Measurement of spontaneous and X-irradiation-induced 6-thioguanine-resistant human blood lymphocytes using a T-cell cloning technique

Lymphocytes separated from human peripheral blood were cultured in vitro, in the presence of 6-thioguanine (TG), to select and clone rare TG-resistant (TGr) cells present in the circulation in vivo. The incidence of such TGr cells ranged from 0.83 X 10(-5) to 2.53 X 10(-5) (mean 1.48 X 10(-5) ) in healthy individuals aged between 19 and 79 years; did not differ between males and females; but increased significantly with age at a rate of 2.4 cells/10(7) lymphocytes/year. Exposure of lymphocytes (G0) in vitro to X-ray doses of upto 200 rad resulted in a dose-dependent increase in TGr cell frequencies. The rates of increase were approximately in proportion to the square of the dose and these rates were closely similar to those obtained in cultured skin fibroblasts and suggest that the bulk of these mutations are a consequence of chromosome structural aberrations. The cloned TGr cells are considered to be HPRT- mutants and the mutation frequencies in lymphocytes determined using this cloning technique were compared with the variant frequencies obtained in earlier experiments utilising an autoradiographic technique to detect azaguanine-resistant (AGr) variant cells. Mutation frequencies with the cloning technique were 10-20-fold lower than variant frequencies with the autoradiographic method.     

Selective decontamination,induced colonization resistance and connected immunological changes in piglets

14-d-old conventional piglets were picked from normal piggery, washed with disinfectants, placed into isolators suitable for germfree work, fed a sterile diet and treated with peroral antibiotics (nalidixic acid, kanamycin, and nystatin). Beginning with day 5 or 7, Enterobacteriaceae were not found in feces. The absence of these bacteria was proved by inoculation of germfree newborn piglets with caecal content. In selectively decontaminated piglets, the white blood cell count in blood had fallen to 6 X 10(9)/L; this decrease was due to an extremely low number of granulocytes (to 0.8 X 10(9)/L). On day 35, IgG-positive cells almost disappeared from the spleen, whereas IgA cells were found in an unusually great amount. Corresponding changes in serum levels were established. The colonization resistance effect in Enterobacteriaceae-deprived piglets was confirmed; settling of introduced various E. coli strains did not occur or was delayed.     

Plating efficiency as a function of time postirradiation: evidence for the delayed expression of lethal mutations

The plating efficiency (PE) of a gamma-irradiated (7 Gy) human cell hybrid line (HeLa X skin fibroblast, designated as CGL1) has been measured as a function of time postirradiation and compared to that of unirradiated cells at similar cell densities and under the same growth conditions. The results indicate that following irradiation, the PE of the irradiated cells initially increases but never returns to that of unirradiated cells during the experimental period that we have examined. Furthermore, after a period of 9 to 10 days (equivalent to at least 10 cell doublings) postirradiation and plating, the PE of the irradiated cells begins to decrease and continues to do so over the next 5 days. A decrease does not occur in unirradiated cells until much later (i.e., Day 15) corresponding to at least 5 additional cell doublings. The data are discussed in terms of a delayed expression of lethal mutations. The possible impact of these observations on the estimation of radiation-induced transformation frequencies is also considered.     

Genetic effect of chronic gamma radiation exposure in male mice

The frequency of reciprocal translocations (RT) in mouse spermatogonia induced by gamma-rays at doses of 1.5 to 4.5 Gy and dose rates of 2.7 X 10(-6), 5.8 X 10(-6), 9.4 X 10(-5) and 4.5 Gy/min was studied. A linear increase was observed in the RT frequency with increasing the dose, at all dose rates. At 9.4 X 10(-5) Gy/min the RT frequency was, on average, 10 times lower, as compared to that for a single acute dose rate of 4.5 Gy/min. Further reduction of the dose rate did not result in a decrease of the RT yield, and at the lowest dose rate of 2.7 X 10(-6) Gy/min (the dose being 3.0 Gy) the RT frequency was higher than using the same dose at dose rates of 5.8 X 10(-6) and 9.4 X 10(-5) Gy/min. Possible reasons for an increase in the RT frequency at low dose rates are considered. A study of the frequency of abnormal sperm heads (ASH) has shown that at the dose rate of 9.4 X 10(-5) Gy/min it is independent of an accumulated dose and is equal to the value obtained when exposing to an acute dose of 3.0 Gy. At dose rates of 2.7 X 10(-6) and 5.8 X 10(-6) Gy/min ASH frequencies were only slightly increased at all doses, as compared to the control level.     

