INTRODUCTION

Top Abstract Introduction Materials and methods Results Discussion References

Established mammalian and other eukaryotic genomes apparently allow the insertion of foreign DNA sequences both naturally, e.g., upon virus infections (5, 33) or via the gastrointestinal tract (34, 35), and under experimental conditions exemplified by the genomic fixation of foreign DNA after the application of various transfection or microinjection protocols. While investigating the oncogenic transformation of hamster cells after infection with human adenovirus type 12 (Ad12), we have studied in considerable detail the insertion of Ad12 DNA into the hamster cell genome and some of its consequences (for recent reviews, see references 6 and 7). Among the sequelae of foreign DNA insertion, we have concentrated on the de novo methylation of the integrated foreign DNA (27, 41, 42) and on alterations in the patterns of methylation in several cellular genes and DNA segments (15). The integration of foreign (Ad12) DNA into the hamster cell genome is not nucleotide sequence or chromosomal site specific (5, 16, 18). Ad12 DNA,  DNA, and probably any other foreign DNA can be inserted in multiple copies at many different sites, sometimes partly fragmented, and often at a single chromosomal location.

The described alterations in cellular DNA methylation patterns upon foreign DNA insertion are conceivably a reflection of more general changes in chromatin structure in the affected cells. Implications of DNA methylation for chromatin structure and vice versa have been investigated in several laboratories (17, 22, 30, 44). We have frequently observed that up to >50 genome equivalents of Ad12 DNA, i.e., an integrate of foreign DNA measuring 1 to 2 megabase pairs in total length, have been inserted into the established hamster cell genome. The addition of such a large segment of foreign DNA could well lead to structural rearrangements in the cellular genome. The extent and nature of such structural chromatin changes are unknown, but analyses of alterations of DNA methylation patterns might be a reliable indicator and motivate more refined analyses.

We now report that the insertion of bacteriophage  DNA, as a paradigm foreign DNA, into an established hamster genome can alter methylation patterns in certain segments of cellular DNA. It is conceivable that the endogenous retroviral intracisternal A particle (IAP) subclone I (IAPI) sequences are particularly susceptible to undergo methylation changes upon eliciting structural perturbances in a mammalian genome. We also demonstrate by Southern blot hybridization and by genomic sequencing experiments that the BHK21 cell population used in all of these experiments shows uniform, and not mosaic, methylation patterns in the IAPI DNA sequences.

	MATERIALS AND METHODS

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Clonal lines of BHK21 cells with integrated bacteriophage  and plasmid pSV2neo DNAs. The BHK21 hamster cell line (ATCC CCL 10) was kept in continuous monolayer culture in Dulbecco medium enriched with 10% fetal calf serum. These cells were transfected with a mixture of 5 µg each of bacteriophage  DNA and of plasmid pSV2neo DNA (38); the latter carries the gene for neomycin phosphotransferase under the control of the simian virus 40 early promoter. The Ca²⁺-phosphate precipitation technique (12) was used for transfection experiments. Neomycin-resistant cell clones were isolated by G418 (Gibco) selection by adding 1 mg of G418 per ml of medium 2 days after transfection. Neomycin-resistant single-cell clones isolated 2 weeks after the start of the G418 selection were recloned twice as single cells on microtiter plates and screened for the presence of integrated  DNA by conventional restriction endonuclease and Southern blot hybridization analyses (19, 37). During all subsequent passages, G418 was omitted from the culture medium. In mock-transfection experiments, DNA was omitted from the otherwise complete transfection mixture. In other control experiments, BHK21 cells were recloned up to three times on microtiter plates, and IAPI methylation patterns were determined for 66 of these BHK21 cell clones as well as for 9 BHK21 cell clones previously subjected to a mock-transfection protocol. In further control experiments, the IAPI segment in nontransgenic, subcloned BHK21 cells was analyzed by the bisulfite protocol of the genomic sequencing method (11).

