In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes

The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes.     

Intercorrelations and sources of variability in three mutagenicity assays: a population-based study

The purpose of this study was to evaluate the intercorrelation between three genetic assays in 112 subjects. The group was pooled from two originally separate but homogeneous subgroups of 56 persons each. Procedures included assays for hprt mutant frequencies, micronuclei in human lymphocytes, and mutations at the glycophorin A (gpa) loci. We found no statistically significant or biologically important intercorrelations among the three biomarkers. We did, however, observe significant correlations between log_e hprt mutant frequency and cloning efficiency (inverse correlation for these 2 variables), age and log_e hprt mutant frequency, an inverse relationship between cloning efficiency and age, and an important differential sex effect favoring a greater micronuclei frequency in females than males. No significant correlations between the covariates of interest and glycophorin A variant frequencies NN or NO were observed. Using multivariable linear regression, age was found to account for the majority of the variability in hprt mutant frequency (greater than sex and/or smoking); for micronuclei data, only sex contributed a statistically significant and biologically important proportion to the total variation. We conclude that despite observing no significant intercorrelations between the three assays performed simultaneously from the same individuals in a large population database, a significant correlation between age and hprt mutant frequency and an inverse association between cloning efficiency and hprt do exist; furthermore, we verified the strong differential sex-specific effect on micronucleus frequencies.     

Genetic instability in human T-lymphocytes

Mutations arising in vivo in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of T-lymphocytes provide a measure of mutation induction in human somatic cells. Studies of measured background HPRT mutant frequency (MF) values show wide inter-individual variation. At the extremes are individual with ‘outlier’ MF values, i.e., non-exposed individuals with MF > 100 × 10⁻⁶ [Robinson et al., Mutation Res. 313 (1994) 227–247.]. The elevated HPRT MF in one well-studied outlier is due to the in vivo expansion of mutant cells possessing an identical T-cell receptor (TCR) gene rearrangement pattern. We report here that this in vivo expanding TCR clone shows multiple different HPRT mutations and thus possesses a mutator phenotype. Other individuals with T-cell mutator phenotypes have been found, suggesting that this phenomenon may contribute to the extremes of variation in HPRT MFs in the human population.     

Low persistence of the induced mutant phenotype in Chinese hamster cells

We have analysed the recovery of individual CHO-derived mutants during the generations immediately following their induction. This characteristic, which we call persistence, was measured by propagating mutagenized cultures in non-selective medium after subdivision into many very small populations, each containing either zero or one mutant. The recovery of most hypoxanthine phosphoribosyltransferase (hprt)-deficient mutants induced by ethyl methanesulphonate was low, and we have previously shown that this was usually due to an apparent rapid loss of the mutant phenotype with continued culture in non-selective medium (Bradley, 1980). A minority of about 15% manifest high persistence. We now show that most adenine phosphoribosyltransferase (aprt)-deficient mutants and some ouabain-resistant mutants had low persistence. Mutants induced by UV irradiation also generally exhibited low persistence but those induced by X-irradiation had significantly higher persistence than what was seen among EMS-induced mutants. Among various sublines of CHO cells which were tested for persistence of induced mutants, only one group consistently yielded mutants of high persistence. These were lines which carried glucose-6-phosphate dehydrogenase mutations which themselves had been originally induced by EMS.     

Shuttle vector system for the analysis of mutational events in mammalian chromosomal DNA

cDNA of the human hprt gene was introduced into the BamHI cloning site of the retroviral shuttle vector pZipNeoSV(X)1. The mouse cell line 2TGOR, a hypoxanthine phosphoribosyltransferase-deficient derivative of Balb/c 3T3, was tranformed with the vector and some stably transformed HAT^rNEO^r clones were established. One of the clones, VH-12, contained a single copy of the vector integrated stably into a chromosome in a proviral form. From this clone, we were able to recover efficiently the vector sequence preserving its intact strucure by use of COS cell fusion. The relatively small size of the hprt cDNA (657 base pairs for the coding region) allowed quick determination of the entire DNA sequence. It was also notable that use of 6TG NEO double selection for mutant isolation could eliminate the 6TG^r derivatives of VH-12 cells which arose from loss of the total vector sequence or from some epigenetic event, because such alterations would lead to inactivation of the neo gene as well as the hprt cDNA. The properties of our shuttle vector system were particularly useful for analysis of the molecular mechanisms of mutational events in chromosomal DNA of mammalian cells.     

