strains

The antimutagenic activity of trans-cinnamaldehyde (C6H5CH = CHCHO) on chemically induced mutagenesis has been shown in E. coli. Using the Ames Salmonella typhimurium tester strains TA1535 (hisG46 uvrB rfa) and TA100 (TA1535/pKM101), the effects of cinnamaldehyde on spontaneous reversions and reversions induced by 4-nitroquinoline-N-oxide(4NQO) and ethyl methanesulfonate (EMS) have been examined. To observe the effect of cinnamaldehyde in the absence of functional muc genes, a third strain, TA1535/pGW201 (pKM101 muc140: :Tn5) was included in the testing. Modifications of the standard Ames test procedures and direct-plating techniques were employed to study the "antimutagenic" response exerted by cinnamaldehyde. In all strains tested, concentrations of cinnamaldehyde up to 25 micrograms/ml slightly decreased the number of spontaneous reversions and induced reversions were more markedly reduced. The decreases in the numbers of 4NQO-induced revertants were greater than those decreases which occurred for EMS-induced reversions. There was no effect on viability in 1% (v/v) nutrient broth supplemented minimal medium containing 5-25 micrograms/ml of cinnamaldehyde. Cinnamaldehyde did not display any mucAB dependent or independent specificity against the mutagens used. On minimal medium supplemented with histidine and biotin, concentrations of cinnamaldehyde above 10 micrograms/ml were lethal for the strains tested. When the test medium was supplemented with 1-5% (v/v) liquid nutrient broth, viability was not affected at concentrations up to 25 micrograms/ml. For both TA100 and TA1535 the presence of 20 micrograms/ml of cinnamaldehyde in 1% (v/v) liquid nutrient broth-supplemented minimal glucose broth extended the lag phase for 2-4 h with no effect on survival. Depending on the test procedure employed, decreases in numbers of revertants may reflect lethality rather than antimutagenesis. When used to test for antimutagenesis rather than mutagenesis, modifications of the standard Ames test procedure may mimic an antimutagenic response due to a decrease in the total number of revertants seen even though enough cells survive to produce a background lawn.     

UV-light induction of "true" revertants to adenine independence in Bacillus subtilis cells]

The UV-irradiation of Bacillus subtilis Mu5u8u16 (met5 leu8 purA) induces with relatively high frequency the revertants to adenine independence (Ade+) which form the rapidly growing morphologically uniform colonies on the solid selective medium. The genetic analysis of a portion of UV-induced Ade+ revertants (crosses in transformation system) has cleared up that their DNA does not contain the original mutation ade16. This means that they arise as the result of "true" reversions. This reversion in purA gene can serve as a good model for the study of UV-induced mutagenesis in a proximal structural locus of Bac. subtilis chromosome.     

Analysis of correlation between some parameters depending on cell proliferation and life span of "stationary phase aging" cultures and maximum life span of mammals

Earlier we developed a "stationary phase aging" model and introduced a definition of life span of "stationary phase aging" cell cultures. In this model the cells grow after seeding in flasks without subcultivation and medium change. They reach cell saturation density, stop dividing, gradually degrade ("stationary phase aging") and perish. By the term "culture life span" we designate the time from cell seeding until culture death. We designate the culture as dead when the number of living cells is less than 10 per cent of their number at saturation density of cell culture. The life span of transformed Chinese hamster cells was found to be proportional to the duration of their growth from cell seeding up to saturation density, as well as to the number of cell culture doublings and to be inversely proportional to the velocity of cell culture doubling for the same growth period. Maximum life span of mammals is known to be proportional to pregnancy duration and to the age at puberty. We found that maximal life span of mammals was proportional to the number of cell population doublings and inversely proportional to the velocity of cell population doubling during embryonal period or for the time from zygote to growth termination. The dependences for cell cultures and for mammals are analogous to each other.     

Effect of cell density and attachment on resuscitation in soil of starved Pseudomonas fluorescens MON787

The effect of cell density and attachment on starvation survival and recovery was determined using luminometry to measure activity of a lux -marked strain of Pseudomonas fluorescens MON787. Bioluminescence was found to be a sensitive indicator of in situ activity of P. fluorescens MON787 in soil. The activity of a bacterial inoculum could be monitored during growth in soil, and was found to correlate with an increase in cell numbers. Luminescence could detect decreasing activity of P. fluorescens during starvation in soil, and recovery of activity and cell numbers following exposure to starvation and matric potential stress. The effect of localised cell density and attachment in soil on recovery from lag phase after nutrient addition was investigated and compared to recovery of starved liquid cultures. Nutrient addition to starved P. fluorescens in soil or liquid medium resulted in an immediate recovery of activity, followed by a second increase in luminescence after 5 h. Cells exposed to both starvation and matric potential stress in soil did not show a detectable immediate increase of activity, but required a 5-h lag phase before recovery of both activity and cell growth. The lag phase values were not significantly different over a range of localised cell densities. This suggests that cell density of P. fluorescens in the range tested is not a factor which affects recovery of soil bacteria from starvation.     