Expression of the fragile (X) chromosome in an interspecific somatic cell hybrid

Interspecific somatic cell hybrids were constructed between a Chinese hamster lung cell line deficient in hypoxanthine phosphoribosyltransferase and two lymphoblastoid cultures (GM 4025 and GM 3200) from unrelated males affected with the fragile (X) syndrome. Thirteen independent colonies survived selection in hypoxanthine-azaserine, while only one colony survived selection in hypoxanthine-aminopterin-thymidine. One hybrid formed from GM 4025 was found to contain a human X chromosome as the only detectable human chromosome in the majority of cells analyzed. Induction of fragile (X) expression in this hybrid at frequencies up to 20% was achieved by treatments with 5-fluoro-2'-deoxyuridine (5 X 10(-8) M or 1 X 10(-7) M) or methotrexate (5 X 10(-6) or 1 X 10(-5) for 12 h. Use of the somatic cell hybrid system may allow study of the fragile (X) from different patients on a homogeneous xenogeneic background and may provide a better system for characterization of the fragile (X) at the biochemical and molecular level.     

A method to quantify spontaneous and in vivo induced thioguanine-resistant mouse lymphocytes

A clonogenic assay to quantify thioguanine (TG)-resistant (TGr) spleen lymphocytes in the mouse has been developed to support studies of in vivo mutation affecting the hypoxanthine phosphoribosyltransferase (hprt) locus. Lymphocytes are cultured in 96-well microtiter plates for 9 days with proliferation initiated by the mitogen concanavalin A and supported thereafter by conditioned medium containing interleukin-2. Lymphocytes are plated at high densities (4-8 X 10(5)/well) with TG and irradiated L5178Y lymphoma cells (10(4)/well) to detect the presence of TGr cells. To determine the cloning efficiency without TG lymphocytes are plated at a low density (10/well) with irradiated L5178Y cells and irradiated lymphocytes (4-8 X 10(5)/well). Proliferation of cells is detected by [3H]thymidine incorporation and scintillation spectrometry. Spontaneous frequencies of TGr clones are independent of TG dose from 0.2 to 10 micrograms/ml and independent of cell density over the range cited. The TGr clones tested have less than 10% hypoxanthine incorporation in vivo relative to unselected clones and have stable phenotypes in the absence of selection. The spontaneous frequency of TGr cells ranged from 1 to 3 X 10(-6). In vivo treatment of mice intraperitoneally with ethylnitrosourea 15 days prior to in vitro culture resulted in a linear dose-related increase of TGr cells, with 70.2 mg/kg inducing a frequency of TGr cells of 2 X 10(-5).     

X-ray-induced mutants resistant to 8-azaguanine. I. Effects of cell density and expression time.

Asynchronous Chinese hamster ovary cells were irradiated and colony survival in Alpha MEM medium with dialyzed serum was determined with or without 15 mug/ml 8-Azaguanine (AG). Data indicated that a reproducible assay for the system was dependent upon controlling cell density at least two days prior to induction as well as throughout the expression period. Generally, spontaneous and radiation-induced mutant frequencies decreased when cell densities exceeded a critical density of 3-6 X 10(4) cells/cm2. Infrequently, the critical density was exceeded by a factor of two with no observed decrease, possibly correlated with a longer cell doubling time. Drug depletion artifacts can occur because of drug degradation, or because wild-type cells utilize the drug or produce conditions which reduce uptake of the drug. Thus, as the effective drug concentration is lowered, the observed mutant frequency increases because a spectrum of mutants resistant to only low concentrations can now survive. In fact, refeeding with AG at intervals during the incubation period lowered spontaneous and radiation-induced frequencies approx. 5-fold. Therefore, to standardize conditions, cells were trypsinized at the end of the expression time and replated at a constant cell number for mutant selection by AG. Over two generations of growth during the expression period were required for optimal manifestation of induced mutants, and when densities were kept below 4 X 10(4) cells/cm2 at all times, observed mutant frequencies did not change significantly over a period between 80 and 140 h post-induction (over 4 generations for irradiated cells and over 6 generations for controls). Previous reports of observed mutant frequencies decreasing beyond three generations may be due to cell interaction prior to mutant selection.     

