Genetic response ofAntirrhinum majus to acute and chronic plant irradiation

Summary Clonal lines ofAntirrhinum majus heterozygous for flower color have been used to study somatic mutations induced by irradiating plants with chronic gamma rays from cobalt-60 and acute X rays. Pink or white flecks and sectors of various sizes on the purple flowers are interpreted as resulting from mutational events at different times in flower development.Acute X-ray treatment of pre-flowering stages caused no visible change in flowers opening prior to the 15th day thereafter. On the 16th to 19th days there was dense mottling, and by the 20th or 21st day numerous eight-cell mutant spots could be counted. The number of spots induced by 1,000 r of X rays was used to obtain a preliminary estimate of an average mutation rate for the three genes studied of 6.33×10^–6 per cell per roentgen.Chronic irradiation in a gamma radiation field and greenhouse showed a linear response of mutation rate to daily dose rate, the actual rate being subject to seasonal variation. There were occasional indications of non-linearity but further tests have failed to show any significantly higher mutagenic efficiency of dose rates below 6 r/day.In plants removed from chronic gamma irradiation the frequency of mutation spots returns to the spontaneous level after about six weeks. The appearance, however, of mutant sectors on plants several months after exposure to both acute and chronic radiation indicates the induction of persistent genetic changes. Progeny tests of seeds and cuttings are planned to test the nature of these genetic events.With 6 Figures in the TextResearch carried out at the Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission.     

Procarbazine-induced specific-locus mutations in male mice.

Procarbazine is used in drug-combination treatment of Hodgkin's disease. The specific locus method was used to test and confirm the ability of procarbazine to induce gene mutations in pre- and post-meiotic germ cells of male mice. The lowest dose of procarbazine that significantly increased the mutation frequency in As spermatogonia over the control frequency was 400 mg/kg (P = 0.003). The corresponding dose for the post-spermatogonial germ-cell stages was 600 mg/kg (P = 0.009). The dose--response was linear for the point estimates of the mutation frequencies after treatment of As spermatogonia with 0, 200, 400 and 600 mg/kg. The point estimate of the mutation frequency at the 800 mg/kg level was one-third of that expected from a linear extrapolation. Variation in mutation rates among the 7 loci between the lowest (a locus) and the highest (p locus) was 12-fold. Only 24% of procarbazine-induced specific locus mutations in As spermatogonia were lethal in the homozygous condition. From the mutation spectra and the viability tests, it is concluded that procarbazine-induced mutations may be mainly due to base-pair changes. Procarbazine-induced specific-locus mutations fulfilled the criteria for the estimation of the doubling dose, the dose necessary to induce as many mutations as occur spontaneously. The doubling dose of procarbazine in As spermatogonia of mice was 114 mg/kg. The therapeutic dose for procarbazine is about 215 mg/kg. If man and mouse were equally sensitive, this dose would induce 1.9 times as many mutations as arise spontaneously. From the incidence of patients with Hodgkin's disease (1 : 42 000) the calculated population dose of procarbazine is 5.12 micrograms/kg. Assuming equal sensitivity between the sexes we can calculate, for an estimated number of 30 000 genes, the induction of about 22 mutations per million children due to procarbazine treatment. The same number of induced mutations can be calculated if the risk of patients is used for the estimation of the genetic hazard.     

Increased inactivation of Saccharomyces cerevisiae by protraction of UV irradiation.

The principle of equi-effectivity of the product of intensity and exposure time (principle of Bunsen-Roscoe) of UV irradiation has been assumed to be valid for the inactivation of microorganisms in general. Earlier studies claimed higher survival of Escherichia coli B/r with fractionated irradiation compared with single-exposure survival. However, data on the inactivation effect of protraction of UV irradiation are not available. By means of a specially designed UV irradiation apparatus which secured absolute UV dose measurements throughout the experiments, the effects of variation of UV irradiation intensities (253.7 nm) and exposure times were tested on the inactivation of a bacterial virus (Staphylococcus aureus phage A994), a vegetative bacterial strain (E. coli ATCC 25922), and bacterial spores (Bacillus subtilis ATCC 6633) as well as three haploid laboratory strains (RC43a, YNN281, and YNN282) and two diploid strains (commercial bakery yeast strain and laboratory strain YNN281 x YNN282) or yeast (Saccharomyces cerevisiae) and spores of the latter diploid yeast strain. Each test organism was exposed to three UV intensities (0.02, 0.2, and 2 W/m2), with corresponding exposure times resulting in three dose levels for each intensity. Differences in inactivation rates were tested by analyses of variance and Newman-Keuls tests. Virus and bacteria showed no differences in inactivation rates by variation of intensities and exposure times within selected UV doses; hence, the principle of Bunsen-Roscoe could not be rejected for these strains. However, in the eukaryotic test strains of S. cerevisiae longer exposure times with lower intensities led to enhanced inactivation in both haploid and diploid strains, with a more pronounced effect in the diploid yeast strains, whereas in yeast spores in this dose rate effect could not be observed.     