High temperature-induced changes in exopolysaccharides, lipopolysaccharides and protein profile of heat-resistant mutants of Rhizobium sp. (Cajanus)

A thermosensitive wild-type strain (PP201) of Rhizobium sp. (Cajanus) and its 14 heat-resistant mutants were characterized biochemically with regard to their cell surface (exopolysaccharides (EPSs) and lipopolysaccharides (LPSs)) properties and protein profile. Differences were observed between the parent strain and the mutants in all these parameters under high temperature conditions. At normal temperature (30 °C), only half of the mutant strains produced higher amounts of EPSs than the parent strain, but at 43 °C, all the mutants produced higher quantities of EPS. The LPS electrophoretic pattern of the parent strain PP201 and the heat-resistant mutants was almost identical at 30 °C. At 43 °C, the parent strain did not produce LPS but the mutants produced both kinds of LPSs. The protein electrophoretic pattern showed that the parent strain PP201 formed very few proteins at high temperature, whereas the mutants formed additional new proteins. A heat shock protein (Hsp) of 63–74 kDa was overproduced in all mutant strains.     

Influence of sex, smoking and age on human hprt mutation frequencies and spectra.

Examination of the literature for hprt mutant frequencies from peripheral T cells yielded data from 1194 human subjects. Relationships between mutant frequency, age, sex, and smoking were examined, and the kinetics were described. Mutant frequency increases rapidly with age until about age 15. Afterward, the rate of increase falls such that after age 53, the hprt mutant frequency is largely stabilized. Sex had no effect on mutant frequency. Cigarette smoking increased mean mutant frequency compared to nonsmokers, but did not alter age vs. mutant frequency relationships. An hprt in vivo mutant database containing 795 human hprt mutants from 342 individuals was prepared. No difference in mutational spectra was observed comparing smokers to nonsmokers, confirming previous reports. Sex affected the frequency of deletions (>1 bp) that are recovered more than twice as frequently in females (P = 0. 008) compared to males. There is no indication of a significant shift in mutational spectra with age for individuals older than 19 yr, with the exception of A:T --> C:G transversions. These events are recovered more frequently in older individuals.     

Analysis of mutational effects at the HPRT locus in human G(0) phase lymphocytes irradiated in vitro with gamma rays

The mutational effects of ionising radiation at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus were studied in human peripheral blood G(0) phase lymphocytes irradiated in vitro with gamma rays. The presence of radiation induced mutants was assessed by selecting the HPRT mutants every week on the basis of 6-thioguanine resistance up to 1 month after irradiation. A dose-related increase of 14.25x10(-6) mutants/Gy was measured after an expression time of 7 days. After 2 weeks from culture starting the fraction of clonable cells in irradiated and control cell populations decreased, limiting the measurements of mutant frequency. The mutational spectrum of the HPRT gene was determined by PCR analyses in a total of 99 mutant clones derived from irradiated lymphocytes. The independent origin of mutant clones carrying the same mutation was assessed by analysing the TCR gamma gene rearrangements. The results showed a dose-related increase of deletion mutants up to 3Gy, whereas point mutation frequency increased only up to 2Gy. Two preferentially deleted regions were identified; one involving the HPRT exon 3, and another one the 3'-terminal and the 3'-flanking region of the gene. One complex mutation involving a non-contiguous deletion of exons 2-5 and 7/8 was observed among the mutants isolated after 3Gy irradiation.     

Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes

T-cell receptor (Ti) gene restriction fragment patterns (RFPs) were determined by Southern blots of genomic DNA obtained from T-lymphocyte colonies isolated from a single normal individual. 4 wild-type colonies and 11 in vivo derived 6-thioguanine-resistant mutant colonies with previously characterized hprt gene structural alterations were studied. Among the hprt mutants, 10 of the 11 showed unique Ti RFPs indicating their origins in different in vivo progenitors. Unique Ti RFPs were also seen among the wild-type T-cell colonies. One, however, shared its Ti RFP with a mutant. These results suggest that mutation in vivo of the hprt gene in human T-lymphocytes occurred after thymic maturation and that the 11 recovered hprt mutants probably resulted from 11 independent mutational events.     

The genotoxic effect of carcinogenic PAHs, their artificial and environmental mixtures (EOM) on human diploid lung fibroblasts

Long-term exposure to synthetic and endogenous estrogens has been associated with the development of cancer in several tissues. One potential mechanism of estrogen carcinogenesis involves catechol formation and these catechols are further oxidized to electrophilic/redox active o-quinones, which have the potential to both initiate and promote the carcinogenic process. Previously we showed that 4-hydroxyequilenin (4-OHEN) autoxidized to an o-quinone and caused a variety of damage to DNA. Since these deleterious effects could contribute to gene mutations, we investigated the Chinese hamster V79 cells to ascertain the relative ability of estradiol, 4-hydroxyestradiol, 17β-hydroxyequilenin, 4,17β-hydroxyequilenin, estrone, 4-hydroxyestrone, equilenin, and 4-hydroxyequilenin to induce the mutation of the hypoxanthine–guanine phosphoribosyltransferase (hprt) gene. All the 4-hydroxylated catechols induced significantly more colony formations in V79 cells as compared to the parent phenols at 100 nM, suggesting that the catechol estrogen metabolites are more mutagenic towards the hprt gene than estrogens. Since 4-OHEN induced the highest mutation frequency, we examined a biomarker for transformation potential of this compound in MCF-10A cells using an anchorage-independent growth assay. Although 4-OHEN induced anchorage-independent growth of these cells, the isolated clones were not able to grow as tumors in vivo when injected into nude mice. These cells were assayed for genetic changes using cDNA microarrays. Real time RT-PCR confirmation of some of the differentially expressed genes showed down-regulation of metallothionein 2A, p53, BRCA1, and c-myc. Moreover, we showed the involvement of other genes important in cell transformation and oxidative stress, strengthening the hypothesis that this mechanism plays a considerable role in 4-OHEN-induced anchorage-independent growth.     

Mutatect: a mouse tumour model for detecting radiation-induced mutations in vivo

A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosphoribosyltransferase) locus is described. It is highly sensitive to detection of mutants induced by clastogenic agents such as ionizing radiation. MN-11 cells are grown as a subcutaneous tumour in C57BL/6 mice for a period of 2 weeks, during which time they can be exposed to mutagenic treatments. Cells taken from the animal are cultured ex vivo and 6-thioguanine (6-TG)-resistant mutant clones can be readily identified and scored. This model system may have special utility for detecting multi-locus deletion events (chromosomal mutations) induced by high LET forms of radiation that might be encountered in space.     

