Mutagenesis, Vol. 14, No. 3, 287-293,
May 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Characterization of color mutants in lacZ plasmid-based transgenic mice, as detected by positive selection
1 Beth Israel Deaconess Medical Center and Harvard Medical School, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115, USA and 2 Novartis Pharma AG, Toxicology/Pathology, Genetic Toxicology, CH-4002 Basel, Switzerland
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Abstract |
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The plasmid-based transgenic mouse model, which uses the lacZ gene as the target for mutation, is sensitive to a wide range of in vivo mutations, ranging from point mutations to insertions and deletions extending far into the mouse genome. In this study, the nature of subtle lacZ mutations, which do not completely abolish ß-galactosidase activity, as detected by positive selection, was investigated. These subtle mutants are called `color mutants' due to their light blue staining on X-gal medium. Replating of color mutants and retransformation of plasmid DNA, purified from individual color mutants, resulted in the same phenotype as the original color mutant. The p-gal positive selection system tolerates ~10% of wild-type activity as indicated by spectrophotometric determination of ß-galactosidase activity of individual color mutants. Restriction digestion and size separation of plasmid DNA revealed no visible change in the size of the plasmid in color mutants. Sequence analysis confirmed the presence of a point mutation in each lacZ gene of nine different color mutants. The results indicate that color mutants are caused neither by the presence of a mixture of wild-type and mutated lacZ plasmids within the same host cell nor by a mixture of cells within the original mutant colony which carry either wild-type or mutated lacZ plasmids. In addition, it was discovered that the mouse line studied harbors four polymorphic base changes among the integrated plasmid copies.
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Introduction |
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Transgenic mouse models, harboring bacterial reporter genes for the quantitative detection of mutations in diverse tissues, are used for the evaluation of mutagenic hazards (Gossen and Vijg, 1993
; Boerrigter et al., 1995; Gorelick and Mirsalis et al., 1996) and for studying the accumulation of spontaneous mutations with age (Martus et al., 1995; Dollé et al., 1997). The first such mouse models developed were based on the recovery of reporter genes as part of chromosomally integrated bacteriophage 
shuttle vectors. Two examples are a lacZ/ (Gossen et al., 1989) and a lacI/ model (Kohler et al., 1991). Although positive selection systems have been developed for both of these models, individual phage particles were screened initially by infecting Escherichia coli host cells in the presence of the chromogenic lactose analog 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal). Mutant lacZ/ phage particles give rise to colorless plaques on a background of blue plaques, whereas mutant lacI/ phage particles give blue plaques on a background of colorless plaques.
Color mutants, i.e. those in which the function of the gene product is diminished but not abolished, have been found in both lacZ/ (Douglas et al., 1994; Katoh et al., 1994) and lacI/ mice (Rogers et al., 1995). These color mutants display a homogeneous light blue color on X-gal, resulting from in vivo mutations that arose in the mouse. Mutant plaques can also appear as color mutants when they have a sectored phenotype or a mosaic genotype. Sectored plaques are defined as plaques of which some portions are blue and others colorless. Mosaic plaques show a homogeneous color, visually indistinguishable from in vivo mutants, but contain both mutant and wild-type phage. Sectored and mosaic plaques raised concerns for ex vivo mutations, which can arise from in vivo DNA damage becoming fixed as a mutation in the bacterium, or in vitro mutations, which arise during phage growth in the bacterium (Nishino et al., 1996; Stuart et al., 1996; Paashuis-Lew et al., 1997).
Color mutants have also been found in the lacZ plasmid-based transgenic mouse model (Dollé et al., 1996). In this system the lacZ reporter gene is recovered as part of the chromosomally integrated pUR288 plasmid by restriction enzyme digestion of mouse genomic DNA. The single linear plasmids are purified by LacI protein-coated magnetic bead affinity binding, followed by ligation. After concentration, the circular plasmids are electrotransferred into E.coli host cells. A positive selection system, similar to that used for lacZ/, allows growth of only those cells with mutated lacZ genes. The resulting colonies can then be analyzed for phenotypic and molecular traits to gain insight in the mutation spectrum of the mouse.