Ionizing radiation and genetic risks. XI. The doubling dose estimates from the mid-1950s to the present and the conceptual change to the use of human data on spontaneous mutation rates and mouse data on induced mutation rates for doubling dose calculations

This paper provides an overview of the concept of doubling dose, changes in the database employed for calculating it over the past 30 years and recent advances in this area. The doubling dose is estimated as a ratio of the average rates of spontaneous and induced mutations in a defined set of genes. The reciprocal of the doubling dose is the relative mutation risk per unit dose and is one of the quantities used in estimating genetic risks of radiation exposures. Most of the doubling dose estimates used thus far have been based on mouse data on spontaneous and induced rates of mutations. Initially restricted to mutations in defined genes (with particular focus on the seven genes at which induced recessive mutations were studied in different laboratories), the doubling dose concept was subsequently expanded to include other endpoints of genetic damage. At least during the past 20 years, the magnitude of the doubling dose has remained unchanged at approximately 1 Gy for chronic low LET radiation exposures.One of the assumptions underlying the use of the doubling dose based on mouse data for predicting genetic risks in humans, namely, that the spontaneous rates of mutations in mouse and human genes are similar, is incorrect; this is because of the fact that, unlike in the mouse, the mutation rate in humans differs between the two sexes (being higher in males than in females) and increases with paternal age. Further, an additional source of uncertainty in spontaneous mutation rate estimates in mice has been uncovered. This is related to the non-inclusion of mutations which arise as germinal mosaics and which result in clusters of identical mutations in the following generation. In view of these reasons, it is suggested that a prudent way forward is to revert to the use of human data on spontaneous mutation rates and mouse data on induced mutation rates for doubling dose calculations as was first done in the 1972 BEIR report of the US National Academy of Sciences. The advantages of this procedure are the following: (i) estimates of spontaneous mutation rates in humans, which are usually presented as sex-averaged rates, automatically include sex differences and paternal age-effects; (ii) since human geneticists count all mutations that arise anew irrespective of whether they are part of a cluster or not, had clusters occurred, they would have been included in mutation rate calculations and (iii) one stays close to the aim of risk estimation, namely, estimation of the risk of genetic diseases in humans.On the basis of detailed analyses of the pertinent data, it is now estimated that the average spontaneous mutation rate of human genes (n=135 genes) is: (2.95+/-0.64)x10(-6) per gene and the average induced mutation rate of mouse genes (n=34) is: (0.36+/-0.10)x10(-5) per gene per Gy for chronic low LET radiation. The resultant doubling dose is (0.82+/-0.29) Gy. The standard error of the doubling dose estimate incorporates sampling variability across loci for estimates of spontaneous and induced mutation rates as well as variability in induced mutation rates in individual mouse experiments on radiation-induced mutations. We suggest the use of a rounded doubling dose value of 1 Gy for estimating genetic risks of radiation. Although this value is the same as that used previously, its conceptual basis is different and the present estimate is based on more extensive data than has so far been the case.     