Induction of mutations in males of the fish Oryzias latipes at a specific locus after gamma-irradiation

We have studied frequencies of mutations induced at the b locus of the fish, Medaka Oryzias latipes, after gamma-irradiation. Homozygotes for the b locus have colorless melanophores whose phenotypic expression can be distinguished from that of the wild type. An advantage of the use of oviparous fish for detection of skin color mutations is that the mutant phenotype can be confirmed as early as 1.5 days after fertilization because of the transparent egg membrane of the embryo. Wild-type (B/B) male fish were exposed to 4.75 or 9.5 Gy of 137Cs gamma-rays at a dose rate of 0.95 Gy/min and then mated with the female testers (b/b). A total of 77,761 F1 offspring were examined for mutation and other abnormalities. In the control, we had 1 mutant among 22,068 offspring, resulting in a mutation rate of 4.53 X 10(-5)/locus/gamete. However, this mutant embryo died before hatching. Therefore, in an attempt to present specific-locus mutation frequencies in the fish, the frequencies of color mutants that survived more than 4 days after hatching were used as frequencies of viable mutants; (number of viable color mutants)/(number of hatched fry that survived more than 4 days after hatching). In the 4.75 Gy-irradiated group the viable mutant frequencies were 45.0 X 10(-5), 69.7 X 10(-5) and 0/locus/gamete, while exposure to 9.5 Gy resulted in mutation rates of 217 X 10(-5), 130 X 10(-5) and 8.06 X 10(-5), respectively, for sperm, spermatids and spermatogonia. In comparison with viable color mutant frequencies those of the total color mutants, which include such mutants as ones that died before hatching (defined as number of total color mutants/number of fertilized eggs minus number of early deaths), were considerably higher. For sperm, spermatids, and spermatogonia after exposure to 4.75 Gy, the frequencies were 1180 X 10(-5), 629 X 10(-5) and 9.90 X 10(-5)/locus/gamete, respectively, and in 9.5-Gy-irradiated fish, the frequencies were 1940 X 10(-5), 953 X 10(-5) and 55.5 X 10(-5). Although our data are incomplete, the present results were compared with mutation induction in mice. We concluded that the frequencies of viable color mutants in the fish can be compared with those in mice.     

Conditions necessary for quantifying ethyl methanesulfonate-induced mutations to purine-analogue resistance in Chinese hamster V79 cells.

We have investigated conditions necessary to quantify the relationship between exposure to a mutagen, ethyl methanesulfonate (EMS), and the frequency of mutation induction at the hypoxanthine-guanine phosphoribosyl transferase locus in V79 cells. Maximal expression of potential mutants has been achieved by either subculturing at fewer than 5 X 10(5) cells/100-mm dish at 2-day intervals or by daily feeding of cultures. An expression period of 5 days (measure from 1 day after the initiation of treatment with the chemical mutagen) should be allowed, since at least 4 days of expression is required to reach to steady maximum of mutation frequency. It appears that there is no concentration dependence of expression time necessary to reach a plateau of mutation frequency with increasing concentrations of EMS up to 1.6 mg/ml. About 1.25 X 10(5) cells/100-mm dish or fewer should be plated for selection to avoid the loss of mutants which occurs at 1.5 X 10(5) cells/dish, presumably through cross-feeding (metabolic cooperation). The use of 6-thioguanine in hypoxanthine-free medium (supplemented with dialyzed fetal calf serum) appears to be a very stringent condition for selection. Mutation induction by EMS as a function of EMS exposure (EMS concentration X treatment time) increases linearly with concentration up to 12 h. For these treatment periods, the observed mutation frequencies for EMS are directly proportional to mutagen exposure regardless of the duration of the treatment.     

Enumeration of 6-thioguanine-resistant peripheral blood lymphocytes in man as a potential test for somatic cell mutations arising in vivo.