Analyses of the BHK21 cell lines carrying integrated  DNA. The presence of -specific sequences in the cellular DNA or RNA was assessed by analyzing cellular DNA or total cellular RNA by conventional Southern or Northern blotting experiments, respectively, using ³²P-labeled  DNA or pSV2neo DNA as the hybridization probe. The cellular DNA or RNA (2) was extracted by standard protocols. The DNA was cleaved with EcoRI or PstI prior to Southern blot hybridization experiments. DNA probes were ³²P labeled by the oligonucleotide labeling procedure (10).

Determination of methylation patterns in cellular IAPI sequences and in integrated  DNA in the  DNA-containing BHK21 cell clones. (i) Restriction enzyme and Southern blot hybridization analyses and quantitation by phosphorimager evaluation. Cellular DNA was cleaved with HpaII, MspI, or HhaI, and the DNA fragments were separated by electrophoresis on 0.8 to 1% agarose gels, Southern blotted onto Qiagen Nylon-Plus membranes, and hybridized to ³²P-labeled  DNA or pSV2neo DNA or to cloned IAPI DNA (15).

(ii) Genomic sequencing method. The genomic sequencing technique based on the bisulfite protocol (11) was applied to determine exactly which 5'-CG-3' dinucleotides in the cellular IAPI segments in the genomes of the  DNA-containing BHK21 clones or the T637 cell line were methylated. Similar experiments were performed with the DNAs from four clonal sublines of nontransgenic BHK21 cells. Details of the bisulfite protocol of the genomic sequencing procedure as used in our laboratory were described previously (24, 36, 45). Briefly, the genomic DNA was alkali denatured in 0.3 M NaOH for 15 min at 37°C and for 3 min at 95°C. The DNA was then treated with sodium bisulfite. The bisulfite solution was prepared by dissolving 8.1 g of sodium bisulfite (Sigma) in 15 ml of degassed water by gently inverting the tube; 1 ml of 40 mM hydrochinone was subsequently added. The solution was adjusted to pH 5 by adding 0.6 ml of freshly prepared 10 M NaOH. The denatured DNA solution (110 µl) was mixed with 1 ml of the bisulfite solution, overlaid with mineral oil, and incubated at 55°C for 16 h in a water bath in the dark. Subsequently, the DNA was purified by using glassmilk (Gene Clean II Kit; Bio 101 Inc.). A selected segment in the IAPI sequence was amplified by PCR with appropriate oligodeoxyribonucleotide primers (see Fig. 6). Reaction products were then cloned into the pGEM-T vector (Promega) and transfected into Escherichia coli XL1BlueMRF' by standard methods (14). A number of clones were isolated, and the nucleotide sequences were determined with an Applied Biosystems 377 DNA sequencer by standard methods. The bisulfite reaction converted all C residues into U residues and then, after PCR amplification, into T residues, whereas the 5-methyldeoxycytidine (5-mC) residues were refractory to this chemical conversion reaction. Thus, a C residue in the eventually determined nucleotide sequence proved the presence of a 5-mC residue in this position in the original genomic nucleotide sequence. All bona fide C residues scored as Ts.

	RESULTS

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Rationale and design of the study. In investigations on integrated Ad12 genomes in Ad12-transformed hamster cell lines and Ad12-induced hamster tumor cells, we found extensive changes in the methylation patterns of cellular genes and cellular DNA segments, notably in the endogenous IAP retrotransposons and in the major histocompatibility complex region (15). Infection of BHK21 cells with Ad12 virions did not elicit such changes. Since in Ad12-infected and Ad12-transformed hamster cells similar patterns of early Ad12 gene transcription were observed (28), it was unlikely that the presence of early Ad12 gene products was responsible for the increases in cellular DNA methylation.

