Transfer, methylation and spontaneous mutation frequency of ΦX174am3cs70 sequences in medaka (Oryzias latipes) and mummichog (Fundulus heteroclitus): Implications for gene transfer and environmental mutagenesis in aquatic species

Marine

Environmental

Research,

Vol. 40, NO. 3, pp. 2477265, 1995 Elsevier Science Ltd Printed in Great Britain

0141-1136(94)00144-S

ELSEVIER

Transfer, Methylation and Spontaneous Mutation Frequency of fDX174am3cs70 sequences in Medaka (Ovyzias latipes) and Mummichog (Fundulus heteroclitus): Implications for Gene Transfer and Environmental Mutagenesis in Aquatic Species Richard N. Winn,” Rebecca J. Van Benedenb & James G. Burkhart”* “Environmental Toxicology Program, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, North Carolina 27709, USA ‘Department of Zoology, University of Maine, Orono, Maine 04469-5751, USA

ABSTRACT This study describes the production of transgenic medaka (Oryzias latipes) and mummichog (Fundulus heteroclitus) containing multiple copies of the bacteriophage @XI 74am3cs70. This work is an initial approach for measuring mutations in aquatic species using the same gene target sequence in fish and laboratory mammals. The @Xl 74 sequence is unique in that there is no detectable homology with chromosomal DNA of medaka, mummichog or mice. The authors have compared cytoplasmic injection of 1-2 cell embryos with linear single copy and catenated constructs of the phage DNA. The catenated construct results in greater efficiency of gene transfer for both species in terms of copies per cell. Analyses of DNA from founder transgenic fish with methylation sensitive (HpaII) and methylation insensitive (154.~~1)restriction enzyme isoschizmers indicates CpG methylation of the integrated @Xl 74 sequence. This study also demonstrates the esJicient rescue of live phage from the chromosomal DNA of founder fish in suficient numbers to determine a spontaneous mutation frequency for reversion of am3. A pooled sample of 20 ug DNA from four fish yielded I .09 x IO7progeny phage with a spontaneous mutation frequency of I .83 x 10e7. This spontaneous mutation frequency is similar to the spontaneous frequency for the same gene indictor recoveredfrom transgenic mice. These results demonstrate that Jish containing multiple copies of @Xl 74 can be produced with no obvious detrimental efects and that the overall approach may be useful in basic and applied studies of environmental mutagenesis.

*To whom correspondence

should be addressed. 247

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INTRODUCTION The potential for adverse health effects in both human and natural populations by pollution of water systems, groundwater, rivers and estuaries is a growing concern. Carcinogenic responses to environmental chemicals have been demonstrated in several species of both wild and laboratory fish (Hawkins et al., 1988; Murchelano & Wolke, 1991; Meyers et al., 1991; Harshbarger et al., 1993; Law et al., 1994). Chemically-induced mutation is an important event leading to cancer and increases in other diseases, yet there are few methods available in aquatic species which can be applied to the assessment of genetic hazard, or focus on the study of gene mutations as they occur at the DNA level in vivo (Kligerman, 1982; Landolt & Kocan, 1983; McMahon et al., 1988; Wirgin et al., 1989; Anderson & Harrison, 1990; Van Beneden et al., 1990, 1993). Effects of X-irradiation on segregation and mutation induction in male and female germ cell stages have been demonstrated in the guppy (Lebistes reticulatis Peters), but the data was insufficient to establish mutation rates for specific loci (Shriider, 1969; Shriider & Holzberg, 1972). Chakrabarti et al. (1983) demonstrated increases in mutation frequencies at the gol-I locus in zebrafish (Brachydanio rerio) sperm after exposure to y-radiation. More recently, tester stocks of medaka (Oryzias latipes) containing visible specific recessive mutations at five loci have been developed and used to demonstrate chemical and y-radiation-induced increases in mutation comparable to specific locus mutation data obtained from mice (Shima & Shimada, 1988, 199 1) A capacity to detect a new mutation as it occurrs on a temporal scale in somatic and germinal tissue would enhance our understanding of the impact of environmental mutagens on individuals and populations. Studies of mutagenesis in aquatic species and mammals have been impeded by the large numbers of observations that are required: in many cases the number of animals and data points required to evaluate mutagenic effects exceed the available resources. There is also a challenge to understand how observations in one species may (or may not) relate to effects in other species. The biological complexity of this problem could be reduced by using the same gene marker for mutation in a variety of species, independent of any requirement for expression, growth or selection in tissues. Analytical constraints are associated with mutations expected to occur in vivo at a frequency in the range lop5 to lo-’ per base pair. This requires: (1) each animal to provide many individual gene markers at the single copy level; (2) efficient recovery of the single-gene copies; and (3) unambiguous detection of mutants among the total number of genes analyzed. At this time technologies do not exist to realistically meet these experi-

