Mutagenesis, Vol. 14, No. 1, 37-42,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Inverse restriction site mutation (iRSM) analysis. Mutation detection involving the formation of restriction enzyme sites in target genes
Centre for Molecular Genetics and Toxicology, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
This paper describes a rapid screening procedure for the detection of DNA sequence changes resulting in the creation of new restriction enzyme sites. The basic methodology involves the identification of the conversion of one restriction site into another by mutagenesis. The selective removal of the wild-type sequences by digestion with a restriction enzyme acting on the wild-type sequence increases the sensitivity beyond that of PCR–RFLP analysis (10–4–10–5 detectable here). In this paper we describe the rapid detection of induced in vivo mutations transforming the ApaI restriction site present in intron 6 of the mouse p53 gene to a unique AvaII site. The potential application of this method in other genes and organisms as a rapid screen for induced mutations is discussed.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The central role that DNA mutations play in carcinogenesis and human genetic disease have led to the development of a wide range of techniques capable of detecting such mutational events in tissues exposed to mutagens (Gossen et al., 1989
; Parry et al., 1990; Kohler et al., 1991; Cerutti et al., 1994). However, these mutation assays all have limitations and none have been specifically designed, as yet, for rapid mutational screening. This report describes the development and use of the inverse restriction site mutation (iRSM) assay as a potential screen for in vitro/in vivo DNA mutation induction. While the restriction site mutation (RSM) assay detects restriction sites made resistant to enzyme digestion by mutation (Parry et al., 1990; Cerutti et al., 1994; Steingrimsdottir et al., 1996; Parsons and Heflich, 1997), the iRSM assay detects the creation of new restriction sites from pre-existing ones. This technique differs from conventional restriction fragment length polymorphism (RFLP) analysis in that rare mutant alleles can be detected, due to selective removal of the wild-type sequences by restriction digestion of the pre-existing site and specific amplification of mutant sequences by PCR. Standard RFLP has been previously employed to detect prevalent mutations. For example, Nakazawa et al. (1990) showed the formation of a XbaI site in codon 61 of H-ras gene upon oncogenic activation. Similarly, the K-ras gene has been shown to contain a BstNI site upon activation (Kahn et al., 1991) and Saiki et al. (1985) showed detection of the sickle cell anaemia genotype by DdeI restriction digestion of the haemoglobin gene. However, these RFLP analyses, as the name suggests, are only capable of detecting polymorphisms, i.e. mutations that are present at a frequency of >1%. Hence, RFLP analysis is not sufficiently sensitive to detect early mutational events in a small number of mutated cells surrounded by an excess of wild-type cells. This study describes application of the iRSM technique to the detection of rare in vivo mutational events induced in mouse spleen by N-ethyl-N-nitrosourea (ENU), in mouse bone marrow by 1,2-dimethylhydrazine (DMH) and in mouse liver by 2-acetylaminofluorene (2-AAF) in the intron 6 region of the mouse p53 gene. However, it should be noted that this technique is readily applicable to any gene, mutagen or organism by selection of appropriate restriction enzyme sites.