The goal of this study was to investigate the nature of color mutants derived by positive selection from brains of lacZ plasmid-based transgenic mice. The results indicate that color mutants are caused neither by the presence of a mixture of wild-type and mutated lacZ plasmids within the same host cell nor by a mixture of cells within the original mutant colony which carry either wild-type or mutated lacZ plasmids. Instead, all of them were found to be genuine mutants selected by the p-gal system below a cut-off value of ~10% of wild-type activity.
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Materials and methods |
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Genomic DNA isolation
Brain tissue samples were obtained from male 4-month-old C57Bl/6J transgenic mice harboring pUR288 plasmids at two integration sites, on chromosomes 3 and 4, respectively (Dollé et al., 1997
). Genomic DNA was isolated as described earlier (Dollé et al., 1996). In short, tissues were homogenized and digested overnight in 10 mM Tris–HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl, 1% SDS, 120 µg/ml RNase A (Boehringer Mannheim, Indianapolis, IN) and 0.5 mg/ml proteinase K (Life Technologies, Grand Island, NY) at 50°C while rotating. Digests were extracted twice with 1 vol phenol:chloroform:isoamyl alcohol (Life Technologies). Following the addition of 1/5 vol of 8 M potassium acetate, the mixture was extracted with 1 vol chloroform. DNA was ethanol precipitated, washed with 70% ethanol and solubilized in TE buffer (10 mM Tris–HCl, pH 8.0, 1 mM EDTA). DNA concentrations were determined by spectrophotometric measurements.
Plasmid rescue
Procedures for plasmid rescue have been described in detail (Dollé et al., 1996). Briefly, 10–20 µg of purified genomic DNA was digested with 40 U HindIII (New England BioLabs, Beverly, MA) in the presence of magnetic beads (Dynabeads M450 sheep anti-mouse IgG; Dynal, Oslo, Norway) that had been pre-coated with a LacZ–LacI fusion protein. Following three wash steps of the magnetic beads with 250 µl 1x binding buffer, plasmid DNA was eluted with isopropylthio-ß-galactoside (IPTG). The retrieved plasmids were circularized with T4 DNA ligase (Life Technologies) and electroporated into E.coli C (
lacZ/galE–) host cells. To determine the number of transformants a small aliquot (0.1%) of the transformed cells was plated in top agar containing 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-gal; Gold BioTechnology, St Louis, MO). The majority (99.9%) of the transformed cells was plated in top agar containing 0.3% phenylgalactoside (p-gal; Sigma, St Louis, MO), allowing growth of those cells harboring mutant plasmids. Mutant frequencies were calculated as the ratio between the number of colonies on selective (p-gal) plates versus the number of colonies on non-selective plates (X-gal) times the dilution factor (1000x).
Phenotypic characterization of colonies
Single colonies were transferred to the wells of a 96-well round-bottom, polystyrene cell culture plate (Falcon, Lincoln Park, NJ) containing 100 µl LB medium supplemented with 75 µg/ml ampicillin (Sigma) and 25 µg/ml kanamycin (Sigma) and grown overnight at 37°C, 140 r.p.m. A plate replicator was used to transfer the cultures as spots onto LB agar plates containing 75 µg/ml ampicillin, 25 µg/ml kanamycin and 0.21% D-galactose (D-gal; Sigma), 75 µg/ml X-gal or 0.3% p-gal. After 2 h incubation at 37°C, X-gal-containing agar plates were examined for blue staining cultures displaying wild-type ß-galactosidase activity. After overnight incubation at 37°C, all cultures were analyzed for growth on p-gal (to confirm wild-type/mutant phenotype) and D-gal (for galactose sensitivity) and color on X-gal (for ß-galactosidase activity).