An autoradiographic method to enumerate variant 6-thiogunanine-resistant (TGr) peripheral blood lymphocytes (PBLs) that occur in vivo in man is described. Variant cells are detected in PBL cultures stimulated to tritiated thymidine (3HTdr) incorporation in vitro with phytohemagglutinin (PHA) in the presence of TG. Cells with the naturally-occurring Lesch--Nyhan (LN) mutation served as prototype-variant cells. PBLs from a LN hemizygous male were found to be resistant to TG inhibition of PHA-stimulated 3HTdr in corporation in vitro while a LN heterozygous female was found to be a mosaic with 2/1000 PBLs resistant to 2 X 10(-4) M TG. Experiments with artificial mixtures of LN and normal PBLs showed that the LN cells were virtually all detectable even when present in low frequency (10(-5)). TGr PBLs were found in healthy non-LN individuals at median frequencies of 1.0 X 10(-4) and 1.1 X 10(-4) when determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG respectively. Their frequencies were not age-related. TGr PBL-variant frequencies (Vf's) were determined in 47 cancer patients who were being treated with cytotoxic agents that are known to be mutagens. The median TGr PBL Vf determined at 2 X 10(-3) M TG in cancer patients was 2.2 X 10(-4) while, when determined at 2 X 10(-4) M TG, it was 8.5 X 10(-4). The distribution of Vf's for the treated cancer-patient group differed from that for the normal control group in that more than half of the treated cancer patients had TGr PBL Vf's greater than the highest seen for controls. Unlike those of the normal controls, the TGr PBL Vf's of treated cancer patients differed if determined at 2 X 10(-3) M TG and 2 X 10(-4) M TG, a behavior that suggested partial resistance and mimicked that seen with LN TGr PBLs. PBLs resistant to 2,6-diaminopurine (DAPr) were not found in two individuals, although the TGr PBL Vf was elevated in one. TGr PBL Vf's were greatly elevated under conditions of in vivo selection in patients receiving purine-analogue immunosuppressive therapy. The TGr PBL enumerative assay system is presented as one of potential value to detect somatic cell mutations occurring in vivo in man.     

Effectiveness of disinfectants in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in a biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Transformation of Xenorhabdus nematophilus

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.     

Transformation of Xenorhabdus nematophilus.

The ability of Xenorhabdus nematophilus 19061/1 to be transformed by pHK17 plasmid DNA was studied and optimized. A number of factors, including culture conditions, stage of growth, transformation buffer pH, cation type and concentration required for the production of competency, washing, heat shock conditions, and cell-DNA ratio, were found to affect transformation significantly. On the basis of these observations, a procedure for the routine transformation of X. nematophilus 19061/1 at frequencies of 1 X 10(5) to 10 X 10(5) transformants per microgram of pHK17 plasmid DNA was developed. Maximum transformation was obtained when cells which had reached the mid- to late-logarithmic growth phase (total counts, 2.5 X 10(8) to 5 X 10(8) cells per ml) within 4.5 to 5.5 h were washed once in cold transformation buffer before they were suspended in the same buffer to 0.1 of their original volume. The highest transformation was obtained when dimethyl sulfoxide was added in two steps to the cells immediately before the DNA was added, after which the cell-DNA mixtures were incubated for 30 min on ice before they were given a 3-min heat shock at 37 degrees C. Following these treatments, the transformed cells were incubated in L broth-60 mM CaCl2 for 1 h before they were plated onto selective medium. We also were able to transform X. nematophilus 19061/1 with plasmid pBR325, and we transformed other species of Xenorhabdus with several common plasmids.     

Mutagenicity of lead chromate in Drosophila melanogaster in the presence of nitrilotriacetic acid (NTA)

By using the sex-linked recessive lethal mutation test in Drosophila melanogaster (standard Basc scheme) we analysed the mutagenic effects of treatments by feeding with nitrilotriacetic acid (NTA: 5 X 10(-2) M), with the insoluble Cr(VI) compound lead chromate, PbCrO4 (supernatant of 4.6 X 10(-4)-M suspension in which the actual concentration was 0.06 gamma/ml as Cr(VI)) and with both compounds preincubated at 3 relative ratios (NTA: 5 X 10(-2) M; PbCrO4: 4.6 X 10(-4), 4.6 X 10(-5) and 9.2 X 10(-6) M, respectively). The estimation of mutation frequencies was done at different developmental stages of the germ cells, namely spermatozoa, spermatids and spermatocytes. Ethyl methanesulphonate (EMS: 5 X 10(-3) M) was used as the reference positive control, with clearly mutagenic results. Treatments with NTA or with PbCrO4 alone did not induce any significant increase of the mutation frequency. PbCrO4 at the 3 concentrations tested was completely soluble in the 5 X 10(-2)-M NTA solution, and the mixture of NTA and PbCrO4 induced significant increases of the frequency of sex-linked lethal mutations, with a significant dose-effect relationship with respect to PbCrO4, apparently as a result of the interaction of the compounds and subsequent release of the genotoxic heavy-metal Cr(VI) ions. This result indicates an important synergistic action of NTA with PbCrO4 under the conditions described.     

