Restriction Site Mutation Assay Research Articles

Identification of Early p53 Mutations in Clam Ileocystoplasties Using Restriction Site Mutation Assay

Because a risk of cancer arising in enterocystoplasties exists, it is necessary to identify which patients are most at risk of tumor formation. The aim of this study was to determine whether rare mutated p53 sequences were more common at the enterovesical anastomosis than in the bladder remnant in patients with a clam ileocystoplasty using the restriction site mutation (RSM) assay. DNA was extracted from endoscopic biopsies obtained from the ileovesical anastomosis and native bladder remnant (control specimens) of 38 patients with a clam ileocystoplasty. The RSM assay was used to study five known hotspots for mutations of the p53 gene using the restriction enzymes Hha I (codon 175), Taq I (codon 213), Hae III (codon 249/250), and Msp I (codons 248 and 282). The mutational events of p53 were confirmed by sequencing the undigested mutated polymerase chain reaction products identified by RSM analysis. We found p53 mutations at the ileovesical anastomosis in 7 of the 38 patients. The mutations were observed at codon 213 (n = 1), codon 248 (n = 3), and codon 250 (n = 3). No p53 mutations were detected in any control specimen. The ileovesical anastomosis is genetically unstable in patients with a clam ileocystoplasty. The p53 mutations identified by the RSM assay at the enterovesical anastomosis could possibly be used as markers of genetic instability to identify patients at risk of developing a tumor. Prospective, randomized longitudinal studies are required to substantiate this hypothesis.

POS-01.57: Sperm chromosomal abnormalities and DNA damage in infertile mustard gas casualties’ men detected by fluorescence in-situ hybridization, inverse restriction site mutation assay, sperm chromatin structure assay and the comet assay

POS-01.57: Sperm chromosomal abnormalities and DNA damage in infertile mustard gas casualties’ men detected by fluorescence in-situ hybridization, inverse restriction site mutation assay, sperm chromatin structure assay and the comet assay

Genetic damage in oligozoospermic patients detected by fluorescence in-situ hybridization, inverse restriction site mutation assay, sperm chromatin structure assay and the Comet assay.

The possibility that oligozoospermic men may have elevated levels of genetic damage in their sperm is of particular concern as they could transmit defects to their offspring. Sperm samples were obtained from 12 infertile, oligozoospermic patients and 12 healthy normozoospermic volunteers. Fluorescence in-situ hybridization (FISH) was used to determine aneuploidy rates in sperm and inverse restriction site mutation (iRSM) assay to determine gene mutations; defective chromatin packaging was quantified by sperm chromatin structure assay (SCSA) and DNA strand breaks by the Comet assay. FISH analysis showed a significant increase in gonosomal X,Y,18 (P < 0.01) disomy and diploid sperm with X,Y,18,18 (P < 0.05) in the infertility patients compared with the controls. A significant increase (P < 0.01) in disturbed sperm chromatin was found in the infertility patients compared with the control group using the SCSA assay. In the Comet assay, a significant increase (P < 0.01) in the tail moment was found in the infertility patients compared with the control group, indicating significantly high levels of DNA strand breaks. There was no significant increase in point mutations detected by iRSM assay. The data indicate that infertile oligozoospermic men have an elevated level of XY aneuploidy and XY diploidy in the germ-line, as well as elevated levels of sperm chromatin disturbances and sperm DNA strand breaks. These data demonstrate that oligozoospermic infertility patients show several different types of genetic damage in their sperm. Thus, such men appear to have defects at a variety of levels of spermatogenesis and their infertility may not just be a result of the oligozoospermia.

