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Deoxyuridine Is Generated Preferentially in the Nontranscribed Strand of DNA from Cells Expressing Activation-Induced Cytidine Deaminase

Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224

	Abstract

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Activation-induced cytidine deaminase (AID) is required for somatic hypermutation and class switch recombination of Ig genes in B cells. Although AID has been shown to deaminate deoxycytidine to deoxyuridine in DNA in vitro, there is no physical evidence for increased uracils in DNA from cells expressing AID in vivo. We used several techniques to detect uracil bases in a gene that was actively transcribed in Escherichia coli cells expressing AID. Plasmid DNA containing the gene was digested with uracil-DNA glycosylase to remove uracil, and apurinic/apryimidinic endonuclease to nick the abasic site. The nicked DNA was first analyzed using alkaline gel electrophoresis, in which there was a 2-fold increase in the linear form of the plasmid after AID induction compared with plasmid from noninduced bacteria. Second, using a quantitative denaturing Southern blot technique, the gene was predominantly nicked in the nontranscribed strand compared with the transcribed strand. Third, using ligation-mediated PCR, the nicks were mapped on the nontranscribed strand and were located primarily at cytosine bases. These data present direct evidence for the presence of uracils in DNA from cells that are induced to express AID, and they are preferentially generated at cytosines in the nontranscribed strand during transcription.

	Introduction

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Activation-induced cytidine deaminase (AID)² initiates somatic hypermutation, class switch recombination, and gene conversion in B cells (reviewed in Ref. 1 and 2). Since its discovery, the precise mechanism of AID action in these cells is still under scrutiny, and two theories have emerged. The homology of AID to APOBEC1, an apolipoprotein-B RNA editing cytidine enzyme, led to the RNA deamination theory that AID deaminates cytidine to uridine in the mRNA of a gene that encodes a B cell-specific endonuclease (3). This putative endonuclease would nick DNA and provide entry points for hypermutation and recombination. In contrast, copious genetic and biochemical evidence supports the DNA deamination theory that AID deaminates cytidine to uridine in DNA. Regarding genetics, overexpression of AID in Escherichia coli (4), yeast (5), cultured cell lines (6, 7, 8, 9, 10), and mice (11) produced mostly C to T transitions in DNA, which likely resulted from a DNA polymerase replicating deoxyuridine (dU). Nonetheless, overexpression of APOBEC1 in E. coli also produced C to T transitions (12), suggesting that this assay is not specific for determining enzyme activity in mammalian cells. The best evidence for uracils as intermediates in hypermutation and switching in B cells is the involvement of the enzyme uracil-DNA glycosylase (UNG), which removes dU to produce an abasic site (13, 14). However, even this role for UNG has been questioned recently (15), implying that AID may not deaminate C in DNA.

Regarding biochemistry, AID has been shown to deaminate deoxycytidine (dC) on ssDNA (16, 17), bubble substrates (16), and supercoiled substrates (18) in vitro. On these artificial substrates, AID is targeted to mutate C in the WRC motif (19, 20, 21), in accord with the specificity of hypermutation in vivo (22). During in vitro transcription, AID is most active on C in the nontranscribed DNA strand (23, 24, 25). It has been proposed that during transcription elongation, a stretch of DNA in the nontranscribed strand can form an exposed single-strand loop, which is a substrate for AID. It is thus reasonable to expect that induction of AID in cells will lead to an increase in dU in the nontranscribed strand of actively transcribed genes.

To obtain direct evidence for DNA deamination in vivo, we examined DNA from E. coli expressing AID. The DNA was digested with UNG to detect dU, and apurinic/apyrimidinic endonuclease 1 (APE1) to nick abasic sites. Nicked DNA was analyzed by gel electrophoresis, and uracils in a specific gene were identified by DNA probes and ligation-mediated PCR (LM-PCR).