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mental conditions with natural chromosomal gene sequences, especially in species where there has been little genetic analysis at the DNA level. Advances in gene transfer technology suggest that transgenic fish may offer research opportunities which can expand their role as environmentally relevant animal models (Chen & Powers, 1990). Several factors related to mosaicism, integration of low transgene copy numbers, and variable gene expression have also influenced development and use of transgenic fish (Stuart et al., 1990; He et al., 1992; Maclean et al., 1992; Ozato et al., 1992a; Vieland, 1992). Our initial efforts in the area of environmental mutagenesis’ in fish have been to develop an in vivo mutation detection system using transgenic fish with a chromosomally integrated and recoverable bacteriophage, QX 174, containing a marker for mutation. Following exposure to a mutagenic agent, the vector DNA sequence is recovered from host genomic DNA, transfected into a specialized strain of E. coli for packaging, then plated with various selective indicator bacteria to determine the total number of progeny phage recovered and the number of mutants. In the work reported here, we have used QX174am3cs70 to produce the transgenic fish; mutants are detected by reversion of am3 to wild-type phage by one transition and two transversions of A:T base pair 587. We have chosen to take advantage of the sensitivity and specificity provided by a reversion assay combined with no requirement for DNA sequencing. The previous use of various @X174 strains to study fidelity of DNA replication and repair in a variety of models provides a solid base for understanding the nature and potential origins of mutations detected (Weymouth & Loeb, 1978; Kunkel & Loeb, 1979; Chambers et al., unpubl., 1988; Malling & Burkhart, 1989; Chambers, 1991; Deschavanne & Radman, 1991). In the development of the @X174 system for mutation detection in cultured mammalian cells, low numbers of phage sequences per host genome contributed to low recovery of the phage (Burkhart & Malling, 1989). Subsequently, in the production of transgenic mice, the copy number was increased by catenating the DNA. However, in generations beyond F2, the efficiency of phage recovery per copy was dramatically reduced (Burkhart et al., 1992, 1993). The loss in efficiency was the result of hypermethylation and selection by the E. coli recovery host against hyper-CpG-methylated DNA. We have recently been able to develop a method for recovery of @X174 that is CpG-methylation independent, but depending on specific target sequences, CpG methylation continues to play a role in the interpretation of mutation data (Burkhart & Malling, 1993). The CpG methylation status of transgenes in fish has been examined in one recent study and found to be variable in tissues (Maclean et al., 1992). The study demonstrated the need for further analysis of the

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role of methylation in the function of transgenes in fish, especially considering the impact of CpG hypermethylation on transgene expression in later (beyond F3) generations of mammals (Doefler, 1983; Cedar & Razin, 1990; Razin & Cedar, 1991). Our efforts to adapt the @X174 system to fish focused on medaka (Oryzius Zatipes) and mummichog (Fundulus heteroclitus). We compared the efficiency of incorporation of the transgene into the nuclear genome via cytoplasmic injection of 1-2 cell embryos with either single gene copy or catenated @X174am3cs70 RFDNA (double stranded replicating form of phage DNA). The CpG methylation was also studied in founder fish. We have established a preliminary spontaneous mutation frequency among phage sequences recovered from the founders which can be compared with mutation frequencies for the same sequence in mammals and in cultured mammalian cells. The production of transgenic fish containing a recoverable marker for mutation seeks to utilize new molecular technologies to ask basic questions about mechanisms of mutagenesis continuously, in all stages of gametogenesis, fertilization, development and aging. At the same time hazard assessment may be possible in aquatic organisms that are relevant endpoints for environmental exposure.