Comparison of Triazine-Resistant and -Susceptible Biotypes of Senecio vulgaris and Their F(1) Hybrids

The relationship of triazine resistance to decreased plant productivity was investigated in Senecio vulgaris L. F₁ reciprocal hybrids were developed from pure-breeding susceptible (S) and resistant (R) lines. The four biotypes (S, S × R, R, R × S) were compared in terms of atrazine response, electron transport, carbon fixation, and biomass production. Atrazine response, carbon fixation rate, and PSII and whole-chain electron transport rates of hybrids were nearly identical to those of their respective maternal parents. Significant differences occurred between the two susceptible (S, S × R) and two resistant (R, R × S) biotypes in atrazine response (I₅₀), carbon fixation rate, and PSII and whole-chain electron transport rates; PSI rates were identical in all four biotypes. Coupled and uncoupled, whole-chain electron transport rates of thylakoids of the two susceptible biotypes were approximately 50% greater than those of the two resistant biotypes at photon flux densities greater than 215 micromoles per square meter per second. Carbon exchange rates of the two susceptible biotypes were 23% greater than those of the two resistant biotypes. Hybrid biotypes (S × R, R × S) were not identical to their maternal parents in biomass production. The S, S × R, and R × S plants all achieved greater biomass than R plants. These results suggest that while the resistance mutation influences thylakoid performance, reduced productivity of triazine-resistant plants cannot be ascribed solely to decreases in electron transport or carbon assimilation rates brought about by the altered binding protein. Since the F₁ hybrids differed from their maternal parents only in nuclear genes, it appears that the detrimental effects of the triazine resistance mutation on plant growth may be attenuated by interactions of the plastid and nuclear genomes.     

Genetic effect of 3-carbethoxypsoralen, angelicin, psoralen and 8-methoxypsoralen plus 365-nm irradiation in Saccharomyces cerevisiae: induction of reversions, mitotic crossing-over, gene conversion and cytoplasmic "petite" mutations.

The genetic effects of two mono-functional photosensitizing furocoumarins, 3-carbethoxypsoralen (3-CPs) and angelicin, were compared with those of two bi-functional furocoumarins, 8-methoxypsoralen and psoralen in Saccharomyces cerevisiae. A drug concentration of 5 X 10(-5) M plus various doses of 365-nm irradiation at a dose rate of 1.2 kJ m-2 min-1 were used. Per dose of 365-nm irradiation, the frequency of induced nuclear events such as gene mutation and mitotic recombination (conversion and crossing-over) is higher for the bi-functional than for the mono-functional compounds. The higher efficiency of the bi-functional furocoumarins is also evident when the frequency of mutants is expressed as a function of survival. However, the photo-addition of the 4 furocoumarins studied leads to the same response for the induction of recombinational events per viable cell. Amongst genetically altered colonies induced in the diploid strains D5 and D7, the colonies corresponding to the induction of crossing-over are effectively produced by bi-functional furocoumarins, but are rare (D7) or even absent (D5) after treatment with monofunctional furocoumarins. This suggests a certain specificity of genetic alterations produced by the bi-functional agents. 3-CPs is the most effective inducer on the cytoplasmic "petite" mutation in stationary phase cells per unit irradiation dose or per viable cell.     

Rates of spontaneous mutation in an archaeon from geothermal environments.

To estimate the efficacy of mechanisms which may prevent or repair thermal damage to DNA in thermophilic archaea, a quantitative assay of forward mutation at extremely high temperature was developed for Sulfolobus acidocaldarius, based on the selection of pyrimidine-requiring mutants resistant to 5-fluoro-orotic acid. Maximum-likelihood analysis of spontaneous mutant distributions in wild-type cultures yielded maximal estimates of (2.8 +/- 0.7) x 10(-7) and (1.5 +/- 0.6) x 10(-7) mutational events per cell per division cycle for the pyrE and pyrF loci, respectively. To our knowledge, these results provide the first accurate measurement of the genetic fidelity maintained by archaea that populate geothermal environments. The measured rates of forward mutation at the pyrE and pyrF loci in S. acidocaldarius are close to corresponding rates reported for protein-encoding genes of Escherichia coli. The normal rate of spontaneous mutation in E. coli at 37 degrees C is known to require the functioning of several enzyme systems that repair spontaneous damage in DNA. Our results provide indirect evidence that S. acidocaldarius has cellular mechanisms, as yet unidentified, which effectively compensate for the higher chemical instability of DNA at the temperatures and pHs that prevail within growing Sulfolobus cells.     