尖孢镰刀菌亚麻专化型Folprp4基因参与调控菌丝生长和分生孢子发生

目的: 通过对尖孢镰刀菌中Folprp4基因的鉴定,揭示其在尖孢镰刀菌中的功能及致病相关性。方法: 基于同源重组原理,根据测定出的Folprp4基因序列,应用Split-Marker重组技术构建含有潮霉素抗性基因(hph)的基因缺失盒。将基因缺失盒经PEG介导转化到野生型原生质体中,在含有潮霉素B的TCC培养基上筛选转化子,通过PCR正负筛查获得Folprp4基因缺失突变株(ΔFolprp4)。构建含有Folprp4基因的载体pZDH1,并将其转化到敲除突变体中进行互补测验。结果: 与野生型(hm)和异位插入突变体(ecFolprp4)相比,敲除突变体菌丝生长受到严重阻碍,当野生型和异位插入突变体长满整个平板时,敲除突变体菌落呈小点状。敲除突变体的另一个显著变化是ΔFolprp4的分生孢子产量显著下降。侵染实验表明,ΔFolprp4对亚麻幼苗的毒力显著降低。互补实验表明,该互补载体的回复子(Folprp4-C)在菌落形态、生长速率、分生孢子产量和毒力方面均恢复到了野生型菌株。结论: Folprp4基因与尖孢镰刀菌的菌丝生长、分生孢子发生和致病性有关。     

Molecular analysis of spontaneous and ethyl methanesulphonate-induced mutations of the hprt gene in hamster cells

Independent spontaneous or ethyl methanesulphonate (EMS)-induced mutants lacking HPRT enzyme activity were analysed for changes in hprt gene structure. Of 21 spontaneous mutants, 6 had total gene deletions, 2 had partial gene deletions, and 13 were indistinguishable from wild-type by Southern analysis. In contrast a sample of 23 EMS-induced mutants, each of which showed potentially interesting characteristics (e.g. high reversion frequency, X-chromosome rearrangement), showed no detectable hprt gene changes. RNA isolated from 59 mutants with presumptive point mutations (13 spontaneous, 46 EMS-induced) was analysed on dot blots for changes in the amount of hprt mRNA. A wide range of mRNA levels was found, from mutants with undetectable amounts to those with more than wild-type amounts. However, Northern blots of all these mutant RNAs revealed only one (EMS-induced) mutation with a change in hprt mRNA size. Taken with our previously-published data on these mutants, it is argued that they represent a broad range of mutational types, and that the hprt gene mutation system provides a sensitive means of distinguishing mutational spectra of different DNA-damaging agents.     

In vivo ionizing irradiations produce deletions in the hprt gene of human T-lymphocytes

The hprt T-lymphocyte cloning assay, which detects mutations occurring in vivo in humans, has been used to examine mutants induced in patients receiving radioimmunoglobulin therapy (RIT) for cancer. Samples from 13 patients before treatment (controls) and 15 samples from 12 patients after treatment were studied for both mutant frequencies and molecular changes in the hprt mutant T-cell clones. Patients were studied up to 48 months after treatment. Post-RIT patients showed increased mutant frequencies as compared to pre-treatment values. T-cell receptor (TCR) gene analysis of mutant T-cell clones demonstrated that 84% arose independently, both pre- and post-treatment, which is the same proportion as seen in normal individuals. However, several individuals did show large sets of mutants with the same TCR gene rearrangement patterns. Molecular analysis of mutants demonstrated a greater proportion of mutations with hprt gene changes on Southern blots after RIT treatment than before (40% versus 20%). RIT increases the proportion of mutations with total rather than partial gene deletions or other gross structural changes compared to normal individuals or pre-treatment patients. These studies are defining the spectrum for radiation-induced hprt gene mutations in vivo in human T-lymphocytes.     

Deletion mutants from R plasmids with increased copy number.

Rms 201-12 and Rms 201-46 are R mutants with increased copy number, and are derived from a conjugative plasmid Rms 201 that encodes resistance to five drugs, ampicillin (Apc), tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), and sulfonamides (Sa). The mutants expressed increased levels of resistance to Apc, Sm, and Cm, and a decreased level of resistance to Tc than those of the parent Rms 201 plasmid. When the Rms 201-12+ or Rms 201-46+ cells were inoculated onto plates containing a high concentration of Tc, colonies developed on the plate at a frequency of 10-3 to 10-4 after overnight incubation. The cells grown on the Tc plate carried a tet (Tc gene)-deleted R mutant besides tet-possessing Rms 201-12 or Rms 201-46, and we isolated the tet-deleted R mutant by purifying the R+ cells on drug-free plates. On the other hand, various deletion mutants possessing tet were isolated by prolonged culture of the cells. We have presented a circular gene order of Rms 201 by comparing the genetic markers of all deletion mutants derived from Rms 201, Rms 201-46, and Rms 201-12. The gene(s) regulating the copy number was closely linked to the rep gene. The gene(s) specifying entry exclusion was jointly lost with the tra region.     