MATERIALS

AND METHODS

@X174 DNA preparation Prior to injection, circular bacteriophage (5.2 kb) was linearized with PstI (cuts once was ligated briefly with T4 DNA ligase (New a ladder of @X174 catenates between 2-10 1993).

QX 174am3cs70 RFDNA within the sequence) and England Biolabs) to form copies (Burkhart et al.,

Culture and micro-injection Adult medaka were obtained from a laboratory-reared population, maintained in freshwater at 22-24°C and placed in a 16 h light/8 h dark photocycle. Newly fertilized eggs were collected from the female’s vents within 30 min post-fertilization. Fundulus heteroclitus adults were obtained from a laboratory-reared population and from local feral fish (Beaufort, NC, USA) and maintained in 20 ppt artificial seawater (Instant Ocean) at 24°C in a 16 h light/8 h dark photocycle. Fertilized eggs were either collected from aquaria filters

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within 30 min post-fertilization, or eggs were stripped from females and fertilized in vitro by mixing with sperm of captive males in a culture dish of seawater. A solution of either single gene copy or catenated @X174 DNA (10-15 mg/ml in TE (1 mM EDTA, 10 mM Tris, pH 7.4) buffer) was injected into the cytoplasm of the embryos at the l-2 cell stage, using a glass injection pipette (2-3 pm) connected to a gas-driven (N2) injection apparatus (PLI 1 Pica Injector, Medical Systems Corp.) under a dissection microscope (25-50 x ) equipped with micromanipulators. Aliquots of eggs were maintained at 4°C for up to 2 h to delay development prior to injection. Hatching of medaka occurred within 10-12 days at 22°C while hatching of F. heteroclitus took place within 10-15 days at 22°C. Analyses of presumptive transgenic fish DNA was obtained from caudal fin clips by homogenization of the tissue in 0.3 ml 1 x SSC (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 0.5% SDS, and 10 mg/ml proteinase K for l-3 h at 55°C. Samples were extracted twice with methylene chloride:isoamyl alcohol (24: 1) containing 0.15M NaCl, precipitated with two volumes 100% ethanol and resuspended in TE buffer. Samples containing @X174 DNA were detected by Southern blot hybridization. Genomic DNA (5-10 pg) was digested with PstI, followed by electrophoresis in a 0.8% agarose gel, transfer to a nylon membrane and hybridizaton to nick-translated biotinylated @X174 RFDNA (PolarPlex Chemiluminescent Kit, Millipore/New England Biolabs). Prehybridization was performed at 42°C for 1 h in a 20 mM sodium phosphate buffer (pH 7.5) containing 50% formamide, 5 x SSC, 5 x Denhardt’s solution, 0.1% SDS and 100 pg/ml denatured calf thymus DNA. Hybridization was performed at 42°C for 16 h with about 20 ng of the biotinylated probe. Membranes were washed twice at room temperature for 5 min in a solution containing 2 x SSC and 0.1% SDS, and twice for 15 min at 42°C in a solution of 0.2 x SSC, 0.1% SDS. Membranes were analyzed according to the manufacturer’s detection protocol with film exposures of 20-60 min. Copy number standards, prepared by adding @X174 DNA (equivalent to l-20 copies per genome) to calf thymus DNA (5 pg), were digested with PstI and loaded on the agarose gel adjacent to the lanes containing DNA from presumptive transgenic fish. Copy number estimates were made by using the DNA content of 2.2 pg (Hinegardner & Rosen, 1972) and 2.72 pg (Dawley, 1992) for the diploid genomes of medaka and mummichog respectively.