The basic methodology is shown in Figures 1 and 2. Figure 1 shows the mutation types detectable by ApaI/AvaII restriction digestion analyses. Figure 2 shows the major steps involved in iRSM mutation analysis. Mutations which transform one restriction site into another can be screened for by two digestion steps interspersed by PCR amplification. In the first step, wild-type restriction sites are destroyed by digestion (ApaI), resulting in selective removal of the majority of these sites (>99% in our hands), allowing the mutants (AvaII sites) to be more easily identified. Regions containing uncut restriction sites are then amplified by PCR, using primers flanking the enzyme sites. Finally, the PCR product, containing a mixed population of uncut ApaI sites and mutated ApaI sites are digested with AvaII. Any mutations which transform the ApaI site into the AvaII site (Figure 1) can be detected by the formation of AvaII restriction fragments. Due to the efficiency of the first restriction enzyme digestion being <100% and the rarity of induced DNA mutations, the PCR product predominantly contains wild-type ApaI sequences. If the ApaI digestion efficiency was 99%, after the first ApaI digestion step of 1 µg genomic DNA (3x105 copies of each p53 gene) any mutant AvaII sites will be present among 103 wild-type sites. If the digestion efficiency was 99.9%, then mutants would be present amongst 102 copies of wild-type and, similarly, 99.99% digestion would leave mutants among 10 copies of wild-type. Therefore, wild-type sequences will persist after digestion, their level being dependent upon the efficiency of the ApaI digestion. However, unlike the RSM assay, where uncut wild-type sites produce false positive results (Steingrimsdottir et al., 1996; Parsons and Heflich, 1997), perhaps by the formation of indigestible heteroduplexes with mutant PCR products, enzyme inefficiency is not a problem for the iRSM assay. This is due to the mutant fraction giving rise to unique AvaII restriction fragments, which are easily separated by electrophoresis. The use of 1 µg DNA (the optimum for PCR amplification) limits the theoretical sensitivity of the analyses to detecting one mutant allele in 3x105 copies of wild-type; this gives an estimated maximum detection limit of 3x10–6. However, in our experience ~10 mutations are necessary to produce enough amplified DNA to be visible on a gel (Steingrimsdottir et al., 1996; Jenkins, 1997) and hence the practical sensitivity of the iRSM assay is of the order of 10–5. This limit can obviously be improved by performing multiple analyses.
|
|
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Mutagen treatment and DNA extraction
Male CD-1 mice were treated in groups of four with ENU, 2-AAF or DMH as previously described (Jenkins, 1997
; Jenkins et al., 1997, 1998). The mutagens were administered on two consecutive days, orally by gavage, the vehicle employed being 1% sodium carboxymethylcellulose (SCMC). The control animals, treated in groups of five, received SCMC only. The dose levels were set as follows: ENU, 275 mg/kg/day; DMH, 20 mg/kg/day; 2-AAF, 200 mg/kg/day. The animals were killed 3 days post-treatment and cadavers stored at –70°C. DNA was extracted from the tissues of interest by use of a high salt kit (Stratagene, Cambridge, UK). The tissues were chosen on the basis of previous mutational data obtained with the RSM assay (Jenkins, 1997). The DNA concentration and purity was assessed by spectrophotometry at 260/280 nm.
The inverse RSM assay
Restriction enzyme digestion. All restriction enzymes were purchased from Promega (Madison, WI). Initial digestions were carried out in Taq polymerase buffer (Promega) with 1.5 mM MgCl2, 1 µg DNA and 60 U ApaI overnight at 37°C, 20 µl final volume. Subsequent post-PCR digestion was performed on 17 µl PCR product along with the manufacturers buffer and 20 U AvaII overnight at 37°C.
PCR amplification. PCR amplifications were performed in an MJ research DNA engine (Watertown, MA), using primers obtained from Cruachem (Glasgow, UK). The intron 6 sequence data was obtained from the literature (Goodrow et al., 1992) and primers were designed using the PRIMER program available at the SEQNET remote site. The forward primer for the intron 6 region was 5'-CCCTACCTCACTACAGGTGACC-3'; similarly, the reverse primer was 5'-CTTTCTAGCAACCCGTTTGC-3'. Amplification was carried out in 50 µl aliquots with the whole 20 µl of the digest, plus 20 pmol each primer, 400 mM dNTP, 1.5 mM MgCl2 in Promega Taq polymerase buffer with 1.25 U Taq polymerase (Promega). Thirty three cycles of 94°C for 30 s, 60°C for 10 s and 72°C for 15 s were performed.
PAGE analysis of mutations
The creation of a unique AvaII site was assessed by PAGE analysis with 6% gels, stained with silver. The presence of the AvaII restriction fragments (124 and 181 bp) was determined by comparison with a DNA molecular weight marker (Research Genetics, Huntsville, AL).