Clonal BHK21 cell lines carrying integrated  genomes. The clonal BHK21 cell lines that had been selected upon cotransfection with pSV2neo DNA and  DNA were analyzed by Southern blot hybridization for the presence and arrangement of the bacteriophage  and pSV2neo plasmid DNAs and by the FISH method for the chromosomal locations of these foreign DNA molecules. A large number of  DNA-containing clonal BHK21 cell lines were generated. The analytical Southern hybridization data from some of these cell lines are presented in Fig. 1a. Comparisons with fragment intensities in the marker lanes of EcoRI- or PstI-cleaved authentic bacteriophage  DNA revealed that the generated  DNA-containing cell lines had less than 1 copy to more than 50 copies of the foreign DNA chromosomally integrated. The cleavage patterns of the cellular DNAs with EcoRI or PstI demonstrated the presence of off-size DNA fragments with homologies to  DNA. These off-size fragments were most likely due to junction fragments representing cellular DNA linked to the integrates or to partly rearranged  DNA, as previously documented with many transformed cell lines and tumors for integrated Ad12 DNA (16, 18, 27, 39).

View larger version (69K): [in this window] [in a new window]   FIG. 1.   Clonal cell lines of BHK21 cells that carry integrated  DNA and pSV2neo DNA. (a) Integration patterns of  DNA in some of the clonal, exemplarily selected  DNA-transgenic BHK21 cell lines. The DNA extracted from BHK21- clones as indicated was cleaved with EcoRI (E) or PstI (P), and the fragments were separated by electrophoresis on a 0.6 or 0.8% agarose gel. The DNA was then transferred by Southern blotting to a Qiagen Nylon-Plus membrane, and the  DNA-specific fragments were visualized by hybridization to 32P-labeled  DNA followed by autoradiography. As size and quantity markers,  DNA cut with EcoRI or PstI was coelectrophoresed. Amounts of 1, 5, or 10 genome equivalents (ge) of  DNA were used. (b) Integration patterns of the pSV2neo plasmid used in cotransfection experiments. Experimental conditions were identical to those described for panel a except that 32P-labeled pSV2neo DNA was used as the hybridization probe. The same Qiagen Nylon-Plus filter as shown in panel a was used in these experiments, after removing the  DNA probe by boiling in 0.1% sodium dodecyl sulfate-0.1 × SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The BHK21-18 and -18* clones are two distinct  DNA-carrying BHK21 cell clones.

View larger version (32K): [in this window] [in a new window]   FIG. 2.   FISH analyses of the chromosomal locations of the integrated  genomes and the integrated pSV2neo plasmids in a series of BHK21 cell lines rendered transgenic for  DNA and pSV2neo DNA. Experimental details are described in the text. (a) Control BHK21 cell devoid of foreign DNA; (b) BHK21-7; (c) BHK21-17; (d) BHK21-18*; (e and f) BHK21-15. (a and f) Biotinylated  DNA alone was used as hybridization probe; (b to e) a mixture of biotinylated  and pSV2neo DNAs was used for hybridization. The finding of a single signal demonstrated that both transgene DNAs were located at one chromosomal site, which was different for each cell line.

De novo methylation of integrated  DNA. Foreign DNA, e.g., Ad12 DNA, covalently inserted into an established mammalian genome became readily de novo methylated (18, 26, 27, 41, 42). Integrated  DNA in the clonal  DNA-transgenic BHK21 cell lines was frequently, but not always, extensively de novo methylated (Fig. 3a). Similarly, the cotransfected and integrated pSV2neo DNA could also be de novo methylated (Fig. 3b). The DNA extracted from the  DNA-carrying cell lines was cleaved with HpaII, MspI, or HhaI, and the fragments were separated by electrophoresis on 1% agarose gels, blotted by the Southern procedure, and hybridized to ³²P-labeled  DNA. Routinely, 10 U of restriction endonuclease per µg of DNA was used. Control experiments using 5 or 30 U per µg of DNA yielded identical results. The autoradiograms for the DNAs from several of the  DNA-transgenic BHK21 cell lines demonstrated marked de novo methylation of the transgenic  and pSV2neo DNAs (Fig. 3). With continuous passage of the cells, the extent of transgene methylation increased, probably due to the spreading of de novo methylation (27).