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Determination of CpG methylation

The potential for CpG methylation of the @X174 sequences was evaluated by using the restriction enzyme isoschizmers HpaII (methylation sensitive) and MspI (methylation insensitive). Both enzymes recognize a CCGG sequence and @X174 contains five sites for these enzymes. HpaII will not cleave if the second cytosine is methylated at the 5’ position. MspI will cleave the sequence independent of methylation. Genomic DNA (S-10 pg) from founder transgenic fish was digested simultaneously with either HpaII or MspI and PstI, followed by electrophoresis, and Southern blot hybridization as described above. Control @Xl 74 RFDNA (completely unmethylated) equivalent to 20 copies per fish genome was digested with either PstI or HpaII and MspI. Recovery of the @X174 sequence from genomic DNA

DNA was prepared as previously decribed and resuspended in TE to a final concentration of 0.25 pg/ml as determined by OD260. The @X174 phage sequence was partially purified from the fish genomic DNA as described by Burkhart et al. (1993). In brief, the tissue DNA (also contains RNA) was digested overnight at 37°C with 5 U Pst I (one restriction site in QX) and 3 U Sau3A (does not cut QX) per pg DNA in a volume of 500 ml of 10 mM MgC12, 1 mM dithiothreitol, 10 mM bis tris-propane HCl pH 7.0 (NEB). Digests were heated to 65°C for 5 min, incubated 1 h with 10 pg proteinase K at 55°C phenohchloroform and chloroform extracted, ethanol precipitated and resuspended in TE to a concentration of 0.125 pug DNA/ml; 40 ml was loaded onto minicolumns (Isolabs) containing TE equilibrated Sephacryl S-1000 (Pharmacia) and eluted with 350ml TE. Monitoring of the 0D260 indicated that greater than 99% of the genomic DNA and RNA was removed by the S-1000 gel filtration. Recovered DNA was ligated overnight at 12°C in a total volume of 500 ml with 400 U E.coli DNA ligase (NEB) with the supplied buffer and then heated to 90°C for 10 min. The ligated samples were dialyzed four times with Hz0 in 100000 MW cutoff filters (Ultrafree, Miilipore) that were previously blocked by incubation with 1 mg/ml salmon sperm DNA and rinsed with H20. Final volumes were reduced to 25-50 ~1. Samples were then mixed with 350 ml of competent DXRl cells and electroporated (BTX model 600) at 2.0 kV and 720 (Burkhart et al., 1992). Electroporated cells were incubated with 10 ml SOC medium (BRL) at 37°C and 300 rpm for 2 h. Packaged phage were released by addition of 1 ml of 10 mg/ml lysozyme (sigma), 50 mm tris-HCl pH 8.0 and 3 ml of 30 mM edta, 30 mm borate, 30 mm tris-HCL pH 8.0 and incubated at 37°C and

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300 rpm for 30 min. Finally, 1 ml of chloroform was added, the sample was vortexed thoroughly, and the chloroform allowed to separate from the aqueous phase. Determination of spontaneous mutation frequencies The DHXRl cell line was used as the competent cell line for packaging the electroporated RFDNA sequences. DXRl does not select against DNA on the basis of eukaryotic CpG methylation and has been shown to be resistant to any subsequent infection by intact @X174 phage. The total number of packaged phage recovered from a sample of transgenic fish DNA was determined by plating small aliquots (50 ml) of serially diluted phage suspension on E. coli CQ2 which contains the suppressor for the amber @X174am3 mutation. The frequency of am3 reversion was determined by plating the remainder of the phage suspension from a given fish sample on E. coli C which does not contain the am3 suppressor at a density of approximately 5 x lo5 phage/ml culture (OD600 = 0.5). The frequency of revertants (mutants) among the recovered packaged phage was calculated as the ratio of the number of plaques appearing on E. co/i C divided by the estimated total number of phage in the sample derived from titration on E. coli CQ2.

RESULTS

AND DISCUSSION

Oryzias latipes and Fundulus heteroclitus as transgenic models for in vivo

mutagenesis Medaka and mummichog as experimental fish have the potential for use in a variety of field applications and laboratory experiments (Yamamoto, 1975; Courtney & Couch, 1984; Atz, 1986; Eisler, 1986; Egami et al., 1990; Vieland, 1992). Medaka is an excellent animal model used extensively for studies of chemical carcinogenesis and toxicology in laboratory settings (Hoover, 1984; Hawkins et d., 1986; Van Beneden et al., 1990). Medaka is used for transgenic fish development because of desirable features such as small size, manipulatable eggs, short embryogenesis and generation times, and ease of culture (Ozato et al., 1989; Ozato et al., 1992b). Various foreign genes, including chicken delta-crystallin, bacterial chloramphenicol acetyl transferase, rainbow trout growth hormone, luciferase, and human growth hormone have been introduced into medaka (Inoue et al., 1989, 1990; Ozato et al., 1989; Tamiya et al., 1990; Lu et ai., 1992). Expression and/or germ-line expression was demonstrated in several laboratories and