DNA sequencing
DNA sequencing, in order to confirm the mutations, was carried out with an ALF DNA sequencer (Pharmacia, Uppsala, Sweden) using an Autoload DNA sequencing kit (Pharmacia, Uppsala, Sweden).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Figure 3
shows an illustrative gel photograph of the iRSM analysis of untreated and ENU-treated mouse spleen DNA. Lanes 1 and 2 show the AvaII digestion of DNA amplified from an untreated sample, showing that no AvaII site was present in the p53 intron 6 region. Lanes 3 and 4 show the application of ApaI digestion prior to amplification plus AvaII digestion, showing no AvaII sites (mutations) in the untreated samples. Lanes 5 and 6 show the AvaII digestion of PCR products amplified from ENU-treated spleen DNA, showing that mutations (AvaII sites) are not detectable by conventional RFLP. However, lanes 7 and 8 show that ApaI digestion before selective PCR amplification and AvaII digestion allows the detection of such ENU-induced mutations (particularly in lane 7). The PCR band intensities of the amplified samples are similar in Figure 3, regardless of whether restriction digestion (ApaI) was performed before amplification or not. This may be a consequence of the 33 cycles of amplification performed, where undigested DNA (high copy number) reaches the plateau stage of amplification before the digested DNA (low copy number) and hence the digested DNA amplifies more efficiently in the final cycles. In addition, digested DNA amplifies more efficiently due to better thermal characteristics, which may also help to explain the similar band intensities in Figure 3.
|
Figures 4 and 5
show representative results of AvaII mutational analysis in ENU-treated spleens, DMH-treated bone marrows and 2-AAF-treated livers of mice. Table I also shows the data obtained. These figures demonstrate the creation of AvaII sites through mutations induced by the three chemicals in the tissues under test. Mutations are visible by the appearance of AvaII restriction fragments (124 and 181 bp). The control samples show AvaII digestion of PCR products amplified from untreated tissue samples, demonstrating that no intrinsic AvaII sites were present. Overall, the ENU-treated spleen samples were found to contain AvaII sites (GC
AT and GCTA mutations) in 33% of analyses (40 samples screened, four spleens 10 times each). The DMH-induced AvaII sites were detected in 13% of bone marrow samples (24 samples, four bone marrows six times each) and the 2-AAF-treated liver samples contained 25% AvaII sites (24 samples, four livers screened six times each). These percentage positive values provide an idea of the prevalence of the mutations in these particular tissues and echo results obtained by RSM analysis of these tissues, i.e. that ENU-treated spleens display more mutations than 2-AAF-treated livers and that DMH-treated bone marrow possess the least number of mutations (Jenkins, 1997). No single animal was shown to be significantly sensitive to mutation. The mutations were detected in all individual mice screened (results not shown).
|
|
|
There is substantial variation in the intensity of the AvaII fragments visible in Figures 4 and 5
, even between samples of the same treatment group. This variation is a result of the differing restriction enzyme efficiencies and prevalence of particular mutations in different samples. In order to obtain accurate quantitative mutation frequency data, mutant standards are required to assess the PCR amplification efficiency, using methodologies similar to those discussed previously (Jenkins et al., 1997). No AvaII sites were detectable in the same numbers of analyses performed on DNA from untreated tissue DNA (30–50 samples/chemical). PCR products containing AvaII sites were sequenced in parallel in order to ascertain the particular mutations induced and to confirm that AvaII sites were present. Figure 6 shows the sequence obtained from an AvaII-sensitive PCR product, detailing the GT spleen mutation induced, in this case by ENU. The mutant fraction present in this particular PCR product comprised 37% of the base sequence, with the wild-type fraction comprising 63%, based on measurements of peak heights on the