View larger version (98K): [in this window] [in a new window]   FIG. 3.   De novo methylation of the integrated  DNA (a) or pSV2neo DNA (b) in several clonal BHK21 cell lines transgenic for  and pSV2neo DNAs. The DNAs from the  and pSV2neo DNA-transgenic cell lines were isolated and cleaved with HpaII (H), HhaI (Hh), or MspI (M). Subsequently, the DNA fragments were separated by electrophoresis in 1.0% agarose gels and analyzed as described in the legend to Fig. 1. 32P-labeled  DNA (a) or 32P-labeled pSV2neo DNA (b) was used as the hybridization probe.

The integrated  and pSV2neo DNAs in the clonal BHK21 cell lines are not detectably transcribed. Total RNA was extracted from several of the  DNA-transgenic clonal BHK21 cell lines by published methods (2) or by using extraction kits (Qiagen), electrophoresed on 1% agarose gels containing 2.2 M formaldehyde, and blotted onto Qiagen Nylon-Plus membranes. Subsequently, the RNA was analyzed for the presence of  DNA-specific sequences by hybridization to ³²P-labeled  DNA. In the RNA preparations from 20 different BHK21 cell lines (8 are shown in Fig. 4) carrying integrated  DNA,  DNA-specific transcripts were not detectable (Fig. 4a). Similarly, pSV2neo-specific signals could not be detected on Northern blots (data not shown). The same RNA blots yielded RNA signals when the single-copy cellular gene for serine proteinase (Fig. 4b) or the multi-copy IAPI segment was used as the ³²P-labeled hybridization probe. We therefore conclude that the integrated  and pSV2neo DNA molecules in the different hamster cell lines are not transcribed as detectable by RNA blot analyses. Transcription of a single-copy cellular gene, however, is readily detected by Northern blotting.

View larger version (68K): [in this window] [in a new window]   FIG. 4.   BHK21 cells transgenic for bacteriophage  DNA do not detectably transcribe this DNA. An autoradiogram of an RNA (Northern) blotting experiment in which 30 µg of total RNA was electrophoresed on a 1% agarose gel containing 2.2 M formaldehyde is shown. Upon transfer of the RNA to a Qiagen Nylon-Plus membrane, the RNA was hybridized to 32P-labeled  DNA (a) or to the 32P-labeled pBluescript-cloned serine proteinase gene from Syrian hamster (b). In this autoradiogram, RNA samples from only eight of the BHK21 cell lines transgenic for  DNA were analyzed. When 32P-labeled pSV2neo DNA was used as the hybridization probe, results similar to those in panel a were obtained (not shown).

Altered methylation patterns in the cellular IAPI segments of several  DNA-transgenic clonal BHK21 hamster cell lines. (i) Restriction analyses with methylation-sensitive endonucleases and phosphorimager quantitations. The cellular DNAs from several of the  DNA-transgenic BHK21 cell lines were isolated from early passages (passages 6 to 8) and were cleaved with the methylation-sensitive restriction endonuclease HpaII or HhaI or, as a control, with MspI. Routinely, 10 U of restriction endonuclease per µg of DNA was used. Control experiments using 5 or 30 U per µg yielded identical results. The fragments were separated by electrophoreses on 0.8 to 1% agarose gels, and the DNA fragments were transferred by Southern blotting to Qiagen Nylon-Plus membranes and hybridized to ³²P-labeled IAPI DNA cloned in plasmid pBR322. The origin of the IAPI subclone (25) was described elsewhere (15). Some of the autoradiograms of these Southern blot hybridization experiments are shown in Fig. 5. The data demonstrated that the levels of methylation of the IAPI segments in the DNAs from the cell lines BHK21-7, -10, -12, -18, and -27 were markedly increased in the 5'-GCGC-3' (HhaI) and also in the 5'-CCGG-3' (HpaII) sequences in comparison to the same DNA segments and sequences in the original, nontransgenic BHK21 cells. In a total of 13  DNA-transgenic cloned BHK21 cell lines, similar changes in the methylation of the IAPI segment were observed. In 64 additional  DNA-transgenic BHK21 cell lines tested, similar alterations in the methylation of the IAPI DNA segments were not apparent. Thus, methylation changes in the IAPI segment could be documented in about 17% of the cell lines investigated. Of course, in the clonal lines without changes in the IAPI segment, different parts of the hamster genome might be affected.