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Medaka

Catenated Construct

Single Copy Construct w

McP64Mc075

(a> Lane

QX Copy Standards

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FQ8

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Fm49

F@55

F@66

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Fig. 1. Southern blot analyses of fin clip DNA (10mg) from selected medaka and mummichog digested with PstI and hybridized with a a biotinylated +X174 probe. (a) Medaka injected with a single copy 9X174 construct carried < 1 @Xl74 copy/diploid genome (lanes l-3) while those injected with catenated @X174 (lanes 47) carried > 5 copies by comparison with prepared copy number standards of 1, 5 and 10 copies (lanes 8-10). (b) Mummichog injected with single copy +X174 carried < 1 copy/diploid genome (lanes 510) and representative fish injected with catenated @Xl74 (lanes 5-10) carried > 5 copies by comparison with the prepared copy number standards (lanes 12-15).

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in aquatic species

genetically defined stocks were produced. F. heteroclitus has been a valuable research animal for more than 100 years and may be the subject of more interdisciplinary contaminant risk assessment studies than any other marine organism (Atz, 1986; Eisler, 1986). F. heteroclitus is a natural estuarine species capable of survival and propagation under a wide range of environmental conditions. Individuals tend to be territorial and therefore subject to chronic exposure to contaminants. These characteristics make it an environmentally realistic animal for hazard assessment and ecotoxicological study (Lotrich, 1975; Vogelbien et al., 1990). Despite extensive use in other research, we have not found evidence for F. heteroclitus as a subject for transgenic development. Like medaka, F. heterocfitus has many characteristics including an extensive literature on oogenesis, fertilization and embryology, relatively small adult size (adults 7-10 cm), controlled year-round spawning, manipulatable egg (diameter 1.8 mm) with a transparent chorion, short embryogenesis (10-15 d at 22”C), and short generation time (3-5 months). Inbred lines have not been reported but the ease of culture indicates that development of lines should not be an obstacle. Production of transgenic founders: Single copy and catenated constructs Southern blot analysis of genomic DNA from injected survivors (> 2 months old) indicated that cytoplasmic injection of either the single copy or catenated linear constructs resulted in detectable @X174 sequences in both species (Fig. 1). Differences in survival and efficiency of incorporation could not be directly attributed to the form of the construct. Transgenic founders occurred with higher frequency in F. heteroclitus than in medaka for both forms of the construct (p < 0.05; Table 1). Injection of the single TABLE 1 Survival to Hatching, Percent Positive for Integration and Estimated Copies per Diploid Genome Detected in Medaka and Mummichog Produced from Injection of Single Copy and Catenated @X174 Constructs @Xl 74 construct

% Survival (hatched/injected)

(a) Medaka (Oryzias latipes) Single copy 18% (63/347) Catenated 35% (44/127) (b) Mummichog (Fundulus heteroclitus) Single copy 53% (18/34) Catenated 22% (82/380)

% Positive (positive/screened)

Copy no./genome

6% (4/63) 9% (4/44)


16% (3/18) 20% (16/82)