sequencing gel. This is in agreement with the relative intensity of the AvaII cut bands and the uncut wild-type PCR product (results not shown). All AvaII-sensitive PCR products, upon sequencing, were shown to contain mutational events capable of creating the unique AvaII sites. However, due to the prevalence of the wild-type DNA and the background noise of the sequencing gel, some of these mutations were barely identifiable, hence AvaII digestion may prove to be more sensitive (and substantially less expensive and time consuming) than direct sequencing. Mutations induced by the chemical and tissue combinations tested here have previously been shown to be present at mutation frequencies of 10–4–10–5 (Jenkins, 1996; Jenkins et al., 1997, 1998), indicating the practical detection limit of a single iRSM analysis.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
This report describes the successful detection of rare in vivo mutations modifying the ApaI (GGGCCC) restriction sites of intron 6 of the mouse p53 gene, creating unique AvaII sites in their place (GGa/tCC). Due to the non-specific sequence requirement of AvaII at the middle base of its recognition sequence, AvaII digestion allows the screening of GC
AT and GCTA mutations at both the third and fourth bases of the ApaI site (Figure 1). These four mutation types may only represent a third of the 12 possible base changes, but they account for over a half of the recorded non-CpG site mutations found in human genetic disease (Cooper and Krawczak, 1993). In addition, the types of mutations detectable by ApaI/AvaII iRSM are characteristically induced by many mutagenic chemicals. For example, alkylating agents and aromatic amines are known to preferentially mutate guanine bases at the 3'-end of a run of guanines (Sendowski and Rajewski, 1991; Lambert et al., 1992), probably due to the charge accumulation in the case of alkylating agents (Richardsen and Richardsen, 1990). Indeed, previous mutational analyses at the ApaI site in intron 6 of the mouse p53 gene, using the RSM assay, have shown that the third and fourth bases of the ApaI site represent hotspots for mutation by the chemicals under test here (Jenkins, 1996; Jenkins et al., 1997, 1998). As well as alkylating agents and aromatic amines, the GGGCCC sequence has been shown to be a hotspot for other DNA damaging agents, including Cu-H2O2-induced reactive oxygen species (Rodriguez et al., 1995). Therefore, the ApaI site and, in particular, the bases involved in conversion of the ApaI site to an AvaII site apparently represent hotspots for mutation induction by a wide range of genotoxic chemicals. Hence, the ApaI site appears to be an extremely useful target for mutational analysis by similar techniques to those outlined in this paper.
Figure 7 details other restriction site combinations which may potentially be used in mutation screening as alternatives to ApaI/AvaII. These include the HaeIII site present at codon 250 of the human p53 gene (Figure 7A). This HaeIII site contains an extra 3' C base (GGCCC). Mutation at the first C to either A or T will result in the creation of a unique AvaII site similarly to the above report (GGa/tCC). The iRSM technique may also allow the study of mutations induced at highly mutable CpG dinucleotides (Cooper and Krawczak, 1993) when they fall within an MspI restriction site (CCGG) with either a 5' C or a 3' G (Figure 7B). Mutations introducing an A or T at the middle base (CpG site) will create a unique EcoRII site (CCa/tGG) which may be detected by analyses similar to those described above. The employment of MspI/ EcoRII digestion allows the detection of both GCAT and GCTA mutations within CpG sites. GCAT mutations have been heavily implicated in spontaneous deamination-mediated mutation (Cooper and Krawczak, 1993). CpG dinucleotides have also recently been shown to be preferentially targeted by benzo[a]pyrene DNA adducts (Denissenko et al., 1997), hence they appear to play a central role in mutagenesis, by both deamination-mediated processes and perhaps as targets for some genotoxic chemicals. Two such MspI sites (with a 3' G base) are available for study in the mouse p53 gene, in exons 6 and 10 (codons 193 and 338).