View larger version (102K): [in this window] [in a new window]   FIG. 5.   Increases in DNA methylation in the 5'-CCGG-3' (HpaII [H]) and 5'-GCGC-3' (HhaI [Hh]) sequences in the IAPI segments of five cloned BHK21 cell lines with integrated  DNA in comparison to DNA from the non--DNA-transgenic BHK21 cell lines. Experimental procedures were similar to those described in the legend to Fig. 3, except that 32P-labeled IAPI DNA was used as the hybridization probe. The data for the BHK21-7 clone have been shown previously (15). The results of phosphorimager analyses are presented in Table 1.

(ii) Analyses of changes in DNA methylation by using the bisulfite protocol of the genomic sequencing technique. Since the restriction and Southern blot hybridization data on increases in IAPI segment methylation revealed rather congruent changes in many of the IAPI genome copies, it appeared to be feasible to document these changes even more precisely by applying the genomic sequencing procedure (11). By this method, each 5-mC residue in a sequence can be determined independently of its location at a specific restriction endonuclease site. Experimental details are described in Materials and Methods. The map in Fig. 6 designates the locations of the 28 5'-CG-3' dinucleotides in the p3-p4 segment of the IAPI region (25). The map also indicates the primers used in the PCR amplification step prior to sequencing of the bisulfite reaction products. The primers were selected in a segment of the IAPI region that had shown the most obvious changes in DNA methylation as determined by HpaII or HhaI cleavage and Southern blot hybridization (Fig. 5; Table 1). The 5'-CG-3' dinucleotides were investigated for the presence of 5-mC residues in DNA from the cell lines BHK21, T637, BHK21-7, and BHK21-10 in 123, 79, 58, and 73 cloned PCR products, respectively. In Tables 2 and 3 the percentages of 5-mC residues in the 5'-CG-3' positions 1 to 34 were calculated for the DNAs from individual cell lines. These values were the averages of >330 independently sequenced cloned, PCR-generated molecules. Values can be compared only between different DNA sources for a given 5'-CG-3' position; hence, lines in Tables 2 and 3 have to be read across. Table 2 presents the percentages of cloned PCR products that are methylated at the individual 5'-CG-3' clones for each of the four cell lines (BHK21, T637, BHK21-7, and BHK21-10) investigated. For more immediate comparisons, the values for each 5'-CG-3' site were normalized relative to the level of methylation at the same site in the reference cell line BHK21, for which the values were arbitrarily set to 1.000 (Table 3).

View larger version (12K): [in this window] [in a new window]   FIG. 6.   Methylation analyses of the IAPI segments in the BHK21 and T637 cell lines and in the  DNA-transgenic clonal BHK21 cell lines BHK21-7 and BHK21-10 by using the bisulfite protocol of the genomic sequencing procedure. A map of subclone I in the IAP retrotransposon in hamster cells (25) is shown. The locations of the primers used in the amplification step of the genomic sequencing procedure following the bisulfite reaction with clonal DNAs are designated with horizontal arrows. Several segments were genomically sequenced. The data shown in Tables 2 and 3 were derived from the primer p3-p4-flanked subsegment of the IAPI region, which contains 28 5'-CG-3' dinucleotide sequences (vertical lines), as published previously (25). For experimental details see the text and the footnotes to Tables 2, 3, and 4. HpaII (stars) and HhaI (circles) sites are indicated. Numbers refer to the published nucleotide sequence (25).