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copy linear construct in medaka resulted in 6% of the surviving fish positive for the QX, while 9% of the fish injected with the catenated form were positive. For I;. heteroclitus, 16 and 20% of the survivors injected with the single copy or catenated form respectively showed incorporation. The number of copies of @X174 per diploid genome was estimated by comparing the intensities of copy number standards (copies of @X174 per diploid genome) with bands of genomic DNA digested with PstI and hybridized to a @X174 probe (Table 1; Fig. l(a) lanes l-3 and Fig. l(b) lanes l-3). Both medaka and F. heteroclitus founders injected with the single copy construct carried relatively low numbers of gene copies (1 to less than 1 copy per diploid genome). In contrast, fish injected with the catenated construct carried multiple copies with approximately five @X174 copies per diploid genome in medaka (Fig. l(a) lanes 4-7) and 515 copies per genome in F. heteroclitus (Fig. 1(b) lanes 5-10). The majority of the positive F. heteroclitus (75%; 12/16) injected with the catenated construct had > 5 gene copies per genome. In the production of transgenic fish where the foreign DNA is injected into the cytoplasm, and chromosomal integration occurs after subsequent cell divisions, most of the transgenic founders are mosaics (Stuart et al., 1990; Fletcher et al., 1992). Although the degree of mosaicism makes the true copy number indeterminate in founders, the data presented here indicates transfer of multiple copies of @X174 into both species. Survival to hatching in medaka injected with catenated construct was higher than that in those injected with the single copy construct (18 versus 35%; Table 1) while the opposite was true for F. heteroclitus (53 versus 22%). A greater proportion of positive fish was produced in mummichog compared with medaka independent of the construct. For both species, catenated constructs provided slightly (but not statistically significant) higher proportions of founders. One explanation for the differences in survival among species may be the variability of F. heteroclitus eggs collected from feral fish late in the normal spawning season (September-October). Reduced hatching from late spawners has been observed previously. Alternatively, the interaction of DNA concentration and differences in the eggs may have contributed to the combinations of lowered survival and the apparent higher number of copies observed in mummichog compared to medaka. Injection of high concentrations of DNA has been shown to produce higher proportions of transgenic fish while also increasing mortality (Penman et al., 1990). Despite the use of similar injection procedures for each species (DNA preparation, injection apparatus, injection at l-2 cell stage) a larger and more systematic analysis is necessary to evaluate the effects of these factors on differential survival and integration.

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Our objective in using a catenated construct was to increase the number of phage genomes that could be recovered per unit of host genomic DNA and make the analysis of mutation feasible. The use of catenated constructs with sequences such as @X174 may have additional utility for gene transfer in fish or other species where introduction of foreign genes via cytoplasmic injection is the most common method (Moav et al., 1992). DNA introduced into the cytoplasm is subject to degradation by exonucleases and the potential for chromosomal integration of single copy constructs is subsequently reduced (Chong & Vielkind, 1989). Injection of catenated constructs may permit a higher proportion of intact sequences to be integrated. Non-expressed sequences such as @X174 could also be ligated to flank a single copy of a functional insert. This would protect the internal construct from degradation. In addition, the complete lack of detectable homology between @X174 and a host species might serve as a simple and reliable marker for integration and linkage of genes that would normally be difficult to distinguish because of homology with the host. An approach to insertional analysis was attempted in transgenic fish using the zebrafish A/u1 repetitive sequences, but the numbers and significant homologies of the various repetitive elements made the interpretation complex (He et al., 1992). Integration Evidence for chromosomal insertion can be inferred from Southern blot analysis or by stable Mendelian inheritance of the transgene (Stuart et al., 1990; Lu et al., 1992). Genomic integration of @X174 into the chromosoma1 DNA of the many founders is supported by analysis of the restriction patterns of genomic DNA isolated from fin clips and probed with the @X174 sequence. The @X174 sequence (5 kb) migrated with high molecular weight (> 50 kb) when the genomic DNA was not digested by restriction enzymes (data not shown). When genomic fish DNA was digested with Suu3A which does not cut within the @X174 sequence (or an array), @X174 was detected as a unique band containing single or multiple copies with no complex patterns indicative of rearrangements or multiple insertions. Digestion of genomic DNA with PstI (single recognition site in @X174) patterns produced single bands of approximately 5 kb (see Fig. 1). There were no significantly smaller bands which would have indicated the insertion of small fragments as previously seen with mouse L-cells transgenie for @X174 (Burkhart et al., 1993). Plasmid and viral vectors do not contain origins of replication that would permit any nonintegrated episomal replication or amplification of prokaryote genomes. Identification of full length phage DNA by non-PCR