	Materials and Methods

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E. coli growth and plasmid DNA isolation

Two strains of E. coli, ung-1 (BW310), referred to as ung^–, and wild type (KL16), referred to as ung⁺, were transformed with plasmid vectors containing human AID cDNA (pSPM111) or the vector only (pTrC99A), as previously described (4). The E. coli strains and plasmids were generous gifts from M. Neuberger (Medical Research Council Laboratory of Molecular Biology, Cambridge, U.K.). Transformed E. coli was grown overnight with shaking at 37°C in Luria-Bertani medium with 100 µg/ml ampicillin (Sigma-Aldrich). The overnight stock was diluted 1/50 the next day in fresh medium with 100 µg/ml carbenicillin (Sigma-Aldrich). It was regrown at 37°C to OD₆₀₀ of 0.8–1 before the culture was diluted further into prewarmed Luria-Bertani medium with 100 µg/ml carbenicillin to give an OD₆₀₀ of 0.25. After reincubating the diluted culture at 37°C for 15 min, isopropyl--D-thiogalactopyranoside (IPTG; ICN Pharmaceuticals) was added to the media to a final concentration of 1 mM. The culture was grown for either 20 or 90 min more at 37°C before isolating the plasmids using a Qiagen minispin column. DNA preparations were used only once after being stored at –20°C.

Rifampicin mutational assay

E. coli were collected at indicated time points, and the bacteria were plated overnight on rifampicin-ampicillin (100 µg/ml each; Sigma-Aldrich) or ampicillin (100 µg/ml) plates. Both large and small rifampicin-ampicillin resistant colonies were counted as resistant colonies. A mutation frequency was obtained by dividing the number of rifampicin-ampicillin-resistant colonies by the number of viable bacteria present in the culture as assessed by the number of colonies grown on ampicillin plates.

Alkaline agarose electrophoresis of plasmid DNA

Equal amounts of plasmid (0.5 µg) were used for UNG/APE1 treatment and for mock-treated controls. The UNG and APE1 preparations used in this assay were gifts from S. Wilson (National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC). UNG digestion was done in base excision repair (BER) buffer without magnesium (50 mM HEPES, pH 7.5, 20 mM KCl, 2 mM DTT) for 1 h at 37°C. APE1 digestion was performed in the same buffer after addition of 10 mM MgCl₂ and 5 mM ATP for 1 h at 37°C. For mock digestions, both enzymes were replaced with H₂O. Digestions were stopped by addition of 10x alkaline loading buffer (0.3 M NaOH, 2 mM EDTA, 10% glycerol, and 0.25% bromphenol blue), and then incubated at 37°C for 30 min before loading onto a gel. pSPMIII was linearized to 4.5 kb with HindIII. Alkaline agarose gels were prepared by soaking a 0.8% agarose gel made in H₂O twice in alkaline running buffer (30 mM NaOH, 2 mM EDTA) for 30 min each. Electrophoresis was performed in running buffer at 2.5 V/cm with buffer recirculation. After electrophoresis, the gel was washed extensively in 0.1 M Tris-Cl, pH 8.0, and the DNA bands were visualized by ethidium bromide staining. The bands were quantified using ImageQuant software. Normalized intensity ratios were calculated as follows. For each time point, the intensity of the 4.5-kb linear band (or background in untreated lanes) was divided by the sum of the intensities of the upper and lower circular bands. This ratio was calculated for both untreated and UNG/APE1-treated lanes. Then the ratios for each time point were normalized to the ratio at 0 min.