Influence of endogenous Thy1.1 cells upon the efficacy of an anti-Thy1.1 antibody-diphtheria toxin conjugate.

The chemical conjugation of antibodies to protein toxins results in cell-specific cytotoxic agents that can be defined in terms of in vitro potency and efficacy; however, it is the in vivo utilities that are largely being pursued in clinical trials. The nature of in vivo target cell depletion by toxin conjugates is largely unknown. The anti-murine Thy1.1 antibody-diphtheria toxin conjugate possesses high in vitro efficacy, and because mice are remarkably resistant to the native toxin, the conjugate possesses in vivo efficacy. When administered intravenously, the conjugate is shown to deplete peripheral blood Thy1.1+ target cells in a concentration-dependent fashion. When the log kill of Thy1.1+ tumor cells was analyzed by the life span extension method, it was determined, however, that the log kill is inversely proportional to the number of target cells. That is, the presence of an endogenous cell population, which is expressing the same surface antigen targeted by the antibody conjugate as on the pathological cell, may drastically lower the clinical efficacy of the immunotoxin. Thus, the greatest potential for antibody-toxin conjugates will be for low target cell burdens and for pathogenic cell populations expressing unique surface antigens. These are important considerations in the design of bioconjugates to insure high in vivo efficacy in elimination of intended target cells.     

Spontaneous Point Mutations That Occur More Often When Advantageous than When Neutral

Recent reports have called into question the widespread belief "that mutations arise continuously and without any consideration for their utility" (in the words of J. Cairns) and have suggested that some mutations (which Cairns called "directed" mutations) may occur as specific responses to environmental challenges, i.e., they may occur more often when advantageous than when neutral. In this paper it is shown that point mutations in the trp operon reverted to trp+ more frequently under conditions of prolonged tryptophan deprivation when the reversions were advantageous, than in the presence of tryptophan when the reversions were neutral. The overall mutation rate, as determined from the rates of mutation to valine resistance and to constitutive expression of the lac operon, did not increase during tryptophan starvation. The trp reversion rate did not increase when the cells were starved for cysteine for a similar period, indicating that the increased reversion rate was specific to conditions where the reversions were advantageous. Two artifactual explanations for the observations, delayed growth of some preexisting revertants and cryptic growth by some cells at the expense of dying cells within aged colonies, were tested and rejected as unlikely. The trp+ reversions that occurred while trp- colonies aged in the absence of tryptophan were shown to be time-dependent rather than replication-dependent, and it is suggested that they occur by mechanisms different from those that have been studied in growing cells. A heuristic model for the molecular basis of such mutations is proposed and evidence consistent with that model is discussed. It is suggested that the results in this and previous studies can be explained on the basis of underlying random mechanisms that act during prolonged periods of physiological stress, and that "directed" mutations are not necessarily the basis of those observations.     

Elimination of lethal and pre-mutational DNA lesions during the photoreactivation of UV-irradiatedEscherichia coli

The comparison of the frequency oftrp ⁺ revertants ofEscherichia coli B/r Hcr⁺ thy trp after UV-irradiation on the one hand and after UV-irradiation plus photoreactivation on the other showed that both photoreversible pyrimidine dimers of the cyclobutane type and the non-photoreversible DNA lesions cause, at equal lethal effects, alsotrp ⁺ reversions with the same efficiency. If lethal, the pyrimidine dimers may thus be conceived as primary pre-mutational lesions.     

Isolation of the thymidylate synthetase gene (TMP1) by complementation in Saccharomyces cerevisiae.