Phenotypic characterization of individual cells and plasmids
Small aliquots of the overnight cell cultures of recovered colonies were plated at limiting dilution in 15 ml top agar containing 75 µg/ml ampicillin and 25 µg/ml kanamycin. After overnight incubation at 37°C, eight single colonies of each replated original culture were individually characterized on agar plates containing p-gal, D-gal or X-gal, as described above. The same overnight cell cultures of recovered colonies were used to inoculate 3 ml cultures in LB medium, containing 75 µg/ml ampicillin and 25 µg/ml kanamycin. Purified plasmid DNA was isolated from the 3 ml cell cultures, incubated overnight at 37°C and 225 r.p.m., using the Wizard 9600 miniprep kit (Promega, Madison, WI). Electrocompetent E.coli C (lacZ/galE–) cells were transformed with small aliquots of purified plasmid DNA and plated in 15 ml top agar containing 75 µg/ml ampicillin, 25 µg/ml kanamycin. After overnight incubation at 37°C, eight single colonies of each retransformed original culture were individually characterized on p-gal-, D-gal- and X-gal-containing agar plates, as described above.
Quantification of ß-galactosidase activity
Determination of the enzymatic activity of ß-galactosidase in the various cultures was done essentially as described by Miller (1972). In brief, 15 ml of LB broth containing 75 µg/ml ampicillin was inoculated with 0.5 ml of an overnight culture of the clone and grown to a density (OD600) of 0.28–0.70 (equivalent to 2–5x108 cells/ml; Miller, 1972). Then 1 ml of the culture was withdrawn and placed on ice to inhibit further growth. The bacterial cells were lysed by adding 2 drops of chloroform and 1 drop of 0.1% SDS solution, vortexing thoroughly and centrifuging (Eppendorf centrifuge at Vmax) for 1 min. In a centrifuge tube, 500 µl of Z buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM ß-mercaptoethanol) was added to 500 µl of the supernatant of the bacterial homogenate or 250 µl supernatant and 250 µl LB broth and allowed to equilibrate in a 28°C water bath. The blank standard used contained 500 µl Z buffer and 500 µl LB/ampicillin. The reaction was started by adding 200 µl of o-nitrophenyl-ß-D-galactoside (ONPG) solution (4 mg/ml, in Z buffer) to each tube. After a yellow color became clearly visible, the reaction was terminated by adding 1 ml of 1 M Na2CO3 solution and vortexing. The reaction time of each sample was recorded. The absorption of the solutions was determined spectrophotometrically at 420 nm versus the blank. The amount of o-nitrophenol (ONP) produced was calculated by assuming that under the above conditions 1 nmol/ml ONP has an optical density of 0.0045 (Miller, 1972). The enzymatic activity was normalized to the number of bacterial cells used, as determined from the OD600, under the assumption that a density of 1.4 corresponds to 109 cells/ml.
Molecular characterization
Purified plasmid DNA was digested with PstI and AvaI (New England Biolabs) and size separated on 1% agarose, 1x TBE gels, allowing for simple classification of the recovered plasmids into no-change plasmids, those displaying a wild-type restriction pattern, and size-change plasmids, those deviating from the wild-type restriction pattern. lacZ genes of selected miniprepared plasmids were sequenced entirely (from base 1 to 3309, with reference to the pUR288 plasmid sequence available in GenBank under the name SYNPUR288V) using the following primers (5'
3'): pUR5101-F (TCGCCACCTC TGACTTGAGC), pUR0050-F (TGCAGCTGGC ACGACAGGTT), pUR0324-F (TGGCGTAATA GCGAAGAGGC), pUR0623-F (GGAAGGCCAG ACGCGAATTA), pUR0928-F (GCGCTGTACT GGAGGCTGAA), pUR1189-F (AAGCAGAAGC CTGCGATGTC), pUR1474-F (GCATGGTGCC AATGAATCGT), pUR1694-F (CACCACGGCC ACCGATATTA), pUR2005-F (CGAACGATCG CCAGTTCTGT), pUR2293-F (GGCAACTCTG GCTCACAGTA), pUR2596-F (TTGGCGTAAG TGAAGCGACC), pUR0841-R (ATGCCGCTCA TCCGCCAC) and pUR3367-R (GCGTATCACG AGGCCCTTTC). Cycle sequence reactions were performed with the d-Rhodamine terminator ready reaction mix (Perkin-Elmer, Foster City, CA), according to the manufacturer's standard protocol, and analyzed with an ABI Prism 310 automatic sequencer (Perkin-Elmer).