|
In addition to inducing mutations in the ApaI site, creating new AvaII sites, as shown above, 2-AAF is known to specifically induce adducts at the third G base of the NarI restriction site (GGCGCC) and such adduction has been shown to result in a characteristic –2 frameshift mutation (the NarI site is 100-fold more mutagenic than other sequences) (Tebbs and Romano, 1994
). This specific deletion mutation creates a HaeIII site (GGCC) (Figure 7C) and hence NarI/HaeIII restriction analysis may be used to screen for these specific 2-AAF-induced mutations. Another possible restriction enzyme combination includes the +1 frameshift mutation transforming the HaeIII site (GGCC) into an AsuI site (GGNCC) (Figure 7D) It is possible that polymerase errors introduced during the first few cycles of amplification could lead to false positive mutations using the iRSM assay. In this report no mutations were detectable in any of the untreated samples, hence these errors were probably not significant here. However, in order to reduce the possibility of this occurring, proof-reading high fidelity enzymes could be employed; this may be especially important if the sensitivity of the assay was increased. In order to improve the sensitivity of the iRSM assay, multiple analyses could be performed; analyses could be performed in a microtitre format, facilitating rapid in vitro screening of mutagens. Additionally, enrichment of the DNA for the target gene (p53 in this case) would substantially improve the sensitivity of the assay. These possibilities are currently being investigated.
In summary, this technique allows the rapid screening of mutations induced in restriction sites through their conversion into alternative unique restriction sites. This preliminary data provides an encouraging glimpse of the potential usefulness of this assay in mutation screening. Further validation experiments are required along with further developmental work before routine application of the iRSM assay is envisaged.
|
Acknowledgments |
|---|
This work was funded by a studentship from the European Union Environmental Program and a case award from SmithKline Beecham Pharmaceuticals. We thank Sally James for preparation of the figures. The dosed animals were kindly provided by SmithKline Beecham.
|
Notes |
|---|
1 To whom correspondence should be addressed. Tel: +44 1792 205678. Fax: +44 1979 295447; Email: g.j.jenkins{at}swansea.ac.uk
2 Present address: Otsuka Pharmaceutical Co. Ltd, 463-10 Kagasuno Kawauchi-cho, Tokushima 771-01 Japan
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
-
Cerutti,P., Hussain,P., Pourzand,C. and Aguilar,F. (1994) Mutagenesis of the H-ras protooncogene and the p53 tumour suppressor gene. Cancer Res., 54, 1934s–1938s.
Cooper,D.N. and Krawczak,M. (1993) Human Gene Mutation. Bios Scientific Publishers Ltd, Oxford, UK.
Denissenko,M.F., Chen,J.X., Tang,M. and Pfeifer,G.P. (1997) Cytosine methylation determines hot spots of DNA damage in the human p53 gene. Proc. Natl Acad. Sci. USA, 94, 3893–3898.
Goodrow,T.L., Storer,R.D., Leander,K.R., Prahalada,S.R., van Zwitten,M.J. and Bradley,M.J. (1992) Murine intron sequences 5–8 and their use in polymerase chain reaction/direct sequencing analysis of p53 mutations in CD-1 mouse liver and lung tumours. Mol. Carcinogen., 5, 9–15.[Web of Science][Medline]
Gossen,J.A., de Leeuw,W.J.F., Tan,C.H.T., Zwarthoff,E.C., Berends,F., Lohman,P.H.M., Knock,D.L. and Vijg,J. (1989) Efficient rescue of integrated shuttle vectors from transgenic mice: a model for studying mutations in vivo. Proc. Natl Acad. Sci. USA, 86, 7971–7975.
Jenkins,G.J.S. (1997) The molecular mechanisms of mutagenesis, PhD thesis, University of Wales, UK.
Jenkins,G.J.S., Mitchell,I.deG. and Parry,J.M. (1997) Enhanced Restriction Site Mutation (RSM) analysis of 1,2-dimethylhydrazine induced mutations, using endogenous p53 intron sequences. Mutagenesis, 12, 117–123.
Jenkins,G.J.S., Chalestori,M.H., Song,H. and Parry,J.M. (1998) Mutation analysis using the Restriction Site Mutation (RSM) assay. Mutat. Res., in press.