The detection of mutations induced in vitro in the human p53 gene by hydrogen peroxide with the restriction site mutation (RSM) assay

We have analysed five mutation hotspots within the p53 gene (codons 175, 213, 248, 249, and 282) for mutations induced by hydrogen peroxide (H 2O 2), employing the restriction site mutation (RSM) assay. In addition, four other restriction sites covering non-hotspot codons of exons 5–9 of the p53 gene (codons 126, 153/54, 189 and the 3′ splice site of exon 9) were analysed by the RSM assay for H 2O 2-induced mutations. Two cell types were concurrently analysed in this study, i.e. primary fibroblast cells and a gastric cancer cell line. Using the RSM assay, H 2O 2-induced mutations were only detected in exon 7 of the p53 gene. This was true for both cell types. These mutations were mainly induced in the Msp I restriction site (codon 247/248) and were predominantly GC to AT transitions (71%). Hence these GC to AT mutations were presumably due to H 2O 2 exposure, possibly implicating the 5OHdC adduct, which is known to induce C to T mutations upon misreplication. Importantly, this study demonstrates that the RSM methodology is capable of detecting rare oxidative mutations within the hotspot codons of the p53 tumour suppressor gene. Hence, this methodology may allow the detection of early p53 mutations in pre-malignant tissues.

Extraction of DNA from oral cytological samples by scraping and smear method suitable for restriction site mutation analysis: a pilot study.

The restriction site mutation assay (RSM) can be used to measure base changes which occur in the DNA coding for bacterial restriction enzymes. The aim of this study was to investigate whether DNA, of sufficient quantity and quality for analysis by RSM, could be extracted from cells collected from precancerous lesions using a cytological smear technique. Six smears were collected from each of five lesions of leukoplakia displaying a variety of clinical appearances. Three methods for the extraction of DNA were compared. The commercial extraction method was shown to be most convenient and reproducible, routinely providing 1-5 microg of DNA per sample. Cell populations collected by a cytological smear technique can provide DNA of sufficient quantity and quality for analysis of RSM.

The restriction site mutation assay: a review of the methodology development and the current status of the technique.

The restriction site mutation (RSM) assay has been employed in our laboratory, as a mutation detection system, since its first description in 1990. In principle the technique is capable of detecting mutations in ubiquitous restriction enzyme sites and is, therefore, readily applicable to any sequenced gene and/or organism. The RSM assay has been applied in our laboratory in various species, detecting rare mutations induced in mouse, rat, Xenopus, flatfish and human cells and tissues. This paper reviews the data accumulated by the RSM methodology in our hands and charts the developmental processes which have steadily improved the technique such that it is now applicable as a sensitive genotypic mutation detection system. This paper also includes PCR primer sequences and restriction enzymes employed in mutational analyses performed in the various species studied. We detail a variety of problems associated with the assay and the steps taken to solve them. The specific hurdles which have been overcome include the lack of quantitative data, the question of the contribution of DNA adducts to the induced mutation profile and the presence of false positives. Finally, the methods which have been developed to increase the sensitivity of the assay are also detailed. This paper describes our recommended RSM methodology, as it is routinely employed in our laboratory, which enables the analyses of mutations induced by chemical exposures and spontaneous endogenous processes. Our aim in presenting the developmental data on the RSM assay is to provide other researchers with sufficient information about the RSM methodology to facilitate its application in mutation analysis in other genes and organisms.

A study of ENU-induced mutagenesis in the mouse using the restriction site mutation (RSM) assay

We report here the application of the restriction site mutation (RSM) assay to study the induction of mutations by the alkylating agent ENU. Specifically, mutations were sought in the spleen and bone marrow of mice 3, 10, and 100 days after being treated with ENU; this was compared to data previously published from our laboratory on ENU-induced testes mutations. It was found that the ENU-induced mutations were all at GC bases implicating the O(6)-ethylguanosine adduct. The mutations detected reached a peak at day 10 in the spleen and were detectable to a lesser extent at 100 days, which is similar to the testes data. In the bone marrow, the mutation level rose until day 100, although the level remained below that of the spleen and testes. However, by studying the mutations detected in control animals, it was found that spontaneous mutational events were detectable at the day 100 time point in all three tissues. Hence the spleen, testes, and bone marrow mutations at day 100 in the ENU-treated samples were probably spontaneous mutational events with very few genuine ENU-induced mutations remaining in any of these tissues after 100 days. This paper also demonstrates the applicability of the inverse RSM methodology in the detection of ENU-induced mutations, whereby mutations can be detected by the conversion of one restriction site to another. The iRSM assay appears to be particularly suitable to studying alkylating agents due to their known sequence specific mutation induction. We also show a comparison of the bone marrow micronucleus data with the RSM assay and show that both assays are capable of detecting the genotoxicity of ENU to the mouse bone marrow in vivo.