View larger version (25K): [in this window] [in a new window]   FIG. 7.   Alterations in DNA methylation at 28 5'-CG-3' sites in the p3-p4 segment (Fig. 6) of the IAPI retrotransposon sequence in the Ad12-transformed hamster cell line T637 and in two  DNA-transgenic BHK21 hamster cell lines, BHK21-7 and -10. Experimental details are described in the text. The numbering of the 5'-CG-3' dinucleotides corresponds to that in Fig. 6. During the genomic sequencing experiments, 123 DNA clones from the reference BHK21 cell line, 79 DNA clones from the Ad12-transformed T637 cell line, 58 DNA clones from the BHK21-7 cell line, and 73 DNA clones from the BHK21-10 cell line were sequenced. The percentage values represent the average of methylated 5'-CG-3' dinucleotides at each site for DNA from each of the cell lines. In many of the clones, 5'-CG-3' positions 23 to 27 were in a deleted segment as part of a naturally occurring polymorphism in this region compared to the published nucleotide sequence (25). Furthermore, 5'-CG-3' positions 19 and 35 were altered to 5'-TG-3' in many clones and were therefore omitted from this analysis.

(iii) Genomic sequencing control experiments with the IAPI p3-p4 segments from four nontransgenic, subclonal BHK21 cell lines. Since methylation at 5'-CG-3' sites in the 900 haploid IAP copies in hamster cells is expected to be polymorphic, a control analysis was performed. The levels of 5'-CG-3' methylation at the 28 sites of the IAPI p3-p4 segments in the BHK21 reference cell line and four subclones of this cell line, sublines BHK21-1, BHK21-2, BHK21-3, and BHK21-4 (the latter mock transfected), were compared to assess the naturally occurring polymorphic fluctuation of methylation in this segment. The data in Table 4 were obtained by the same experimental methods of genomic sequencing with the bisulfite protocol and of normalization as described for Table 3 (also compare Fig. 7). However, the sequence determinations for PCR products cloned upon bisulfite treatment of DNAs from the BHK21 reference cell line, from the Ad12-transformed hamster cell line T637, and from the four different subclonal cell lines BHK21-1, BHK21-2, BHK21-3, and BHK21-4 were completely independent of the experiments described for Tables 2 and 3 and Fig. 7. Totals of 68, 43, 50, 60, 54, and 60 ( = 335) clones, respectively, were sequenced. As expected, the four BHK21 subclones showed some polymorphism in the levels of 5'-CG-3' methylation in the IAPI p3-p4 segment, but practically all values were similar to the equivalent values in the BHK21 nontransgenic reference cell line (compare Tables 3 and 4). In contrast, the values for cell line T637 were again decisively higher than those for the BHK21 reference cell line (Table 4).

The changes in cellular DNA methylation patterns are not due to the Ca²⁺-phosphate precipitation protocol used for BHK21 cell transfection. The standard Ca²⁺-phosphate transfection procedure is toxic for mammalian cells in culture. We therefore had to ascertain that the application of this technique was not responsible for the observed alterations in DNA methylation patterns in the IAPI segments of the BHK21 cells. BHK21 cells were mock transfected by the Ca²⁺-phosphate precipitation protocol, i.e., DNA was not added to the transfection mixture. The cellular DNA was then isolated either from the total cell population or from BHK21 cell clones after three rounds of single-cell cloning. Subsequently, the cellular DNA was cleaved with HpaII, MspI, or HhaI, and the fragments were separated by electrophoresis on 0.8% agarose gels, transferred to Qiagen Nylon-Plus membranes, and hybridized to the ³²P-labeled IAPI DNA clone. The autoradiograms from DNA samples derived from nine different pretreated BHK21 cell clones and from three different uncloned BHK21 cultures provided no evidence for differences in DNA methylation patterns in the IAPI segments of BHK21 cells compared to the DNA samples from untreated control cultures. (The data are not shown but were similar to those in Fig. 8). Apparently, the Ca²⁺-phosphate transfection protocol by itself, including biochemical selection and single-cell cloning, does not cause changes in the methylation patterns of the cellular IAPI segment.

