Iptg

T4 ligase

Electroporation into E.coli galE'/LacZ-

Dilution plate Mutant selection with X-gal in P-gal

Fig. 4. In vivo mutagenesis assay, using pUR288. (A) Plasmid-derived shuttle vector pUR288. CRP, cAMP receptor protein; ori, origin of replication; lacZ, gene encoding /¡-Gal; Hindlll, restriction site used to release the plasmid from genomic DNA. (B) Principle of the assay: LacZ- mutants are positively selected on P-Gal plates. See text for additional details.

mutagenesis assay is illustrated in Fig. 4B. Briefly, shuttle vectors are excised from the transgenic concatemer by restriction with ffindlll. Linearized plasmids are then separated from bulk genomic DNA with the help of magnetic beads that are coated with a LacI fusion protein that can bind to the lacO sequence of the plasmid.4 Elu-tion from the beads is achieved by adding isopropyl-/5-D-thiogalactopyranoside (IPTG), an inactivator of LacI that induces an affinity decreasing conformational change in the protein for lacO. Plasmids are circularized at the cohesive Hin&lW sites by ligation with T4 ligase and then electroporated into E. coli that is (1) deficient in /¡-Gal (lacZ~), (2) galactose intolerant due to the absence of galactose epimerase (galE~), and (3) restriction negative to prevent the degradation of incoming methylated plasmid DNA. The galE~ mutation is key12 because it allows for the positive selection of lacZT mutants in the presence of the lactose analog P-Gal (phenyl-/?-!) galactoside), a substrate for /¡-Gal. LacZ" mutants are unable to cleave P-Gal; in contrast, wild-type LacZ+ cells are able to cleave it, and thereby release galactose. Galactose is converted to UDP-galactoside, which cannot be further metabolized on the galE~ background; instead, it is accumulated intra-cellularly to toxic and eventually lytic concentrations. Thus, whereas LacZ+ cells are prevented from growth on P-Gal-supplemented agar plates, LacZ" cells are not. To determine the rescue efficiency of plasmids from genomic DNA, a small aliquot (usually 2 ¡jl\) of a 2-ml suspension of pUR288-transfected E. coli is plated on a titer plate that has been supplemented with 5-bromo-4-chloro-3-indolyl-/3-D-galactopyranoside (X-Gal). X-Gal is cleaved by /¡-Gal, which produces a blue halo around growing LacZ+ colonies. Note that LacZ" mutants will be missed on the dilution plate (no blue halo), but this is negligible because the ratio between LacZ+ to LacZ" colonies is on the order of 104:1. Note further that the cleavage of X-Gal releases galactose, which is converted to the same toxic UDP-galactoside derived from P-Gal; however, the amounts of galactose liberated from X-Gal are small and therefore compatible with the growth of LacZ+ colonies [the molar concentration of X-Gal (183 pM; 75 /¿g/ml) is 64 times lower than that of P-Gal (11.7 mM, 3 mg/ml)]. The remaining part of the suspension (1998 ¡i\) is plated on a single P-Gal plate to select for mutants. Mutants grow as small, red, formazan-stained colonies in the presence of a tetrazolium salt that should be added for improved visibility of the sometimes tiny colonies. The mutant frequency is calculated as the ratio of mutants to nonmutants; that is, the number of colonies on the P-Gal selection plate to the number of colonies (xlOOO) on the X-Gal titer plate. To characterize the mutational spectrum, mutant colonies can be picked and used directly as templates in long-range PCR amplifications of the entire lacZ gene. The PCR fragments are useful for restriction analysis and DNA sequencing. Plasmid DNA minipreparations are equally useful if it is decided not to use PCR for template preparation. Excellent reviews of the pUR288 assay including detailed protocols

12 J. A. Gossen, A. C. Molijn, G. R. Douglas, and J. Vijg, Nucleic Acids Res. 20, 3254 (1992).

and sections on troubleshooting are available.13-15 More specialized articles on the detection of so-called color mutants,16 the nature of background mutations,17 and sources of assay variability18 have also been published. In addition, a commercial kit-based version of the assay—together with technical support, a step-by-step protocol, and accessory services, such as mutational analysis by DNA sequencing and two-dimensional electrophoresis—is being offered by Leven (www.leveninc.com). Our overall experience with the assay is positive, but attention must be paid to preparing DNA of high quality [even small amounts of impurities (proteins, salts, organic solvents) can sometimes decrease the rescue efficiency of the shuttle vector] and avoiding contamination with unrelated plasmids that are lacZT, lacO+, and ampicillin resistant. Plasmids of this nature (e.g., derivatives of pBluescript) are in widespread use in many laboratories, and they can easily show up as false "pUR288 mutants" after finding their way into reagents or laboratory space where the mutagenesis assay is performed.