Fast neutron r.b.e. for lethality and genotoxicity in a wild-type and a repair-deficient strain of yeast

The relative biological effectiveness (r.b.e.) of cyclotron-produced fast neutrons (11 MeV) in relation to 60Co gamma-rays, was studied in a wild-type and a DNA repair-deficient yeast strain for cell killing and genotoxicity. In the wild-type (D7) strain the r.b.e. varied from 2.7 to 4.1 for lethality, 2.8 to 7.1 for reverse mutation and 3.5 to 7.8 for mitotic gene conversion. At different survival levels, the repair deficient strain (D7 rad 52/rad 52) generally showed a lower r.b.e. for both cell killing and genotoxicity (25.2 to 37.2 per cent reduction for the cell death and 24.8 to 70.6 per cent for mutation and gene conversion) compared to the wild type. Except at very low dose levels, the r.b.e. values for cell killing and genotoxicity were similar within a given strain. At similar survival levels, neutrons were no more genotoxic than gamma-rays.     

The effect of dose rate on the frequency of specific-locus mutations induced in mouse spermatogonia is restricted to larger lesions; a retrospective analysis of historical data

A series of 19 large-scale germ-cell mutagenesis experiments conducted several decades ago led to the conclusion that low-LET radiation delivered to mouse spermatogonia at dose rates of 0.8 R/min and below induced only about one-third as many specific-locus mutations as did single, acute exposures at 24 R/min and above. A two-hit origin of the mutations was deemed unlikely in view of the then prevailing evidence for the small size of genetic lesions in spermatogonia. Instead, the dose-rate effect was hypothesized to be the result of a repair system that exists in spermatogonia, but not in more mature male reproductive cells. More recent genetic and molecular studies on the marker genes have identified the phenotypes associated with specific states of the mutant chromosomes, and it is now possible retrospectively to classify individual past mutations as "large lesions" or "other lesions". The mutation-frequency difference between high and low dose rates is restricted to the large lesion mutations, for which the dose-curve slopes differ by a factor exceeding 3.4. For other lesion mutations, there is essentially no difference between the slopes for protracted and acute irradiations; induced other lesions frequencies per unit dose remain similar for dose rates ranging over more than 7 orders of magnitude. For large lesions, these values rise sharply at dose rates >0.8 R/min, though they remain similar within the whole range of protracted doses, failing to provide evidence for a threshold dose rate. The downward bend at high doses that had been noted for X-ray-induced specific-locus mutations as a whole and ascribed to a positive correlation between spermatogonial death and mutation load is now found to be restricted to large lesion mutations. There is a marked difference between the mutation spectra (distributions among the seven loci) for large lesions and other lesions. Within each class, however, the spectra are similar for acute and protracted irradiation.     

The effects of three rad genes on UV induced mutation rates in haploid and diploid Saccharomyces cells

Effects of the rad 2-20, rad 9-4, r1s, and the corresponding wild type RAD alleles in haploid and homozygous diploid Saccharomyces strains on UV induced mutation rates from adenine, lysine and histidine dependence to independence are reported. The UV induced mutation rates were similar for the RAD, r1s, and rad 9-4 haploids, whereas the rad 2-20 mutation causes a marked increase in the UV induced mutation rates. The diploid rad 2-20 strain also exhibits a marked increase in the UV induced mutation rates, whereas the rad 9-4 diploid has reduced mutation rates when compared to the wildtype. The UV induced mutation rates of haploid and diploid RAD strains are almost identical. For the rad 2-20 and rad 9-4 diploids, however, these rates are smaller than in the corresponding haploid strains. Differential effects of the rad genes on the ratio of locus to suppressor mutations were found. The implications of these findings on possible repair processes in yeasts are discussed.     

The nature of floral signals in Arabidopsis. I. Photosynthesis and a far-red photoresponse independently regulate flowering by increasing expression of FLOWERING LOCUS T (FT)