Denaturing Southern electrophoresis to detect the AID gene

For Southern blot analysis of the AID gene, plasmid DNA was isolated from ung^– E. coli after 90 min of induction of AID. After restriction digestion with NcoI and HindIII to produce a 627-bp fragment, the DNA was quantified, and equal amounts were treated with UNG and APE1, or APE1 alone, in BER buffer as described. The UNG and APE1 preparations used in this assay were obtained from Epicentre Technologies and D. Wilson (National Institute on Aging, National Institutes of Health, Baltimore, MD), respectively. Reactions were stopped by heating to 95°C for 10 min, put on ice, and added to 10x alkaline loading buffer. Around 2.5 ng of either UNG/APE1- or APE1-treated DNA were loaded in each lane for electrophoresis. The gel was made by dissolving 1% agarose in H₂O, cooling it to 50°C, and adding 10 N NaOH and 0.5 M EDTA stock to a final concentration of 30 mM NaOH and 2 mM EDTA. The gel was poured in the cold room to prevent escape of NaOH from the gel. The gel was run in a buffer of 30 mM NaOH and 2 mM EDTA, with recirculation of the buffer at 2.5 V/cm for 1.5 h, and immediately transferred to positively charged nylon membrane (Hybond-N⁺; Amersham Pharmacia Biotech) using 0.4 M NaOH. The membrane was washed in 2x SSC phosphate/EDTA (SSPE) and cross-linked by UV before prehybridization in Hybrisol I (Serologicals). DNA probes were made by amplifying the AID gene from pSPM111 using the following primers: forward, 5'-ATGGACAGCCTCTTGATG-3' and reverse, 5'-AAAGTCCCAAAGTACG-3'. For synthesis of the nontranscribed probe, the reverse primer was used, and for synthesis of the transcribed probe, the forward primer was used. The single-strand probes were labeled using Redivue [-³²P]dCTP (Amersham Biosciences) and the Megaprime DNA Labeling System (Amersham Biosciences). After hybridization for 20 h at 42°C, the membrane was washed in 2x SSPE-0.1% SDS for three times at room temperature and 0.1% SSPE-0.1% SDS twice at 42°C. The membrane was exposed to a phosphorimager screen, and the bands were quantified using ImageQuant software.

LM-PCR of the nontranscribed strand

Plasmid DNA was isolated as described, and treated with T4 DNA ligase (New England Biolabs) at 16°C overnight to ligate spontaneous nicks. The DNA was then treated sequentially with UNG (Epicentre) in BER buffer overnight at 37°C, and then a combination of APE1 and human DNA polymerase (both from D. Wilson) in BER buffer with addition of Mg and ATP as previously described for 3 h at 37°C. After UNG/APE1/polymerase or APE1/polymerase treatments of the DNA, a biotinylated primer specific for nontranscribed strand of the AID gene (5'-Biotin-Biotin-Biotin-Biotin-CAAAACAGCCAAGCTTGTCG-3'; Midland Certified Reagent) was annealed to the DNA by heating to 95°C for 5 min and cooling to 43°C for 30 min. The primer was extended with Sequenase 2.0 T7 DNA polymerase (USB) for 30 min at 45°C. The linker DNA consisted of a duplex prepared by Midland of two complementary strands, 5'-ATGCACTACATACAGTCATCCGGAGATCTGAATTC-3' and 5'-GAATTCAGATCTCC-3' with an EcoRI site, in italics, on both oligonucleotides. The linker was ligated at 16°C overnight. Magnetic streptavidin beads (Dynabeads M-280 Streptavidin; Dynal Biotech) were washed twice in 2x bind/wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 mM NaCl), and 10 µl was added to each ligation reaction. The reactions were incubated using a rotary device at room temperature for 1 h to promote binding of streptavidin beads to biotinylated DNA. The beads were then washed twice with 1x bind/wash buffer, and once with 1x PCR buffer, and then resuspended in 10 µl of 1x PCR buffer. A limited PCR for two cycles was first done on the bead suspension using Platinum Pfx DNA polymerase (Invitrogen Life Technologies) and a forward primer, 5'-CAGTCATCCGGAGATCTGAATTC-3', to detect the linker, and a reverse primer, 5'-CTTACTCGAGTCAAAGTCCCAAAGTACG-3' with a XhoI site, in italics, to detect the AID gene. PCR conditions were 95°C for 45 s, 55°C for 1 min, and 68°C for 2 min. After removing the beads from the reactions, PCR was continued for 35 more cycles at 95°C for 30 s, 55°C for 30 s, and 68°C for 30 s. PCR products were then digested with EcoRI and Xhol (New England Biolabs), cloned into pBS-SK (Stratagene), and sequenced using a Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (USB) and the T7 primer.