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techniques in small amounts of fish genomic DNA isolated from fin clips becomes evidence for integrative replication. @X174 does not contain any eukaryotic origin of replication, and expression of @X174 would likely be lethal to the host cell because the A* protein blocks DNA synthesis in host cells (for review see Baus, 1985). In cases where apparent amplification of foreign genes has been observed in fish at very early stages, only a very small proportion of the molecules have been replicated by gastrulation on a copy number per cell or per round of replication basis. The analysis of chromosomal integration in fish is made more complex by the typically large amount of DNA injected into the cytoplasm (10-20 times the entire fish genome), the indeterminate persistence of unincorporated DNA (or fragments), and the potential for significantly delayed integration at later stages of embryonic development. It is difficult to distinguish the mechanisms associated with incorporation, replication, or loss due to the mosaic nature of the original incorporation combined with the normal programmed cell death in developmental cell lineages (Bowen & Bowen, 1990; Tomei & Cope, 199 1). Methylation of @X174

We evaluated the CpG methylation of @X174 because of its significance in sequence analysis of cloned genes from higher eukaryotes. Previous studies with @X174 also demonstrated that extensive CpG methylation after the F2 generation in transgenic mice resulted in a reduced recovery of the phage (Burkhart et al., 1992, 1993). The genomic DNA from selected founder fish which were simultaneously digested with restriction endonucleases PstI and &a11 (the latter does not cleave methylated CCmGG) and hybridized to a biotinylated @X174 probe are shown in Fig. 2. Comparison of the fish DNA restriction patterns (lanes l-7) with those of control unmethylated @X174 digested with MspI (methylation insensitive HpaII isoschizmer), HpaII and PstI (lanes g-10), indicates that the phage sequences were CpG methylated in fish. Digestion of fish DNA with PstI and MspI gave patterns consistant with the control digest in lane 8 (not shown). Although there are five HpaII/MspI recognition sites in the @X174 sequence, only the two largest fragments are readily observable in this analysis. Figure 2 shows methylation analysis performed on medaka (lanes l-3) and mummichog (lanes 4-7). The extent of methylation did not vary among fish, and was independent of differences in gene copy numbers, mosaicism, sex and species. The methylation of @X174 in founder fish contrasts with that of @Xl 74 among transgenic mouse founders where CpG methylation was more variable and increased in succeeding generations (Burkhart et al., 1993). These results also indicate that there may be

Gene transfer and environmental mutagenesis in aquatic species

h&27 Lane

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@X Copy Standard

FCWO

F@74

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Fig. 2. Methylation of chromosomally integrated @X174 from selected founder medaka (lanes 1-3) and mummichog (lanes 47) evaluated by simultaneous digestion with HpaII and PstI. PstI cuts at one point in the @X174 sequence to generate full-length linear sequences. HpaII recognizes CCGG sequences but will not cleave the DNA if the cytosine in the CpG pair is methylated. The single bands (- 5 kb) in lanes l-7 indicate that the @X174 phage sequence is methylated in both medaka and mummichog. For comparison, lanes 8-10 contain prepared unmethyiated @X174 RFDNA digested with PSI and &a11 as well as MspI, which is the methylation insensitive isoschizmer of HpaII.

differences in the methylation of TDX174 in medaka and mummichog when compared to the variable patterns of methylation observed between tissues in transgenic rainbow trout (Maclean et al., 1992). Analysis of CpG methylation in subsequent generations leading to fish that are homozygous for the phage will provide more insight into the role of CpG methylation in other gene transfer studies. One obvious question is how hypermethylation may relate to changes in expression of inserted sequences that are duplicates or close homologues to endogenous genes. CpG methylation may also play a role in how expressed genes respond to xenobiotics (Gruenbaum et al., 1982; Bestor & Ingram, 1983; Reik et al., 1987; Silva & White, 1988; Belinsky et al., 1990; Cedar & Razin, 1990; Bartlett et al., 1991; Frank et al., 1991) Mutagenesis of @X174am3 recovered from transgenic fish We selected the @X174 bacteriophage as a recoverable mutation target because it has several features related to integration, mutation detection and analysis which are well-suited to the study of comparative mutagenesis. +X174 is a small bacteriophage which permits insertion of many copies in the host genome with lower potential for chromosomal