Denaturing gradient gel electrophoresis
PCR amplification was carried out using 200 ng of genomic DNA, 1 (D/pUR#1) or 2 mM (D/pUR#7) MgCl2, 0.25 mM each dNTP, 0.25 µM each primer and 5 U Amplitaq Gold Taq polymerase (Perkin-Elmer) in a total of 50 µl in Amplitaq Gold PCR buffer. The following primer pairs (5'3') were used (one primer of each pair was coupled to a 40 bp GC clamp): D/pUR#1-F (GTGAGCGAGG AAGCGGAA), D/pUR#1-R (CGCCCGCCGC GCCCCGCGCC CGTCCCGCCG CCCCCGCCCG AATGAGTGAG CTAACTCA), D/pUR#7-F (CGCCCGCCGC GCCCCGCGCC CGTCCCGCCG CCCCCGCCCG TGACTACCTA CGGGTAAC) and D/pUR#7-R (TGACGGTTAA CGCCTCGA). Amplification for D/pUR#1 was carried out in a PTC-100 thermocycler (MJ Research) at 95°C for 12 min, followed by 35 cycles of 94°C for 40 s, 50°C for 50 s, 72°C for 1 min and a final extension of one cycle of 72°C for 10 min. Amplification for D/pUR#7 was carried out using the same PCR program but annealing was at 53°C. Subsequently, PCR fragments were subjected to one round of complete denaturation and renaturation, i.e. 98°C for 10 min, 50°C for 30 min and 37°C for 30 min, to create heteroduplex molecules. Ten microliters of PCR products were combined with loading buffer (0.25% xylene cyanol, 0.25% bromophenol blue, 15% Ficoll and 100 mM Na2EDTA) and loaded directly onto a 10% polyacrylamide (acrylamide:bisacrylamide 37.5:1), 40–70% UF denaturing gradient gel (100% UF = 7 M urea and 40% formamide). Electrophoresis was for 15 h at 100 V in 1x TAE buffer at 60°C. The gels were stained with ethidium bromide and photographed under UV light using the imager from Appligene.
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Phenotypical characterization of colonies
Mutant colonies were obtained from brain genomic DNA of two untreated 4-month-old male lacZ transgenic mice using the p-gal positive selection system. The average spontaneous mutant frequencies for these samples were 5.67x10–5 and 5.10x10–5 (Table I
). In order to confirm the mutant phenotype of spontaneous mutants, small aliquots of 90 mutant cell cultures (derived from single mutant colonies on the selective plate) and six wild-type cell cultures (derived from single blue colonies on the non-selective titer plate) from each sample were replated on LB agar plates supplemented with the appropriate antibiotics and X-gal (to test for ß-galactosidase activity), D-gal (to test for galactose sensitivity) or p-gal (to confirm mutant phenotype).
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After overnight incubation at 37°C a notable portion of the replated mutant colonies displayed a discernable ß-galactosidase activity that was less than that exhibited by wild-type colonies on X-gal medium (Figure 1A
). Note that the blue phenotype of the wild-type colonies appear different from that of the color mutants. This is due to slower growth of wild-type colonies, which becomes apparent in the decreased cell density of the wild-type cultures, compared with mutant cultures. The slower growth of wild-type cultures is likely to be due to trace amounts of lactose analogs in standard LB medium. Consequently, on standard LB agar plates, wild-type colonies form a culture that is less dense than that formed by mutant colonies. Nevertheless, the blue staining of wild-type cultures can be observed as early as 1 h after plating whereas visualization of the blue phenotype of color mutants requires at least 12 h (results not shown).