	Results

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Increase in mutated colonies early after AID induction

Uracils in DNA may be removed by endogenous bacterial UNG or diluted after chromosomal replication. To ensure that enough uracils remained, we tried two conditions. First, plasmids were transformed into both ung⁺ and ung^– E. coli strains, to see whether the presence or absence of bacterial UNG made a difference in detection. Second, bacteria were harvested early after induction of AID, to see whether dU can be found before many rounds of replication and repair. The presence of uracils was monitored by a mutation assay described by Petersen-Mahrt et al. (4), in which the dU lesion produces mutations in the rpoB gene located in chromosomal DNA, and mutants are positively selected on agar plates containing rifampicin. To see how early dU can be detected, bacteria were tested 20 and 90 min after IPTG induction of AID (Fig. 1). As expected, the frequency of rifampicin-resistant colonies was around 10-fold higher in ung^– bacteria compared with ung⁺ bacteria because uracils would remain in the DNA due to lack of the UNG enzyme. An increase in mutant colonies in both strains was seen as early as 20 min after IPTG, indicating that the DNA could be examined for the physical presence of uracils soon after induction. The frequency may have decreased after 90 min in ung⁺ DNA due to repair of the dU lesion.

View larger version (16K): [in this window] [in a new window]   FIGURE 1. Increase in rifampicin-resistant colonies early after AID induction in ung+ (A) and ung– (B) E. coli. Rifampicin- and ampicillin-resistant colonies were obtained from different cultures after AID induction with IPTG. Mutation frequency represents the number of rifampicin-resistant colonies divided by ampicillin-resistant colonies. Note the different scale in ung+ (A) and ung– (B). Different symbols represent three different experiments; the assays were repeated two to four times for each experiment. Bars denote the median number of resistant colonies.

To directly assay for dU, plasmids containing the AID gene were transformed into bacteria, isolated after AID induction, and digested with UNG/APE1. The DNA was separated on a denaturing alkaline agarose gel as described in Fig. 2A, and bands were visualized by staining with ethidium bromide. The 4.5-kb plasmid migrates as two bands in the alkaline gel, which may represent single-strand (Fig. 2A, upper band) and double-strand (Fig. 2A, lower band) circular forms of the plasmid. Nicked DNA would be detected as an increase in the linear form and smaller fragments and a corresponding decrease in the circular forms of the plasmid.

View larger version (42K): [in this window] [in a new window]   FIGURE 2. Detection of dU in plasmid DNA by linearization. A, Schematic diagram of assay. UNG/APE1 treatment generates nicks at dU in the circular double-stranded plasmid. Using denaturing alkaline electrophoresis, the intact circular plasmid migrates as upper and lower forms. Nicked plasmid contains both linear and smaller fragments. Bands are visualized with ethidium bromide. B, Plasmids were isolated from both ung– and ung+ E. coli after AID induction, treated with UNG/APE1, and separated by electrophoresis in alkaline gels. The 4.5-kb bands (arrows) correspond to plasmid DNA linearized with a restriction enzyme. C, Bands were quantified and intensity ratios calculated as described in Materials and Methods. Error bars represent the SD from three different experiments. Untreated () and UNG/APE1 treated () are shown.

Intensities of the bands were quantified for three experiments (Fig. 2C). There was more than a 2-fold increase in the linear band after UNG/APE1 digestion at 20 min in both ung⁺ and ung^– backgrounds. This increase agrees well with the rifampicin mutational assay, which also shows an increased mutation frequency after 20 min. The linear form may have decreased at 90 min due to repair in the ung⁺ strain, and/or increased nicking producing smaller fragments in the ung^– strain.

dU is more prevalent in the nontranscribed strand of a transcribed gene

To detect dU in a specific gene, we modified an established Southern blot technique, which has been used previously to study gene-specific DNA repair in mammalian cells (26, 27). Uracils were measured in the AID gene itself because it is actively transcribed after IPTG induction and can be easily isolated on a plasmid. Experiments were performed on DNA isolated after 90 min of AID induction from ung^– E. coli because this would contain high amounts of dU, as determined from the previous experiments. pSPMIII DNA was digested with restriction enzymes, and then treated with UNG/APE1 or APE1 alone. The denaturing Southern blot technique is described in Fig. 3A. The 627-bp fragment containing the AID gene was separated on an alkaline agarose gel, transferred to a membrane, and the Southern blot was hybridized with radioactive probes for either the transcribed or nontranscribed strand of the AID gene. Nicked, ssDNA from UNG/APE1 treatment would migrate as smaller fragments, and the intensity of the intact 627-bp fragment would be decreased compared with DNA treated with APE1 alone.