Arabidopsis flowers in long day (LD) in response to signals transported from the photoinduced leaf to the shoot apex. These LD signals may include protein of the gene FLOWERING LOCUS T (FT) while in short day (SD) with its slower flowering, signalling may involve sucrose and gibberellin. Here, it is shown that after 5 weeks growth in SD, a single LD up-regulated leaf blade expression of FT and CONSTANS (CO) within 4-8 h, and flowers were visible within 2-3 weeks. Plants kept in SDs were still vegetative 7 weeks later. This LD response was blocked in ft-1 and a co mutant. Exposure to different LD light intensities and spectral qualities showed that two LD photoresponses are important for up-regulation of FT and for flowering. Phytochrome is effective at a low intensity from far-red (FR)-rich incandescent lamps. Independently, photosynthesis is active in an LD at a high intensity from red (R)-rich fluorescent lamps. The photosynthetic role of a single high light LD is demonstrated here by the blocking of the flowering and FT increase on removal of atmospheric CO(2) or by decreasing the LD light intensity by 10-fold. These conditions also reduced leaf blade sucrose content and photosynthetic gene expression. An SD light integral matching that in a single LD was not effective for flowering, although there was reasonable FT-independent flowering after 12 SD at high light. While a single photosynthetic LD strongly amplified FT expression, the ability to respond to the LD required an additional but unidentified photoresponse. The implications of these findings for studies with mutants and for flowering in natural conditions are discussed.     

Detection of mosaicism in lymphocytes of parents of free trisomy 21 offspring

Genetic selection assays were developed to measure rates of deletion of one or more (CAG).(CTG) repeats, or an entire repeat tract, in Escherichia coli. In-frame insertions of >or=25 repeats in the chloramphenicol acetyltransferase (CAT) gene of pBR325 resulted in a chloramphenicol-sensitive (Cm(s)) phenotype. When (CAG)25 comprised the leading template strand, deletion of one or more repeats resulted in a chloramphenicol resistant (Cm(r)) phenotype at a rate of 4 x 10(-2) revertants per cell per generation. The mutation rates for plasmids containing (CAG)43 or (CAG)79 decreased significantly. When (CTG)n comprised the leading template strand the Cm(r) mutation rates were 100-1000 lower than for the opposite orientation. As an initial application of this assay, the effects of mutations influencing mismatch repair and recombination were examined. The methyl directed mismatch repair system increased repeat stability only when (CTG)n comprised the leading template strand. Replication errors made with the opposite repeat orientation were apparently not recognized. For the (CAG)n leading strand orientation, mutation rates were reduced as much as 3000-fold in a recA- strain. In a second assay, out-of-frame mutation inserts underwent complete deletion at rates ranging from about 5 x 10(-9) to 1 x 10(-7) per cell per generation. These assays allow careful quantitation of triplet repeat instability in E. coli and provide a way to examine the effects of mutations in replication, repair, and recombination on repeat instability.     

The inactivation of dilute solutions of crystalline trypsin by x-radiation. I. Kinetics and characteristics

The activity of dilute solutions of crystalline trypsin is destroyed by x-rays. The inactivation is an exponential function of the radiation dose. The reaction yield of inactivation is independent of the intensity at which the radiation is delivered or the quality of the x-rays. The reaction yield increases with increasing concentration of trypsin, varying from 0.06 to 0.7 micromoles per liter per 1000 r for trypsin solutions ranging from 1 x 10(-7) to 2 x 10(-4)M.     

Identification of three MADS-box genes expressed in sunflower capitulum

Three cDNA clones, HaPI, HaAG and HaAP3, were isolated from sunflower inflorescences at the R2 stage of development. The cDNAs share high sequence similarity with the PISTILLATA, AGAMOUS, and APETALA3 genes from Arabidopsis, respectively, which contain a MADS-box and are involved in floral organ development. Expression of the corresponding genes was analysed by northern blots and in situ hybridization. They are expressed preferentially in the R3 and R4 stages of capitulum development. HaAG accumulates in fertile flowers, mainly in stamens, while HaPI and HaAP3 are preferentially expressed in ray (sterile) flowers and more weakly in petals and stamens of fertile flowers.     

Somatic cell mutation. Detection and quantification of x-ray-induced mutation in cultured, diploid human fibroblasts