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A small fraction of the tested colonies was galactose-insensitive, indicated by growth on D-gal medium (Figure 1B
). These cells have lost their galactose sensitivity presumably due to a forward mutation in their galK or galT gene or a reverse mutation creating a functional galE gene and most likely contain wild-type plasmid, as is indicated by their blue staining on X-gal (Figure 1A). Moreover, galactose-insensitive colonies can be recognized on X-gal plates where they stain blue as soon as 1 h after plating, as do `normal' wild-type colonies (results not shown). Since galactose-insensitive colonies are not inhibited by lactose analogs in standard medium, these cultures containing wild-type plasmid do not show an inhibited cell growth like `normal' wild-type cultures.
All mutant colonies were able to grow to some degree on LB agar plates containing 0.3% p-gal, indicating that these were true mutants (Figure 1C). Note that with an increase in the residual ß-galactosidase activity of mutant colonies, as based on their color phenotype (Figure 1A), there was a decrease in their ability to grow on the p-gal plates. Wild-type colonies were not able to grow in the presence of p-gal (Figure 1C).
A summary of the 90 characterized mutant colonies per DNA sample is given in Table I. By visual inspection of the relative color phenotypes on X-gal medium, each mutant was subdivided into one of five classes, ranging from colorless (–) to dark blue (++++). The classification shows that all color mutants combined for these two samples make up on average 50% of the mutation spectrum in brain.
Verification of characterized color mutants
The ability to accurately measure the spontaneous mutant frequency in any tissue of the mouse depends critically upon all the cells in each colony being of the same clonal origin. In principle, it is possible that during the first rounds of replication a mutation in lacZ, either arising `spontaneously' or as the consequence of DNA damage, will give rise to a colony with a `mosaic' phenotype, i.e. a mixture of cells with either blue or white phenotypes. For example, if a mutation in lacZ occurs in E.coli during the first replication round, then half of the cell's progeny will exhibit no blue color. Such mutations are commonly referred to as ex vivo mutations and are not to be confused with color mutants.
To investigate whether or not mutant colonies of light blue color had such a mosaic phenotype, eight individual cell cultures per original colony were grown on X-gal and visually inspected. A total of 41 color mutants, seven white mutants, two galactose-insensitive colonies and six wild-type colonies, obtained from brain sample 1 (see Table I) were examined. Within each group of eight cultures blue color and growth characteristics were similar to each other and to the original colony from which they were derived (results not shown). Since the possibility could not be excluded that individual cells of each colony contained a mixture of wild-type and mutated lacZ plasmids, plasmid DNA purified from cultures of the same 56 original colonies used for the replating experiment above was electroporated into E.coli host cells. Eight individual colony cultures from each transformation were examined on X-gal. Also in this case, each set of eight cultures was identical in color and growth characteristics to the original colony (a sample is shown in Figure 2). Exceptions were the retransformed plasmids from galactose-insensitive colonies that now showed the inhibited growth characteristics of `normal' wild-type cultures (Figure 2, column 5). Occasionally, blue colonies became overgrown with `white' cells (see colonies in column 6 and colony f9 in Figure 2). It was demonstrated that these cells had lost their plasmids and were permitted to grow due to exhaustion of the ampicillin. Thus, it can be concluded that the contribution of ex vivo mutations to the spontaneous mutant frequency in the mouse genome is negligible.