The IAPI segments in the DNAs from 66 isolated and propagated nontransgenic BHK21 cell clones exhibited the same methylation patterns as that from untreated BHK21 cells. BHK21 cells were recloned up to three times on microtiter plates by seeding the cells at <1 cell per 10 wells. Clones derived from single cells were propagated to the level of one 75-cm² monolayer culture, and the DNA was extracted and investigated for changes in IAPI segment methylation patterns by cleavage with the methylation-sensitive restriction endonucleases as described above. The results from 66 different single-cell clones showed identical methylation patterns in the IAPI segments and were identical to those from the bulk culture of BHK21 cells. Representative data of HpaII cleavage patterns of DNAs from 16 different clones are shown (Fig. 8). Similarly, the DNAs from all 66 different BHK21 clones showed identical HhaI cleavage patterns (data not shown).

View larger version (128K): [in this window] [in a new window]   FIG. 8.   The IAPI segment methylation patterns obtained upon HpaII (5'-CCGG-3') cleavage were identical in 66 nontransfected and 9 mock-transfected individual BHK21 cell clones and did not differ from those in DNA preparations from unselected, uncloned BHK21 cells. As examples, the IAPI DNA hybridization results with HpaII (H)-cleaved DNAs from unselected BHK21 cells and from 16 individual BHK21 cell clones are shown. M, MspI control pattern.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

Alterations in cellular DNA methylation patterns. This study has been undertaken to determine whether changes in DNA methylation patterns of cellular genes and DNA segments observed in Ad12-transformed hamster cells (15) are due to the transformed state of the cells, to the insertion of foreign DNA into an established mammalian genome, to a combination of both events, or to preexisting mosaics in the methylation patters in the cellular IAPI segments. The data presented in this report support the interpretation that the integration of nontranscribed bacteriophage  DNA in the hamster genome is associated with such changes in some of the clonal transgenic BHK21 cell lines, notably in the IAPI retrotransposon sequences. It is conceivable that these repetitive sequences, which were integrated into the hamster genome probably several million years ago (43), may be particularly susceptible to alterations in DNA methylation patterns subsequent to the insertion of foreign DNA. Moreover, the sites and the extent of changes in methylation patterns of cellular DNA may be dependent on the sizes and locations of the foreign DNA integrates.

Foreign DNA integration and cellular chromatin structure: possible mechanisms. We have set out to investigate whether foreign DNA insertion into an established hamster cell genome can be related to detectable structural changes in cellular chromatin. Given these premises, certain cellular DNA segments might be exposed more directly to the cellular DNA methyltransferase systems from which they might have been protected prior to the insertion of foreign DNA. Since both after viral infection and after the transfection of foreign DNA, multiple copies of the foreign DNA can become inserted into the recipient genome, major structural perturbations due to the addition of large DNA blocks may ensue. Preliminary data (not shown) are consistent with this interpretation but need further refinement.

General implications. In many experimental procedures, the insertion of foreign DNA into an established mammalian or plant genome has become everyday practice, usually with the goal of expressing a foreign gene or of eliminating or restoring the function of an endogenous gene. The possibly farther-reaching sequelae of such manipulations have frequently not been contemplated. Changes in cellular DNA methylation patterns as a consequence of foreign DNA insertion would call for a precise, case-by-case analysis of alterations in cellular DNA methylation and transcription patterns with the potential for functional consequences. It has been known for almost two decades that DNA methylation and transcription patterns are functionally related (3, 4, 8, 29, 41). In Ad-transformed cells or in Ad12-induced tumor cells, altered transcription patterns in 5 of 40 investigated (in part randomly selected) cellular DNA segments and genes have been described (32).

(i) Studies on the mechanism of viral oncogenic transformation. The process of viral oncogenic transformation might be causally related to changes in cellular DNA methylation and transcription patterns.

(ii) Studies on transgenic organisms. The most obvious interpretations of data gleaned from such studies on transgenic organisms may not always coincide with reality. The insertion of foreign DNA in these experiments could influence the activity of many more genes than the ones directly affected by the knock-in or the knockout procedure.

(iii) Schemes developed for human somatic gene therapy. The stable insertion of foreign DNA into the human genome could have far-reaching consequences with undesirable side effects.