We describe a system for detecting somatic cell mutation to 8-azaguanine (8AG) resistance in cultured, diploid human fibroblasts. Hypoxanthine-guanine phosphoribosyltransferase (HG-PRT)-deficient, AG-resistant fibroblasts from boys with the X-chromosomal, Lesch-Nyhan (L-N) mutation served as one type of prototype mutant cells. Both spontaneous and X-ray-induced mutation were studied. Recovery of L-N cells was a function both of density of normal cells and of the AG concentration used for selection. Optimum recovery was achieved at an initial inoculum of 2·10⁴ normal cells per 60 mm diameter culture dish and an AG concentration of 8·10^?6M. Efficiency of recovery was between 39 and 90% and controls to determine this efficiency were included in mutagenesis experiments.Attempts to free normal cell populations of pre-existing AG-resistant mutant cells by pregrowth in HAT medium failed because, unlike L-N mutants, most spontaneous AG-resistant mutants can grow in HAT medium. Although pre-existing mutants probably caused overestimation, the average spontaneous mutation rate derived from our experiments was 4.5·10^?6 per cell generation. Eliminating one large-yieldv experiment reduced this estimate to 1.9·10^?6. Clonal survival of cultured human fibroblasts as a function of X-ray dose was studied. X-Irradiation increased the mutation rate above spontaneous background. Minimum estimates of the increases were 1.13·10^?9 per R per cell at 75 R, 7.49·10^?8 per R per cell at 125 R, 6.87·10^?8 per R per cell at 150 R and 2.16·10^?7 per R per cell at 250 R. The total mutagenic effect and the induced mutation rate appeared to be dose-dependent. Normal parental cell strains and their derived AG-resistant mutants had similar X-ray sensitivities indicating that X-rays induced mutations rather than selected for pre-existing mutants.Because of the realism of the cultured diploid, human fibroblast model vis-a-vis in vivohuman cellular events, the mutation detection system described herein is proposed as being potentially useful for environmental monitoring.     

A simple method for estimating the intensity of purifying selection in protein-coding genes.

We propose a method by which the intensity of purifying selection on a functional protein-coding gene is estimated by using three aligned homologous sequences: a processed pseudogene (psi), a functional paralog from the same species (g), and a functional ortholog from a different species (o). For each such trio, we calculate the numbers of nucleotide substitutions along the branches leading to psi and g, i.e., K psi and K(g). If we assume that the mutation rates are the same in the genes and the pseudogenes and that mutations occurring in a pseudogene do not affect the fitness of the organism, we can show that the fraction of mutations that are selectively neutral, fg, is equal to the ratio K(g)/K psi. Since advantageous mutations occur only very rarely, such that they do not contribute significantly to the rate of molecular evolution, the fraction of deleterious mutations that are subject to purifying selection is 1-fg. Therefore, the K(g)/K psi ratio can be used directly to estimate the intensity of purifying selection, thereby isolating its effects on the rate of evolution from those of mutation. We compared the selection intensities of 12 orthologous protein-coding pairs from humans and murids. As expected, the fraction of mutations that are subject to purifying selection is strongest in the second codon position and weakest in the third. Interestingly, the mean fractions of effectively neutral mutations in the third codon position were only 41% and 42% for murids and humans, respectively, indicating that many synonymous mutations are subject to selective constraint. In several orthologous genes, we found that the intensity of purifying selection is very different between murid and human orthologous genes. There was no statistically significant difference in overall intensity of purifying selection between humans and murids. Thus, purifying selection does not seem to be an important factor contributing to the observed differences in the rates of evolution between these two taxa.     

X-ray induced mutation in Syrian hamster fetal cells

Transabdominal X-rays are a risk factor for childhood leukemia, and X-ray exposure of mouse fetuses has led to increases in both mutations and initiated tumors in offspring. However, fetal sensitivity and dose-response characteristics with regard to transplacental mutagenesis by X-rays have never been quantified. In the current experiment, pregnant Syrian hamsters at day 12 of gestation were irradiated with 300-kV X-rays. Twenty-four hours later, the fetuses were removed and their cells were allowed a 5 day expression time in culture. They were then seeded for colony formation and also for mutation selection by 6-thioguanine (6-TG). Mutation frequency was linear over the entire dose range, 10-600 R. The average induced 6-TG mutant frequency was 4.7 x 10(-7) per R. These results suggest that fetal cells are highly sensitive to induction of mutations by X-rays, and that a no-effect threshold is not likely. The 10 R dose caused a 25-fold increase in mutation frequency over the historical control, 45 x 10(-7) versus 1.8 x 10(-7), an increase per R of 2.5-fold. Increased risk of childhood cancer related to obstetrical transabdominal X-ray has also been estimated at 2.5-fold per R. Thus, our results are consistent with mutation contributing to this effect.     