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Quantification of ß-galactosidase activity
To obtain an indication of the stringency of the p-gal positive selection system, ß-galactosidase activity was quantitatively measured for the set of 56 cell cultures. Crude protein extracts were obtained from culture lysates. After normalization for cell number, the ß-galactosidase activity was spectrophotometrically determined by the rate of ONPG conversion into ONP. The results are shown in Figure 3
. The mutant cultures show a range of activities from not detectable to a level ~10% of that found in wild-type cultures. As expected, the galactose-insensitive cultures show similar activity levels as `normal' wild-type cultures.
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Molecular characterization
The mutant colonies displaying some residual ß-galactosidase activity most likely represent base substitutions. Indeed, the 41 color mutants that were analyzed by AvaI/PstI double digestion of purified plasmid DNA followed by agarose gel electrophoresis displayed a pattern similar to that obtained with purified plasmid DNA from wild-type colonies (results not shown). These findings are consistent with an earlier report (Dollé et al., 1996
) and spectral analyses of >2400 spontaneous mutants (unpublished), showing that size-change mutants, defined by those deviating from the wild-type restriction pattern on agarose gel, are only to be found among colorless colonies.
To confirm the presence of a base change in color mutants, the entire lacZ gene (3309 bp) of two randomly selected mutants per color class as well as two wild-type colonies, one galactose-insensitive colony and one colorless no-change mutant was subjected to sequence analysis. Compared with the deposited pUR288 sequence in GenBank, the consensus sequence showed four polymorphisms, present in all 13 lacZ genes (Table II). In addition, four other polymorphisms, two of which were linked (Table II, base positions 81 and 507), were found in only two of the 13 sequenced lacZ genes. These uncommon polymorphisms were found in both wild-type and mutant plasmids. In mutant plasmids such a polymorphism was always accompanied by a unique mutation (compare Tables II and III). At least one unique mutation was found in each color mutant; three mutants showed two or three unique base changes per lacZ gene (Table III). The one colorless mutant sequenced was found to be a frameshift mutation, due to a single base deletion (Table III, 42). With the exception of mutant 22, which appeared to be a single base frameshift deletion, all color mutants showed base substitutions. The frameshift mutant, 22, was confirmed to be a genuine color mutant by analyzing the colony color after transformation of E.coli host cells with three independently sequenced different plasmid preparations (results not shown).
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The mutation spectrum found for the color mutants (Table III
) had the following characteristics. The two AG transitions (20 and 8) occurred at identical sequences, changing the second adenosine in a run of four, flanked by a guanosine upstream and a cytidine downstream. One of the two base pair deletions occurred in a run of six adenosines (42). Eight out of 12 base pair substitutions involved G:CA:T transitions, most of which resulted in termination codons (see Table III).
Confirmation of lacZ sequence polymorphisms
To confirm that the polymorphisms found among the sequenced lacZ genes, as shown in Table II, are present in the genomic DNA of the transgenic mouse strain studied (line 60), genomic DNA from three individual mice was subjected to analysis by denaturing gradient gel electrophoresis (DGGE). Using this method, in combination with heteroduplexing, mixtures of two sequence variants are detected as two homoduplex molecules and two earlier melting heteroduplex molecules. It has been demonstrated that the method can detect as little as one sequence variant per 100 wild-types (van Orsouw et al., 1998). Plasmid preparations with the polymorphic and the consensus sequence and genomic DNA from a pUR288 transgenic strain with a different integration site (line 30, chromosome 11) were used as controls. The migration patterns for polymorphisms at base positions 81 and 1230 are shown in Figure 4A and B, respectively. The first two lanes in each figure show one of the two homoduplex molecules amplified from the consensus (W) and the polymorphic (P) sequence. A mixture (M) of both templates results in both homoduplex molecules and the addition of two heteroduplex molecules. Genomic DNA templates from line 60 mice (601–603) resulted in a pattern showing both heteroduplex molecules, but only one detectable homoduplex molecule. The absence of one homoduplex variant is caused by an excess of amplified products from the consensus template annealing with the majority of the amplified products from the polymorphic template forming both heteroduplex molecules. In the other independent transgenic founder line, line 30, these same polymorphisms as discovered in line 60 were not detected. This conclusively demonstrates the occurrence of sequence variants in the lacZ gene among the different plasmid copies integrated head to tail in the line 60 mouse.