An investigation of male-mediated F1 effects in mice treated acutely and sub-chronically with urethane

In order to investigate the alleged potential of paternally administered urethane to cause foetal abnormalities and heritable tumours, male CD-1 mice were treated with urethane, either acutely by intraperitoneal injection at doses of 1.25 and 1.75 g/kg bodyweight (bwt) or sub-chronically in the drinking water at 1.25 for 10 weeks, and 3.75 mg/ml for 9 weeks or vehicle for the control groups. They were mated to untreated females 1 week later. Uterine contents of half the pregnant females were examined just before full term, while the remaining females were allowed to deliver their litters. The resulting F1 offspring were observed for approximately 18 months and 12 months for acute and sub-chronic exposures respectively and subjected to necropsy examination. Some of the mice treated acutely with 1.75 g/kg bwt exhibited partial infertility but none of those treated with 1.25 g/kg bwt had an adverse effect on their reproductive ability. There was no genetic effect of acute urethane treatment on male germ cells as indicated by dominant lethality. After birth, there was an increase (P < 0.05) in post-implantation deaths possibly due to perinatal mortality. There was an increased incidence and earlier onset of liver tumours induced in F1 male offspring from F0 males treated with 1.75 g/kg bwt, (20.7% vs. 10.1%, P = 0.026) but not in the female offspring. F1 males from both treatment groups had mean bodyweights significantly higher than controls (P < 0.01). Some males from each dose group of the acute study were examined using the restriction site mutation assay involving analysis of exon sequences. No mutations were identified in testes, liver or spleen of DNA isolated from the urethane-treated animals. No reproductive or genetic effects were seen with sub-chronic treatment at either 1.25 or 3.75 mg/ml urethane nor was there any predisposition of F1 animals to tumours although observation times were shorter.

Mutation analysis using the restriction site mutation (RSM) assay

The restriction site mutation (RSM) assay (see Steingrimsdottir et al. [H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 (1996) pp. 109–121] for a review) has been developed as a genotypic mutation detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes [G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced restriction site mutation (RSM) analysis of 1,2-dimethylhydrazine-induced mutations, using endogenous p53 intron sequences, Mutagenesis 12 (1997) pp. 117–123]. This increased sensitivity was examined by applying the RSM assay to analyse the persistence of N-ethyl- N-nitrosourea (ENU)-induced mutations in mice testes. Germ line mutations were sought in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide (4-NQO)-induced DNA mutations in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and 32 P post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts.

Application of the restriction site mutation technique toN-methyl-N-nitrosourea-induced mutations in the rat

The restriction site mutation (RSM) assay was developed in this laboratory for the detection of point mutations that occur within restriction endonuclease recognition sequences in the genomic DNA of the rat. Mutations were detected and identified in a number of tissues from N-methyl-N-nitrosourea (MNU)-treated rats. Resistant restriction enzyme products were detected in 5 of the 13 restriction endonuclease recognition sequences tested (NcoI, BslI, CfoI, DdeI, and HindIII). These mutations were detected in the p53 tumor suppressor gene and the H-ras protooncogene. No resistant RSM products were detected in any of the samples taken from untreated animals. The MNU-induced mutations were identified as G to A and A to G transitions. Our results describe the first successful application of the RSM assay in detecting induced mutations in the rat and highlight the usefulness of the RSM assay in the analysis of mutagen-induced base changes without the requirement for selection of a mutant phenotype. Given the increasing use of the rat as an animal model in genotoxicity studies, the development of such tests is essential for future genotoxicity investigations.

Frequency of Ki-ras mutations and DNA alkylation in colorectal tissue from individuals living in Manchester.