The structural gene (TMP1) for yeast thymidylate synthetase (thymidylate synthase; EC 2.1.1.45) was isolated from a chimeric plasmid bank by genetic complementation in Saccharomyces cerevisiae. Retransformation of the dTMP auxotroph GY712 and a temperature-sensitive mutant (cdc21) with purified plasmid (pTL1) yielded Tmp+ transformants at high frequency. In addition, the plasmid was tested for the ability to complement a bacterial thyA mutant that lacks functional thymidylate synthetase. Although it was not possible to select Thy+ transformants directly, it was found that all pTL1 transformants were phenotypically Thy+ after several generations of growth in nonselective conditions. Thus, yeast thymidylate synthetase is biologically active in Escherichia coli. Thymidylate synthetase was assayed in yeast cell lysates by high-pressure liquid chromatography to monitor the conversion of [6-3H]dUMP to [6-3H]dTMP. In protein extracts from the thymidylate auxotroph (tmp1-6) enzymatic conversion of dUMP to dTMP was barely detectable. Lysates of pTL1 transformants of this strain, however, had thymidylate synthetase activity that was comparable to that of the wild-type strain.     

Sequence Analysis of Mutations Arising during Prolonged Starvation of Salmonella Typhimurium

We have examined the effects of prolonged histidine deprivation on the reversion of Salmonella typhimurium histidine auxotrophs containing either hisG46, a missense mutation (CTC----CCC), or hisG428, an ochre mutation (CAA----TAA). Both of these mutants can revert to His+ via intragenic and extragenic mechanisms. Whereas the hisG46 mutant site consists of G/C base pairs, extragenic suppression of hisG46 requires mutation at an A/T site. Conversely, the hisG428 site itself contains only A/T base pairs, and extragenic suppression of hisG428 occurs principally at G/C sites. Thus, by examining the mutational spectrum of hisG46 and hisG428 revertants that occurred in the presence and in the absence of histidine, it was possible to determine the effects of histidine starvation on mutations at G/C vs. A/T sites as well as on intragenic sites vs. extragenic suppressor sites. Using DNA-colony hybridization, we determined the DNA sequences of over 1300 hisG46 and hisG428 revertants. Histidine-independent revertants that arose during growth in liquid medium that contained histidine included both intragenic and extragenic suppressor mutations. The relative frequency of such extragenic suppressors was greatly reduced among the His+ revertants that were isolated after 5-10 days of histidine starvation on agar medium. Moreover, DNA sequence analysis revealed striking differences in the distribution of particular transversions at the hisG428 locus in revertants arising after prolonged histidine starvation as compared to those arising after growth in the presence of histidine.     

The influence of temperature on the ultraviolet induced revertant frequencies of two auxotrophs ofNeurospora crassa

Summary The frequency of ultraviolet-induced adenine reversions relative to inositol reversions in the strainK3/17 ad-3A 38701: inos 37401 is influenced by temperature. Fewer adenine revertants are obtained from a given ultraviolet dose adminstered at 30°C than from the same dose administered at 2°C. The inositol reversion frequency and the percentage survival are not greatly affected by these differences in temperature. Temperature differences are only effective if a) applied during irradiation b) higher temperatures above 15°C are used. Possible explanations for these results are considered.With 2 Figures in the Text     

The nature of reverse mutations in Drosophila melanogaster

A selection system for the screening of reversions has been constructed and used to test reversions of lethals located in the proximal region of the X chromosome of Drosophila and of Kpn mutations.Spontaneous and induced reversions have been screened, X-rays and ethyl methanesulphonate (EMS) being the mutagens used in the induction experiments.No genuine back-mutation was found in 6·10⁵ gametes scored. Sterile reversions of all four lethals tested were obtained. Their frequency suggested that at least in three of the lethals the sterile reversions represented “escapers” of the lethal effect rather than true revertants.Three fertile reversions of l^x4 were found and analyzed. All three were autosomal suppressors, located on the second chromosome, allelic to each other, dominant in males and recessive in females.One fertile reversion of l^3DES was found to be an X-linked suppressor. It is suggested that this suppressor is a Y-suppressed lethal, showing a V-type position effect, resulting from an aberration included in the proximal heterochromatin of the X chromosome.Reversions of Kpn were obtained at a similar rate to that found in previous reports²².The absence of true back-mutants in our experiments, in contrast to findings in previous reports, is discussed. From the existing literature on spontaneous and induced back-mutations in Drosophila melanogaster it appears that for several mutations the rates of forward and back-mutation are of the same order of magnitude. It is suggested that reported cases of back-mutations represent mainly inter- and intrachromosomal recombination in duplicated regions rather than mutational events and that the frequency of true back-mutation in Drosophila is usually of an order of magnitude, similar to that known for microorganisms and fungi.