Mutagenesis in cultured mammalian cells. II. Induction of gene mutations in Chinese hamster cells

Mutations controlling the resistance to 6-mercaptopurine (6-M) and the ability to multiply in a medium with a low concentration of glucose (“glucose-independent” mutants) were induced in cultured Chinese hamster cells by N-nitrosomethylurea (NMU), 5-bromodeoxyuridine (BUdR), UV and X-rays. The chemical agents were found to be very active in induction of mutations to 6-M resistance (NMU and BUdR) and mutations of “glucose independence” (NMU). These agents increase the yield of mutations as compared to the spontaneous mutation rate by about two orders of magnitude. The induced rate of 6-M-resistant mutations by X-rays was 2.0 ? 10⁻⁷ per viable cell per roentgen. BUdR approximately equally increases the cell's sensitivity to both inactivating and mutagenic action of X-rays. The maximum induction of mutations to 6-M resistance by UV was observed at 100 erg/mm². This dose leads to 1 16-fold increase of the mutation frequency as compared to the spontaneous rate. Further increase of the UV dose up to 200 erg/mm² resulted in a lower yield of mutations per dose unit. The highest yield of mutations to 6-M resistance induced by NMU, BUdR and X-rays was observed if cells were plated in selective medium several generations after the mutagenic treatment. The maximum yield of mutations to 6-M resistance induced by UV and of glucose-independence induced by NMU was recorded if cells were transferred to selective media immediately after treatment. The kinetics of expression of mutations and the decline of their number observed after prolonged incubation of treated cells in nonselective conditions are discussed.     

Do rates of intercellular adhesion measure the cell affinities reflected in cell-sorting and tissue-spreading configurations?

These experiments constitute the first experimental test of the hypothesis that the rates of adhesion between cells measure the intensities of adhesion or tissue affinities that could explain cell sorting and tissue spreading. For any set of relative adhesive intensities between cells in a heterogeneous population, a corresponding minimal free energy configuration can be calculated. This is the cell distribution toward which both cell sorting and tissue spreading should lead. Equilibrium configurations were determined for combinations of 7-day embryonic retina (R) with liver (L) and heart (H), both of which became completely enveloped by R. To produce these results, the adhesive intensities would have to fall in the sequences: L-L > L-R > R-R; and H-H > H-R > R-R. To determine whether the rates of adhesion fall into these same sequences, we have devised a new technique which measures the rates of adhesion between pairs of already-formed cell aggregates of like and unlike kinds. These fall in the sequence L-L > or = H-H > L-H > R-R > H-R > L-R. If these rates paralleled the corresponding intensities of adhesion at configurational equilibrium, both L and H should have become only partially enveloped by R. Thus the rates at which adhesions are initiated do not predict the relative adhesive intensities that could explain the observed tissue configurations.     

Culture conditions and the development of the photosynthetic mechanism; influence of light intensity on photosynthetic characteristics of Chlorella

1. Chlorella pyrenoidosa has been grown in a continuous-culture apparatus under various light intensities provided by incandescent lamps, other conditions of culture being maintained constant. Light intensity curves for cells immersed in the No. 11 Warburg buffer and in Knop''s solution + 4.4 per cent CO₂ at a saturating light intensity were determined as characteristics of the photosynthetic mechanism. These characteristics were referred to the centrifuged cell volume as an index of quantity of cellular material. 2. Cells grown at intensities in the range of about 35 f.-c. develop a capacity for a high rate of photosynthesis (c.mm. O₂/hour/c.mm. cells). At culture intensities above or below this range the cells produced have a lower capacity for photosynthesis. A similar effect is observed for rate of photosynthesis per unit dry weight or rate per unit cell nitrogen. 3. The rate of photosynthesis per cell or rate per unit chlorophyll shows no maximum at any light intensity of culture but increases continuously throughout the range of light intensities studied. 4. Maximum rate of growth is attained at a light intensity of about 100 f.-c. The hypothesis is advanced that at culture intensities above that needed to give maximum rate of growth (100 f.-c.) a mechanism is developed which opposes the photosynthetic process and removes the photosynthetic products. 5. The low capacity for photosynthesis shown by cells grown at culture intensities below 35 f.-c. finds no immediate explanation. 6. The shape of the light intensity curve is markedly affected by the light intensity at which the cells have been cultured. Cells grown at lower intensities give light intensity curves approaching the Blackman type with a short transitional region between light limitation and light saturation.