Most human colorectal cancers arise through the accumulation of a series of genetic alterations such as point mutations within the Ki-ras and p53 genes, but the chemical carcinogens that may be implicated in these events are still unidentified. In a previous study, we showed that DNA from human colorectal tissue contained O6-methyldeoxyguanosine (O6-MedG), a promutagenic lesion arising from exposure to as yet unidentified methylating agents. To address whether such exposure may result in oncogene activation in human colorectal tumors, we examined another series of paired normal and tumor DNA samples from the lower intestinal tract for the presence of O6-MedG in DNA (as a marker of exposure) and for mutations within the Ki-ras gene. After isolation by high pressure liquid chromatography, O6-MedG was quantified by a radioimmunoassay with a limit of detection of 0.01 mumol O6-MedG/mol dG. The frequencies of methylation were 33%, 52%, and 48% for normal DNA and 58%, 32%, and 63% for tumor DNA isolated from the cecum, sigmoid colon, and rectum, respectively. Overall, 35% of the individuals had no detectable O6-MedG in the DNA from both their tumor and normal tissue. Ki-ras mutations were initially identified by a restriction site mutation assay and then sequenced to ascertain the mutations thus detected. The frequencies of mutations in tumor DNA isolated from the cecum, sigmoid colon, and rectum were 28%, 29%, and 42%, respectively. DNA isolated from macroscopically normal tissue was found to contain Ki-ras mutations in 14% of sigmoid colon samples and 12% of rectal samples. Most base mutations were in codon 12 (72%), and 64% were GC-->AT transitions: 28% and 8% were GC-->TA and CG-->CG transversions, respectively. All mutations were at the second base of either codon 12 or codon 13 except for a single GC-->TA transversion at the first base of codon 13 in a rectal tumor sample. There was no association between the presence of O6-MedG in DNA from either normal or tumor tissue or both normal and tumor tissue and the incidence of Ki-ras mutations or GC-->AT transitions in mutated Ki-ras genes. It remains to be determined, however, whether there is a relationship between methylating-agent exposure and Ki-ras mutations, as (i) the presence of O6-MedG in colorectal DNA in these samples may not represent the exposure when Ki-ras mutational activation was occurring (i.e., at some unknown time in the past), (ii) interindividual differences in repair-enzyme activity may alter susceptibility to a mutational event after exposure, (iii) the predominant mutagen in the colon and rectum may not be a methylating agent (e.g., nitric oxide), and (iv) exposure to methylating agents need not result in oncogene activation in human tissues but may perhaps promote the emergence of the mutator phenotype.

The application of the restriction site mutation assay to the study of the induction of mutations in the tissues of rodents.

The restriction site mutation (RSM) assay has been developed as a methodology for the study of base changes induced in restriction enzyme recognition sequences. In principle, the RSM methodology may be used to the study mutation induction in any organ, of any species, for which DNA sequence information is available. This paper describes the analysis of the induction of base sequence changes in restriction enzyme recognition sites present in the p53 and alpha-haemoglobin genes of male mice exposed to 1-ethyl-1-nitrosourea. Our results demonstrate the induction of G-->A transitions and G-->T transversions in a number of restriction enzyme recognition sites (BamH1, Pst1, Ban1, Msp1, Alu1) in DNA extracted from a number of different tissues from animals harvested 2-7 days after exposure. The data presented indicates that the RSM methodology has considerable potential for development for the study of induced base changes in the endogenous genes of a variety of species.

The detection of DNA adducts, DNA base changes and chromosome damage for the assessment of exposure to genotoxic pollutants

The exposure of aquatic species to a variety of genotoxic chemicals raises the question as to the potential effects of exposure upon the health status of both current and future aquatic populations. The induction of DNA lesions in exposed species may initiate the formation of both structural and point mutations in genes, the modifications of which may lead to abnormality in both somatic and germ cells. The assessment of exposure levels of genotoxic agents and the genetic consequences requires the application of a set of inter-related techniques capable of providing both qualitative and quantitative information. In this paper we describe some methodologies available for the (a) assessment of genotosin induced DNA adducts using the 32P-postlabelling procedure, (b) measurement of point mutations using the restriction site mutation assay and (c) the measurement of the production of chromosome structural and numerical aberrations using the micronucleus assay. This package of technologies may be optimised to use with individual species present in the aquatic environment to provide a cost effective package, capable of monitoring genotoxin exposure of individual environments.