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disruption than larger constructs. The ability to insert many copies not only enhances recovery of the phage and the efficiency of mutation experiments, but suggests that additional markers or combinations of markers with defined mutational endpoints may be used in the same animal. Thus a broad range of mutations can be detected and characterized by selective means with a minimum of sequence analysis. In these initial experiments, we have chosen to use a reversion assay of mutation at urn3 which reverts via one transition and two transversions at an A:T pair. The specificity of the target provides for increased sensitivity in mechanistic studies using well characterized mutagens and avoids some of the confounding variables that may be associated with interpretation of mutagenesis data from transgenic approaches (Burkhart & Malling, 1993). The primary objectives have been: (1) to determine whether the phage can be recovered from transgenic fish; and (2) to measure a spontaneous mutation frequency in selected founders. +X174 is a sequence-conservative phage which, under normal circumstances, does not tolerate significant rearrangement. Modification or random recombination events during or after incorporation of the sequence into the host fish genome would result in our being unable to recover live phage. Similarly, if the spontaneous mutation frequency for the incorporated viral sequence were, for some reason, to be extraordinarily high in fish, then the entire approach would have little utility within a context of measuring mutation induced by chemical exposure. Recovery and the spontaneous mutation frequency among progeny phage from a pooled sample of fin clip DNA from four different F. heteroclitus founders are given in Table 2. The indeterminate mosaicism means that the initial number of copies is unknown, but the small amount of DNA required to rescue a significant number (in the range of 107) of phage from the genomic DNA of founders indicates that there are probably no major rearrangements as a function of incorporation into the fish genome. We have also been successful in recovering live phage from transgenic medaka. Equally important is the observation that the spontaneous mutation frequency is 1.83 x 1O-7 among phage recovered from F. heteroclitus; TABLE

2

Spontaneous Mutation Frequency Among @X174am3cs70 Phage Recovered From a Pooled Sampleof ‘rransgenicFun&& heteroclitus DNA Fish sampled

Putative copy no.a ___._~~..__~~_~.~~~ 4

7-10

u Based on comparison of from OD 260 measurements

Total Total phage /*g DNAb recovered .~_ ~__._ _~ ~ ~_ 40

1.09 x 10’

founder DNA of samples

Mutants

Spontaneous mutation frequency

~~~~ ~~_... 2

1.83 x 10’

with known copy number standards. after Sau3A I/PstI restriction digestion.

b Derived

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given this very low background it is unlikely that any one of the four individuals comprising the pooled sample had a de nova mutation of the reversion site before incorporation into the chromosomal genome. The spontaneous mutation frequency for the same marker in transgenic mouse L-cells was 3 x 1O-7 and in transgenic mice the spontaneous mutation frequencies in the liver, brain, spleen, kidney and testes ranged between 1.81 x lop7 and 3.70 x 10e7 (Burkhart et al., 1992, 1993). Because the @X174 sequence is methylated in both mice and the transgenic fish, the extent of methylation in fish probably does not influence recovery or mutation at am3,which is a CpG-independent marker. Parallel experiments have also produced a @X174 transgenic goldfish cell line from which the phage can be recovered (Van Beneden et al., in prep.). Taken together, these results indicate that @X174 may be useful as an identical gene target in aquatic species and in laboratory mammals with a potential for evaluating comparative mutagenesis in both basic and applied research. Advantages of the approach are that it: (1) measures mutagenic effect at the DNA level; (2) is not limited by gene expression to a cell type; and (3) has target specificity combined with a numerical power not practically available with current specific-locus systems. Disadvantages include limitation to model species which carry the marker and the fact that an unexpressed transgene provides little information about the role of mutation and natural selection among endogenous expressed genes. A series of definitive experiments will be necessary to establish some of the genetic and pharmacokinetic parameters associated with response of the transgenic marker to a series of known environmental mutagens. Continuing research will establish multiple defined lines of transgenic fish containing various @X174-based mutation markers. We anticipate that transgenic medaka and Fundulus will be valuable animal models for the study in vivo of spontaneous and induced mutagenesis in a wide range of aquatic environmental studies. ACKNOWLEDGEMENTS The authors contributions

thank Dr H. V. Malling to this work.

for his conceptual

and constructive

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