Phagocyte-Mediated Oxidative Mutagenesis in B Lymphoblasts

The utility of the pUR288 assay for evaluating the impact of defined genetic mutations on the levels of oxidative mutagenesis in B cells is illustrated in Fig. 5. Pristane-elicited peritoneal exudate cells (PECs; mainly macrophages and neutrophils) were utilized as effectors of mutagenesis in coincubation experiments with lipopolysaccharide (LPS)-stimulated proliferating splenic B lymphoblasts, the targets of mutagenesis. The experiment was designed as the in vitro equivalent of the pristane granuloma to test the hypothesis that the close proximity to inflammatory phagocytes may create a mutagenic environment for B cells (Fig. 5A). A benefit of the chosen experimental design was that only mutations in B cells were enumerated. B cells harbored the pUR288 shuttle vector but PECs did not. Two major variables were included in the experiment. First, PECs were obtained from B6 knockout mice that lacked the p47p,"u protein subunit of the NADPH oxidase complex and were thus unable to undergo an oxidative burst,19 or were obtained from normal NADPH-proficient B/c mice. Second, lymphoblasts prepared from normal B/c.pUR288 mice were compared with lymphoblasts from B/c.pUR288 mice that carried the xid mutation, xid is a naturally occurring null mutation in

13 J. A. Gossen, W. J. de Leeuw, and J. Vijg, Mutat Res. 307, 451 (1994).

14 J. Vijg, M. E. Dolle, H. J. Martus, and M. E. Boerrigter, Mech. Ageing Dev. 99, 257 (1997).

15 J. Vijg and G. R. Douglas, in "Technologies for Detection of DNA Damage and Mutations" (G. P. Pfeiffer, ed.), p. 391. Plenum, New York, 1996.

16 M. E. Boerrigter, Environ. Mol. Mutagen. 32, 148 (1998).

17 M. E. Dolle, H. J. Martus, M. Novak, N. J. van Orsouw, and J. Vijg, Mutagenesis 14, 287 (1999).

18 M. E. Boerrigter and J. Vijg, Environ. Mol. Mutagen. 29, 221 (1997).

19 S. H. Jackson, J. I. Gallin, and S. M. Holland, J. Exp. Med. 182, 751 (1995).

A Effector cells Target cell

A Effector cells Target cell

B/c blasts xid blasts

FIG. 5. Phagocyte-mediated mutagenesis in neighboring B cells. (A) Scheme of the coincubation of pUR288~ effectors of mutagenesis, peritoneal exudate cells (PECs), with pUR288+ target cells of mutagenesis, splenic B lymphoblasts. (B) Mutant frequencies were determined in normal B/c lym-phoblasts (triplet of light gray columns to the left) and Btk-deficient B/c lymphoblasts (triplet of dark gray columns to the right) in the absence of PECs (left columns), the presence of normal B/c PECs (middle columns), and the presence of B6 p47phox~l~ PECs deficient in NADPH oxidase (right columns). A total of 4-25 x 105 colonies was screened in the different experimental groups.

B/c blasts xid blasts

FIG. 5. Phagocyte-mediated mutagenesis in neighboring B cells. (A) Scheme of the coincubation of pUR288~ effectors of mutagenesis, peritoneal exudate cells (PECs), with pUR288+ target cells of mutagenesis, splenic B lymphoblasts. (B) Mutant frequencies were determined in normal B/c lym-phoblasts (triplet of light gray columns to the left) and Btk-deficient B/c lymphoblasts (triplet of dark gray columns to the right) in the absence of PECs (left columns), the presence of normal B/c PECs (middle columns), and the presence of B6 p47phox~l~ PECs deficient in NADPH oxidase (right columns). A total of 4-25 x 105 colonies was screened in the different experimental groups.