In vivo hprt mutant frequencies in T-cells of normal human newborns

Mutation at the hypoxanthine-guanine phosphoribosyl transferase locus (hprt; HPRT enzyme) in the human fetus was studied by clonal assay of placental cord blood samples from full-term newborns. Conditions for determining hprt mutant frequencies, as defined for adults, were also optimal for studies in newborns. The mean mutant frequency for 45 normal human newborns (37 male, 8 female) was 0.64 X 10(-6) (SD = 0.41 X 10(-6); median value = 0.58 X 10(-6). These values are approx. 10-fold lower than corresponding adult hprt mutant frequency values. Factors such as limiting-dilution cloning efficiencies, delay prior to study of sample, sex, cryopreservation or technician performing the assay did not significantly affect assay results. Maternal smoking did not result in elevated mutant frequency values. Most wild-type and mutant clones studied were CD4 surface antigen positive (helper/inducer). All hprt mutants analyzed lacked HPRT activity.     

Efficient insertional mutagenesis in group A streptococci mediated by Tn917 transposon

The replication-conditional thermosensitive vector pTV1-OK (repATs, Kan^r) harbouring the transposon Tn917 (Em^r) was successfully used to mutagenise a clinical Streptococcus pyogenes isolate (CS101). In the initial studies, conditions were established for electrotransformation of the pTV1-OK vector into CS101. Transformants selected on media containing Kan at 29°C were shown to become Kan^s at 39°C and to carry the transposon-linked Em^r marker. One such transformant was chosen for transposition studies. Upon temperature shift, transposition was achieved with a frequency of approximately 0.01% with a plasmid curing efficiency of close to 100%. Southern blot analysis demonstrated that the majority of mutants contained a single copy of Tn917 and showed no evidence for preferential sites of integration (“hot spots”). Screening of Tn917 libraries of S. pyogenes has led to the identification of mutants lacking either haemolysing or plasminogen activating activity. Mutants defective in each of these activities were identified at a frequency of approximately one in 1000 to 4000 colonies. These findings suggest that the pTV1-OK vector can be used for transposon mutagenesis of S. pyogenes and that this strategy will be valuable for identifying virulence factors and regulatory mechanisms in these bacteria.     

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of an inhibitor and a mimic of superoxide dismutase on bleomycin mutagenesis in Chinese hamster ovary cells

We have investigated the roles of reactive oxygen species (ROS) in bleomycin (BLM)-induced gene mutations in Chinese hamster ovary (CHO) cells using a superoxide dismutase (SOD) inhibitor, triethylenetetramine (TRIEN), and a SOD mimic, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), to lower and increase intracellular “SOD activity”, respectively. Pretreatment of CHO cells with TRIEN (1 mM) for 1 h enhanced the mutagenic response of BLM (5–50 μg/ml, 1 h treatment) in the hypoxanthine-guanine phosphoribosyltransferase (hprt) locus in CHO cell clone K1-BH4 (CHO/HPRT assay) and the xanthine-guanine phosphoribosyltransferase (gpt) gene in a CHO-K1 cell derivative AS52 (AS52/GPT assay). Pretreatment with TEMPOL (1 mM) for 1 h decreased the BLM (20–100 μg/ml, 1 h treatment) mutagenicity in the AS52/GPT assay. The mutagenic response of BLM appears to be modulated by the intracellular level of ‘SOD activity’ and hence the intracellular level of ROS. These data provide further evidence for the involvement of ROS in bleomycin mutagenesis in mammalian cells.