The effect of divalent metal cations on the inhibition of human coagulation factor Xa by plasma proteinase inhibitors

The inactivation of human coagulation factor Xa by the plasma proteinase inhibitors alpha 1-antitrypsin, antithrombin III and alpha 2-macroglobulin in purified systems was found to be accelerated by the divalent cations Ca2+, Mn2+ and Mg2+. The rate constant for the inhibition of factor Xa by antithrombin III rose from 2.62 X 10(4) M-1 X min-1 in the absence of divalent cations to a maximum of 6.40 X 10(4) M-1 X min-1 at 5 mM Ca2+, 8.10 X 10(4) M-1 X min-1 at 5 mM Mn2+, with a slight decrease in rate at higher cation concentrations. Mg2+ caused a gradual rise in rate constant to 5.65 X 10(4) M-1 X min-1 at 20 mM. The rate constant for the inhibition of factor Xa by alpha 1-antitrypsin in the absence of divalent cations was 5.80 X 10(3) M-1 X min-1. Ca2+ increased the rate to 1.50 X 10(4) M-1 X min-1 at 5 mM and Mn2+ to 2.40 X 10(4) M-1 X min-1 at 6 mM. The rate constant for these cations again decreased at higher concentrations. Mg2+ caused a gradual rise in rate constant to 1.08 X 10(4) M-1 X min-1 at 10 mM. The rate constant for the factor Xa-alpha 2-macroglobulin reaction was raised from 6.70 X 10(3) M-1 X min-1 in the absence of divalent cations to a maximum of 4.15 X 10(4) M-1 X min-1 at 4 mM Ca2+, with a decrease to 3.05 X 10(4) M-1 at 10 mM. These increases in reaction rate were correlated to the binding of divalent cations to factor Xa by studying changes in the intrinsic fluorescence and dimerization of factor Xa. The changes in fluorescence suggested a conformational change in factor Xa which may be responsible for the increased rate of reaction, whilst the decrease in rate constant at higher concentrations of Ca2+ and Mn2+ may be due to factor Xa dimerization.     

Effect of SOS repair in enterobacteria on their interaction with the complement system

The interaction of E. coli (serovar 0124) and its rec A-mutants with serum complement resulting in the alternative pathway activation was studied. Bacteria VT1240 (original smooth strain), VT1241 (rough mutant) and VT 2240 (recA56 mutant) were shown to be complement-sensitive when treated with 1.5 X 10(8)--1.9 X 10(8) cells per ml of normal human serum, while the cells with SOS-activated system (recA441 mutant, strain VT3251) retained their viability. An alternative pathway of complement activation was minimal with E. coli VT1241, while VT3251 demonstrated intermediate activity. To decrease the level of complement components (AH50) and factor B (BH50) by 50%, 3.5 X 10(6)--4.5 X 10(6) cells of VT1240 and VT2240 strains were required. R-mutants and recA441 mutants caused a 50% reduction in AH50, when used in the amount of 6.4 X 10(7) and 2.6 X 10(7), respectively, the same degree of BH50 decrease was achieved with the amounts used equal to 1.1 X 10(8) and 4.3 X 10(6), respectively. C3 conversions caused by 4 X 10(8) cells in I ml of the normal human serum in the four strains tested accounted for 5-15%.     

Mutagenic properties of the 8-amino-2'-deoxyguanosine DNA adduct in mammalian cells.