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Discussion |
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In principle, the light blue phenotype of the color mutants, detected in the pUR288 transgenic mouse system, could be attributed to a mixture of cells containing either a wild-type (blue) or a mutated (white) lacZ plasmid. Alternatively, cells might contain both wild-type and mutated lacZ plasmids, in which case the blue phenotype would be determined by the proportion of fully functional and non-functional ß-galactosidase protein within each cell. The data presented in this paper strongly argue against these possibilities and instead provide evidence for the true nature of these color mutants. The finding that replating a single mutant colony yields daughter colonies with the same color phenotype argues against the possibility that color mutants are a mixture of cells containing either wild-type or mutated lacZ plasmids. Secondly, transformation of host cells with plasmid DNA isolated from a color mutant always yielded daughter colonies with the same color phenotype. This last observation indicates that cells do not contain a mixture of wild-type and mutated lacZ plasmids. Finally, sequence analysis revealed that most spontaneous color mutants yielded single base pair substitutions in the lacZ genes of these mutants. In color mutants with multiple base pair substitutions, the additional mutations were mainly silent mutations, presumably not affecting the enzyme activity. Therefore, the color mutants as detected by positive selection in lacZ plasmid-based transgenic mice represent true mutants.
The determined ß-galactosidase activities of the color mutants show a cut-off value at ~10% of that found in wild-type colonies (Figure 3). The cut-off value suggests that either the p-gal selection system does not tolerate a higher ß-galactosidase activity or that there are no color mutants with a higher activity. It is hard to imagine that in a gene of 3 kb no other subtle mutations can be made, resulting in a higher activity than the 10% cut-off value. Especially when the variation in activity of the color mutants is considered, it seems likely that more subtle mutations would occur. This implies that the p-gal selection system, by suppressing more subtle color mutants, underestimates the true mutant frequency. However, the finding that only 13 of the 48 color mutants had a measurable activity spread out over three log intervals indicates that only a minor contribution is to be expected from the remaining log interval from the cut-off to the wild-type activity. Based on the numbers above and the finding that color mutants only comprise 50% of the total spectrum in brain, one might roughly estimate that p-gal positive selection is responsible for a 5% underestimation of the mutant frequency. A 10% loss of lacZ mutants was reported for the lacZ/ system after replating the X-gal-detected mutant plaques on p-gal (Jiao et al., 1996). The same report also mentions that compared with the X-gal system, there is no commensurate reduction in mutant frequency, possibly because any potential reduction is offset by an increased ability of the p-gal system to capture mutants below a threshold of ß-galactosidase activity. The 2-fold difference between the estimated loss of p-gal-selected color mutants between the lacZ-/bacteriophage and the lacZ/plasmid system might be due to increased selection pressure, caused by overexpression of the galK and galT genes in the E.coli host used in the phage system (Mientjes et al., 1994).
Presumably a far larger underestimation of the mutation frequency comes from undetected silent or neutral mutations not resulting in a mutant phenotype, as is indicated by some of the sequenced plasmids showing multiple base pair changes (Table III) and the polymorphisms found among all sequenced plasmids (Table II). But as far as the lacZ reporter gene is being used as a risk assessment tool for endogenous genes, one might question the effect of silent and neutral mutations on the function and regulation of the protein.
The origin of the polymorphisms is unclear. They might have been present in the original plasmid population used to make the transgenic mice by microinjection. However, the second founder line does not seem to contain these polymorphisms and might therefore have originated from a different plasmid batch. Alternatively, these polymorphisms might have been caused by the integration process, which is still not understood. The polymorphisms are potentially useful to recognize specific plasmids within the concatemer if their relative locations can be resolved. This will permit, for example, discrimination between intra- and inter-plasmid deletions, a distinction that cannot be made thus far.