View larger version (26K): [in this window] [in a new window]   FIGURE 3. Detection of dU in a transcribed gene by a denaturing Southern blot technique. A, Schematic diagram of the assay. Transcription of the AID gene, depicted by a horizontal arrow, was induced with IPTG in E. coli, and the plasmid was digested with restriction enzymes to produce a 627-bp fragment containing the gene. The DNA was then treated with APE1 or UNG/APE1, separated by electrophoresis on an alkaline gel, blotted, and hybridized with probes for the transcribed or nontranscribed strand of AID. The presence of uracils would result in more small fragments and correspondingly less of the intact fragment, as measured by hybridization intensity. B, Denaturing Southern blot of the AID gene in ung– E. coli, hybridized with probes for both strands. Each sample is loaded in duplicate. C, The intensity of bound probe was averaged for duplicate lanes; the value for UNG-treated DNA was divided by the value for untreated DNA and expressed as a percentage of hybridization. Time points at 0 min () and 90 min () after AID induction are shown. Error bars represent the average of two experiments.

Location of dU is primarily at C in the nontranscribed strand

A LM-PCR method to detect ssDNA breaks (28) was modified to map the positions of dU in the AID gene. Plasmid DNA was prepared 90 min after AID induction and treated with APE1 and with or without UNG as before. The 5' deoxyribose phosphate group left after nicking the abasic site was removed by the lyase activity of DNA polymerase (29) (Fig. 4A). The DNA was denatured and annealed to a biotin-labeled primer specific for the nontranscribed strand of the AID gene. The biotin primer was elongated with Sequenase polymerase to create a blunt end corresponding to one base 3' of the abasic site, and the DNA was ligated to a blunt end linker. The DNA was then added to streptavidin beads to purify fragments containing the biotin primer, and amplified with nested primers for the linker and AID gene. Products of different sizes were cloned and sequenced to find the original location of dU on the nontranscribed strand.

View larger version (25K): [in this window] [in a new window]   FIGURE 4. Detection of dU in the nontranscribed strand by a LM-PCR technique. A, Schematic diagram of the assay. Transcription (horizontal arrow) is induced in ung– E. coli, and plasmid DNA is digested with UNG/APE1/polymerase or APE1/polymerase . The DNA is annealed to a biotinylated (BBBB) primer (thick line) specific for the nontranscribed strand of the AID gene, and extended with Sequenase (dotted line) to create a blunt end. A linker (thick lines) is ligated to the end, and purified DNA is amplified with primers for the linker and biotinylated primer. The DNA is then cloned and sequenced. B, Sequence of the nontranscribed strand of the AID cDNA (nt 377–655 from GenBank/EMBL/DDBJ under accession no. NM 020661), showing locations of the linker. UNG/APE1/polymerase -treated (•) and APE1/polymerase -treated () DNA are shown. C, Percentage of breaks at each nucleotide was corrected for the abundance of each nucleotide in the AID sequence (G, 25%; C, 27%; A, 24%; and T, 24%).

	Discussion

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The DNA deamination theory predicts that uracils will be generated at sites of cytosines in DNA from cells expressing AID. However, there are several barriers to detecting dU lesions because they can be avidly recognized and removed by the UNG enzyme, or copied over and diluted during chromosomal replication. To increase the probability of finding dU, experiments were performed in bacteria deficient for UNG, and the DNA was examined soon after induction of AID. Petersen-Marht et al. (4) previously demonstrated that AID produces resistance to the antibiotic rifampicin in cultures grown overnight, as a result of mutations introduced into the rpoB gene. Using the same assay, we were able to detect resistant colonies as early as 20–90 min after AID induction, suggesting that this would be an optimal time to find dU lesions. To detect uracils in a transcribed gene, we studied those generated in the AID gene itself for several reasons: it is mutated in mammalian cells that overexpress AID protein (9), it is highly transcribed in E. coli after IPTG induction, and it can be easily purified as plasmid DNA. The identification of uracil was based on use of both the UNG enzyme, which removes uracil from ssDNA and dsDNA to leave an abasic site (30), and the APE1 enzyme, which cleaves 5' of the abasic site in ssDNA and dsDNA (31). The nicked DNA was then analyzed by three different assays. First, plasmid DNA was separated by electrophoresis on an alkaline denaturing gel and stained with ethidium bromide. The circular plasmid was nicked to a linear form after AID induction, implying that the AID protein generated uracils that were recognized by UNG glycosylase and the abasic site was nicked by APE1.