Kahn,S.M., Jiang,W., Culbertson,T.A., Weinstein,I.B., Williams,G.M., Tomita,N. and Ronai,Z. (1991) Rapid and sensitive nonradioactive detection of mutant K-ras genes via enriched PCR amplification. Oncogene, 6, 1079–1083.[Web of Science][Medline]
Kohler,S.W., Provost,G.S., Fieck,A., Kretz,P.L., Bullock,W.O., Sorge,J.A. Putman,D.L. and Short,J.M. (1991) Spectra of spontaneous and mutagen induced mutations in the LacI gene in transgenic mice. Environ. Mol. Biol., 18, 316–321.
Lambert,I.B., Napolitano,R.L. and Fuchs,R.P.P. (1992) Carcinogen-induced frameshift mutagenesis in repetitive sequences. Proc. Natl Acad. Sci. USA, 89, 1310–1314.
Nakazawa,H., Aguelon,A.M. and Yamasaki,H. (1990) Relationship between chemically induced Ha-ras mutation and transformation of Balb/c 3T3 cells: evidence for chemical specific activation and cell type specific recruitment of oncogene in transformation. Mol. Carcinogen., 3, 202–209.[Web of Science][Medline]
Parry,J.M., Shamsher,M. and Skibinski,D.O.F. (1990) Restriction Site Mutation analysis, a proposed methodology for the detection and study of DNA base changes following mutagen exposure. Mutagenesis, 9, 209–212.
Parsons,B.L. and Heflich,R.H. (1997) Genotypic selection methods for the direct analysis of point mutations. Mutat. Res., 387, 97–121.[Web of Science][Medline]
Richardsen,F.C. and Richardsen,K.K. (1990) Sequence dependent formation of alkyl-DNA adducts: a review of methods, results and biological correlates. Mutat. Res., 233, 127–138.[Web of Science][Medline]
Rodriguez,H., Drouin,R., Holmquist,G.P., O'Conner,T.R., Boiteux,S., Laval,J., Doroshow,J.H. and Akman,S.A. (1995) Mapping of copper/hydrogen peroxide induced DNA damage at nucleotide resolution in human genomic DNA by ligation mediated polymerase chain reaction. J. Biol. Chem., 270, 17633–17640.
Saiki,R.K., Scharf,S., Faloona,F., Mullis,K., Horn,G.T., Erlich,A. and Arnheim,N. (1985) Enzymatic amplification of ß-Globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science, 230, 1350–1354.
Sendowski,K. and Rajewki,M.F. (1991) DNA sequence dependence of guanine O-6 alkylation by the N-nitroso carcinogens N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea. Mutat. Res., 250, 153–160.[Web of Science][Medline]
Steingrimsdottir,H., Beare,D., Cole,J., Leal,J.F.M., Kostic,T., Lopez-Barea,J., Dorado,G. and Lehman,A.R. (1996) Development of new molecular procedures for the detection of genetic alterations in man. Mutat. Res., 353, 109–121.[Web of Science][Medline]
Tebbs,R.S. and Romano,L.J. (1994) Mutagenesis at a site specifically modified NarI sequence by acetylated and deacetylated aminofluorene adducts. Biochemistry, 33, 8998–9006.[Medline]
Received on May 28, 1998; accepted on July 22, 1998.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  T.E. Schmid, A. Kamischke, H. Bollwein, E. Nieschlag, and M.H. Brinkworth Genetic damage in oligozoospermic patients detected by fluorescence in-situ hybridization, inverse restriction site mutation assay, sperm chromatin structure assay and the Comet assay Hum. Reprod., July 1, 2003; 18(7): 1474 - 1480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kaur, Y. Zhang, W.-H. Liu, S. Tetradis, B. D. Price, and G. M. Makrigiorgos Ligation of a primer at a mutation: a method to detect low level mutations in DNA Mutagenesis, September 1, 2002; 17(5): 365 - 374. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G.J.S. Jenkins, H.S. Suzen, R.A. Sueiro, and J.M. Parry The restriction site mutation assay: a review of the methodology development and the current status of the technique Mutagenesis, September 1, 1999; 14(5): 439 - 448. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||