Bruton's tyrosine kinase, an important signaling molecule in B cells that has been shown to play a major role in B/c PCTG.20 Figure 5B shows that normal B/c PECs induced elevations in mutant rates in both normal and xid B cells. In contrast, NADPH-deficient B6 PECs (p41ph"x '' ') were unable to mutagenize neighboring B cells. Furthermore, although the overall response pattern was highly similar in normal and xid B cells, the latter seemed to be more susceptible to phagocyte-induced mutagenesis. Taken together, the results demonstrate that the pUR288 assay is useful for both assessing cell-mediated oxidative mutagenesis in B cells and studying the effect of genetic mutations on this phenotype.

Comparison of kUZ and pUR288

In vivo mutagenesis systems are powerful experimental tools that have revolutionized mutagenicity testing. We believe they are worth the initial investment

20 M. Potter, J. S. Wax, C. T. Hansen, and J. J. Kenny, Int. Immunol. 11, 1059 (1999).

of time and resources required to set them up in the laboratory. On the basis of our own experience, we can recommend both the phage A.-based a LIZ assay and the plasmid-based pUR288 assay. Both assays are thought to reflect accurately the mutation rate in the overall genome.21 To appreciate this point, it is important to realize that transgenic shuttle vectors reside as irrelevant, heavily methylated, nontranscribed passengers in the genome. Mutations in the target genes of mutagenesis, lacl (XLYL) or lacZ (pUR288), are therefore neutral, neither beneficial nor damaging to the cell in which they occur. It follows that both assays offer appropriate indicator systems for evaluating general mutagenesis in vivo in the absence of any selective pressure of the mutated gene. A major additional argument for the usefulness of transgenic shuttle vectors is the remarkable similarity of mutational patterns in reporter genes to mutation profiles observed in endogenous genes, such as Hprt22 and Ha-ra.v and Ape?3 When it comes to choosing between /.LIZ and pUR288 for a particular mutagenicity project, it is necessary to consider some important differences between both assays (Fig. 6). The phage assay detects point mutations, frameshifts, and small deletions (<20 bp) with great efficiency, but it fails to detect larger deletions, the hallmark mutations of oxidative mutagens. However, with the pUR288 system, large deletions of up to 3 kbp, the size of lacZ, can be readily detected,24 as well as recombinations of the reporter gene with mouse genomic DNA.5 There are also important practical ramifications that distinguish both assays. The XLIZ assay requires at least 20 fig of high-quality genomic DNA, the acquisition of which may be difficult when working with rare cell types such as certain subpopulations of B cells. The pUR288 assay gets by with less DNA; 5 fig, corresponding to ~2 x 106 cells, may be enough to obtain reliable data. The XLIZ assay in its original version, which does not utilize a positive selection scheme for mutants, is more labor intensive and time consuming than the plasmid assay, which employs the clever galE~ method for mutant selection. A positive selection method for the A.LIZ ell gene has been developed25 and validated,26'27 but we have not yet implemented this version of the assay in our laboratory. The clear strength of the A.LIZ assay is the ease with which mutational

21 L. Cosentino and J. A. Heddle, Mutagenesis 14, 113 (1999).

22 R. A. Mittelstaedt, M. G. Manjanatha, S. D. Shelton, L. E. Lyn-Cook, J. B. Chen, A. Aidoo, D. A. Casciano, and R. H. Heflich, Environ. Mol. Mutagen. 31, 149 (1998).

23 H. Okonogi, T. Ushijima, X. B. Zhang, J. A. Heddle, T. Suzuki, T. Sofuni, J. S. Felton, J. D. Tucker, T. Sugimura, and M. Nagao, Carcinogenesis 18, 745 (1997).

24 S. Nakamura, H. Ikehata, J. Komura, Y. Hosoi, H. Inoue, Y. Gondo, K. Yamamoto, Y. Ichimasa, and T. Ono, Int. J. Radiat. Biol. 76, 431 (2001).

25 J. L. Jakubczak, G. Merlino, J. E. French, W. i. Muller, B. Paul, S. Adhya, and S. Garges, Proc. Natl. Acad. Sci. U.S.A. 93, 9073 (1996).

26 S. E. Andrew, L. Hsiao, K. Milhausen, and F. R. Jirik, Mutat. Res. 427, 89 (1999).

27 D. M. Zimmer, P. R. Harbach, W. B. Mattes, and C. S. Aaron, Environ. Mol. Mutagen. 33, 249 (1999).

A Effector cells Target celt

ROIs

XLIZ+ LPS blasts pUR288+ LPS blasts

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