Second, to find uracils in a transcribed gene, a quantitative Southern blot technique was used. Biochemical studies have shown that AID acts only on ssDNA, and purified AID protein preferentially deaminates dC on the nontranscribed strand, which would be single-stranded during transcription (23, 24, 25). Therefore, there should be more uracils in the nontranscribed strand compared with the transcribed strand of actively transcribed genes. In vivo evidence for deamination of the nontranscribed strand is limited to genetic findings of increased mutation of dC in the switch region of Ig genes from B cells (32). To physically detect dU in the nontranscribed strand, the plasmid DNA was digested with restriction enzymes to isolate the AID gene, and separated by electrophoresis on an alkaline denaturing gel. Uracils would be recognized by UNG/APE1, and smaller nicked fragments would migrate away from the original band. After Southern blotting and hybridization to radioactive probes for the transcribed and nontranscribed strands, the intensities of the original band were quantified. There was 2-fold less hybridization using the nontranscribed probe compared with the transcribed probe after AID induction, indicating that dU was generated preferentially in the nontranscribed strand. These findings thus confirm the biochemical data and demonstrate the physical presence of dU in a transcribed gene in cells.

Third, a LM-PCR technique was used to map the sites of uracils. LM-PCR has been previously used to detect endogenous single- and double-strand breaks in Ig genes from B cells (33, 34, 35). In this study, the breaks were exogenously created at sites of dU by UNG/APE1, and then analyzed by LM-PCR. The data show a marked preference for breaks at cytosines in the nontranscribed strand from UNG/APE1-treated DNA compared with APE1-treated DNA. In the absence of UNG, the linker ligated at random positions throughout the sequence, which may be due to nonspecific nicks created during handling of the DNA. For UNG-treated DNA, the breaks were more focused at certain cytosines. Although AID recognizes C in the WRC motif in vitro and in B cells, none of the cytosines detected in our study or mutations found in the rpoB gene from bacteria (4) were in such motifs. Lack of targeting in E. coli may be due to overexpression of AID, which directs the protein to different sequence contexts.

Thus, we have obtained direct evidence for in vivo deamination of dC by AID to dU in the nontranscribed DNA strand in E. coli, strongly supporting the DNA deamination theory. It will be most critical to detect dU in Ig genes from B cells. The alkaline Southern blot technique did not confer enough sensitivity to detect dU in these regions from total genomic DNA (N. Joshi, unpublished observation; data not shown). However, the LM-PCR assay as we described is an accurate technique for mapping breaks in a specific gene, and could be an invaluable tool for detecting dU lesions in variable genes and switch regions.

	Acknowledgments

 
We thank Michael Neuberger for the AID plasmid and bacteria, David Wilson and Sam Wilson for enzymes, Jeanine Harrigan for advice, and David Wilson, Michael Seidman, and Vilhelm Bohr for helpful discussions.

	Disclosures

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The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Address correspondence and reprint requests to Dr. Patricia J. Gearhart, Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224. E-mail address: gearhartp{at}grc.nia.nih.gov

² Abbreviations used in this paper: AID, activation-induced cytidine deaminase; dU, deoxyuridine; UNG, uracil-DNA glycosylase; IPTG, isopropyl--D-thiogalactopyranoside; APE1, apurinic/apyrimidinic endonuclease 1; LM-PCR, ligation-mediated PCR; dC, deoxycytidine.

Received for publication February 16, 2005. Accepted for publication March 23, 2005.

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