Of all base pair substitutions in the mutation spectrum for brain color mutants (Table III), 75% consisted of G:CA:T transitions. Only one of the G:CA:T transitions occurred at a 5'-CpG dinucleotide (17), whereas almost all other transitions occur at the middle nucleotide of the 5'-TGG triplet or its reverse complementary sequence (5'-CCA). In both the lacI/ and the lacZ/ system G:CA:T transitions at CpG sites occur at a relatively high frequency and are cited as evidence for their in vivo (in the mouse) origin, by explaining them through the methylation–deamination pathway (Gossen and Vijg, 1993; Knöll et al., 1994). An explanation for the relatively small number of mutations at CpG sites in our present study could be that the mutants analyzed in Table III were derived from the brain, whereas the spontaneous spectra for both systems were obtained from liver, bone marrow, spleen and germ cells. It is not inconceivable that organ-specific mutation spectra play a role. But perhaps the most likely explanation is that the presented spectrum in Table III did not derive from random selected mutant colonies, but rather from selected color mutants. The amino acid change in many of these color mutants resulted in a termination signal. Despite the termination signal, these mutants still displayed a blue phenotype in the presence of X-gal. Apparently, the E.coli host strain used has the tendency to read through these termination signals at a low frequency, resulting in the color mutant phenotype. This relaxation toward termination codons of the E.coli host might very well explain the enrichment of G:CA:T transitions, almost all resulting in termination signals, when color mutants are selected specifically. Indeed, some of these exact mutations have been found before with the lacZ/ system, although there they were typed as clear plaques (see Table III, footnote d). Presumably, the host strain used in those experiments reacted more stringently to termination signals.
The mutation found in mutant 22 (Table III) appeared to be a frameshift (–G) at 412 bp from the translation start site. This type of frameshift results in a drastic change in the downstream amino acid sequence, yet the light blue phenotype as well as the mutational change were confirmed three times. An explanation for this frameshift mutant with some residual ß-galactosidase activity could be that during transcription RNA polymerase allows for slippage with a low frequency near the mutated side, reversing the frameshift. Several reports have shown that RNA polymerase in E.coli allows slippage or reiterative copying in a run of T or A residues (Jacques and Susskind, 1990; Wagner et al., 1990; Jin, 1994; Qi and Turnbough, 1995). Fifteen base pairs downstream of the frameshift a run of five T residues occurs that might be responsible for the proposed slippage event, leaving only six amino acids changed in the full-length transcript. Since these six amino acids are not part of any reported active site of ß-galactosidase (Huber et al., 1994), the resulting protein may still be functional.
In conclusion, the sensitivity of lacZ plasmid-based transgenic mice for true color mutants contributes to the capacity of this system to detect a broad range of mutations ranging from large size changes that inactivate lacZ completely to single base pair changes that only partly inactivate the lacZ gene. The relaxation of the E.coli host strain, even for mutations resulting in a premature termination signal, causing the color mutant phenotype, simplifies the molecular characterization of the mutations, i.e. all mutant colonies with a blue color on the X-gal medium can now be assumed to be point mutations.
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Acknowledgments |
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We thank Wendy Smith for photographic assistance and J.M.LaPlante for critical reading of the manuscript. This work was supported by NIH grants PO1 1801 AG10829-01, 1 P30 AG13314-01, 1 RO1 ES/CA 08797-01 and AG08812.
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Notes |
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3 Present address: Institute for Drug Development, Cancer Therapy and Research Center, 8122 Datapoint Drive, Suite 700, San Antonio, TX 78229, USA
4 To whom correspondence should be addressed. Tel: +1 210 616 5850; Fax: +1 210 692 7502; Email: mdolle{at}saci.org
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References |
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Received on July 17, 1998; accepted on December 7, 1998.
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