The DNA adduct 8-amino-2'-deoxyguanosine (8-amino-dG) is found in liver DNA of rats treated with the hepatocarcinogen 2-nitropropane. Site-specifically modified oligodeoxynucleotides were used to explore the mutagenic potential of 8-amino-dG in simian kidney (COS-7) cells. Oligodeoxynucleotides (5'-TCCTCCTX1G2CCTCTC and 5'-TCCTCCTG1X2CCTCTC, X = dG or 8-amino-dG) with the lesion positioned at codon 60 or 61 of the non-coding strand of the human c-Ha- ras1 gene were inserted into single-stranded phagemid vectors and transfected into COS-7 cells. The progeny plasmid obtained was used to transform Escherichia coli DH10B. Transformants were analyzed by oligodeoxynucleotide hybridization and DNA sequencing to establish the mutation frequency and spectrum produced by the modified base. The correct base, dCMP, was incorporated preferentially opposite 8-amino-dG at X1and X2. When 8-amino-dG was at X1, targeted GNH2-->T transversions were detected, along with smaller numbers of GNH2-->A transitions and GNH2-->C transversions. When the adduct was at X2, only GNH2-->T transversions were observed. The frequencies of targeted mutation at X1and X2were 2.7 and 1.7%, respectively. Mutation frequency and mutagenic spectrum were sequence context dependent. In addition, non-targeted G-->T transversions, accompanied by some G-->A transitions, were detected 5' to 8-amino-dG when the lesion was at X2. We conclude that 8-amino-dG is a mutagenic lesion, generating G-->T and G-->C transversions and G-->A transitions in mammalian cells.     

Unstable Chromosome Aberrations Do Not Accumulate in Normal Human Fibroblast after Fractionated X-Irradiation

We determined the frequencies of dicentric chromosomes per cell in non-dividing confluent normal human fibroblasts (MRC-5) irradiated with a single 1 Gy dose or a fractionated 1 Gy dose (10X0.1 Gy, 5X0.2 Gy, and 2X0.5 Gy). The interval between fractions was between 1 min to 1440 min. After the completion of X-irradiation, the cells were incubated for 24 hours before re-plating at a low density. Then, demecolcine was administrated at 6 hours, and the first mitotic cells were collected for 42 hours. Our study demonstrated that frequencies of dicentric chromosomes in cells irradiated with a 1 Gy dose at different fractions were significantly reduced if the fraction interval was increased from 1 min to 5 min (p<0.05, χ²-test). Further increasing the fraction interval from 5 up to 1440 min did not significantly affect the frequency of dicentric chromosomes. Since misrejoining of two independent chromosome breaks introduced in close proximity gives rise to dicentric chromosome, our results indicated that such circumstances might be quite infrequent in cells exposed to fractionated X-irradiation with prolonged fraction intervals. Our findings should contribute to improve current estimation of cancer risk from chronic low-dose-rate exposure, or intermittent exposure of low-dose radiation by medical exposure.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain.

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.     

Natural plasmid transformation in a high-frequency-of-transformation marine Vibrio strain

The estuarine bacterium Vibrio strain DI-9 has been shown to be naturally transformable with both broad host range plasmid multimers and homologous chromosomal DNA at average frequencies of 3.5 X 10(-9) and 3.4 X 10(-7) transformants per recipient, respectively. Growth of plasmid transformants in nonselective medium resulted in cured strains that transformed 6 to 42, 857 times more frequently than the parental strain, depending on the type of transforming DNA. These high-frequency-of-transformation (HfT) strains were transformed at frequencies ranging from 1.1 X 10(-8) to 1.3 X 10(-4) transformants per recipient with plasmid DNA and at an average frequency of 8.3 X 10(-5) transformants per recipient with homologous chromosomal DNA. The highest transformation frequencies were observed by using multimers of an R1162 derivative carrying the transposon Tn5 (pQSR50). Probing of total DNA preparations from one of the cured strains demonstrated that no plasmid DNA remained in the cured strains which may have provided homology to the transforming DNA. All transformants and cured strains could be differentiated from the parental strains by colony morphology. DNA binding studies indicated that late-log-phase HfT strains bound [3H]bacteriophage lambda DNA 2.1 times more rapidly than the parental strain. These results suggest that the original plasmid transformation event of strain DI-9 was the result of uptake and expression of plasmid DNA by a competent mutant (HfT strain). Additionally, it was found that a strain of Vibrio parahaemolyticus, USFS 3420, could be naturally transformed with plasmid DNA. Natural plasmid transformation by high-transforming mutants may be a means of plasmid acquisition by natural aquatic bacterial populations.