High frequency DNA rearrangements associated with mouse centromeric satellite DNA

J. Mol. Biol. (1986) 187, 547-556

High Frequency DNA Rearrangements Associated with Mouse Centromeric Satellite DNA Karen A. Butner and Cecilia W. Lot Biology Department, Goddard LaboratoryjGS,

University of Pennsylvania Philadelphia, Pa 19104, U.S.A.

(Received 18 June 1985, and in revised form

12 September 1985)

A DNA transformed mouse cell line, generated by the microinjection of a pBR322 plasmid containing the herpes thymidine kinase (tk) gene, was observed to exhibit a high frequency of DNA rearrangement at the site of exogenous DNA integration. The instability in this cell line does not appear to be mediated by the tk inserts or the immediately adjacent mouse DNA, but instead may be a consequence of the larger host environment at the chromosomal site of tk insertion. Results obtained from restriction analysis, in situ chromosome hybridizations, and cesium chloride density-gradient fractionations indicate that the tk inserts are organized as a single cluster of direct and inverted repeats embedded within pericentromeric satellite DNA. To determine the molecular identity of the flanking host sequences, one of the mouse-tk junction fragments was cloned, and subsequent restriction and sequence analyses revealed that this DNA fragment consists almost entirely of classical mouse satellite DNA. On the basis of these observations, we suggest that the instability in this cell line may reflect the endogenous instability or fluidity of satellite DNA.

1. Introduction

suggesting that this class of DNA diverges much more rapidly than many single-copy genes. Various models have been proposed to explain the instability associated with sDNA (for a review, see John & Miklos, 1979) but the molecular mechanism(s) that underly this instability are still not known. The study of this phenomenon has been hampered by the lack of an experimental system that would permit a direct examination of the properties of sDNA in situ. This difficulty results from the highly repetitive nature of sDNA, which has precluded the tracking of one specific subset of satellite repeat above a high background of identical or similar sequences. In this paper, we describe a DNA transformed mouse cell line that may provide a unique opportunity for tracking the movements of sDNA. The cell line used in this study, LC2, is a tk+ transformant, which was obtained (Lo, 1983) by microinjecting tk- mouse L cells with a pBR322 plasmid (ptk) containing the herpes viral thymidine kinase gene (Wigler et al., 1977). Southern (1975) analyses of several DNA samples of LC2 cells grown over a period of a year and a half revealed changes in the ptk-specific restriction pattern. Surprisingly, these changes were detected in cells being propagated in a medium (HAT) that selects for tk expression. Previously we demonstrated that these ptk restriction pattern changes were not due to

The genome of all eukaryotes is composed of a heterogeneous collection of unique and repetitive DNA. One class of repetitive DNA that is found in large abundance in most higher eukaryotes is the satellite DNA, which characteristically fractionates as a separate or “satellite” band in cesium chloride density gradients. This pattern of fractionation arises from the fact that sDNA$ is usually organized as long tandem arrays of simple sequence a substantially different base repeats with composition from that of main-band DNA (Singer, 1982). Satellite DNA has the unusual property of being highly unstable. One indication of this is the finding that the amount and the organization of sDNA in closely related organisms can vary over a wide range, even among different individuals within a single species (Craig-Holmes et al., 1973, 1975; Seabright et al., 1976; Maresca et al., 1984). Moreover, any specific satellite sequence is commonly restricted to a single species only, thus t Author to whom correspondence should be addressed. 1 Abbreviations used: sDNA, satellite DNA; tk, thymidine kinase gene; kb, IO3 bases or base-pairs; base-pairs. 0022-2836/86/040547-10

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alterations in DNA methylation as they were observed with restriction enzymes not inhibited by p”‘CpG modifications (BamHI, PvulI, BglII, Hinff; Lo, 1983). We also determined that incomplete digestion was not involved; rehybridization of the Southern blots with globin, alpha-fetoprotein and actin probes always revealed the standard mouse restriction patterns. The latter observations also suggest that these changes in restriction pattern may be confined to sequences in the vicinity of the ptk inserts. In the present study, we found that the restriction pattern changes in LC2 result from a high frequency of DNA rearrangement at the site of ptk integration. We show that this instability is probably related to the flanking host environment and, after further analyses, we demonstrate conclusively that this environment consists of pericentromeric sDNA. On the basis of these we suggest that the instability observations, associated with the transfected ptk DNA in the LC2 cell line may reflect the endogenous instability of the flanking host sDNA.

2. Materials and Methods (a) Cell culture and subclone isolation LC2 was derived from an LMTKcell iontophoretically injected with HindIII-digested ptk DNA (Lo, 1983). This cell line was maintained in Dulbecco’s modified Eagle’s medium (DME) supplemented with 10% calf serum, HAT medium (14 pg hypoxanthine/ml, 0.15 pg aminopterin/ml, 3 pg thymidine/ml), 50 units penicillin G/ml and 50 pg streptomycin/ml. Single-cell subclones of LC2 were obtained by drawing individual cells into a hand-pulled capillary pipette and releasing each cell into a separate cloning cylinder filled with conditioned HAT medium. (b) Southern blotting analysis High molecular weight DNA was isolated from cultured cells using the method described by Davis et al. (1980). Restriction enzymes were purchased from either New England Biolabs, Bethesda Research Laboratories digestions were or Amersham Corp., and restriction carried out according to manufacturers’ specifications. Reactions were routinely incubated overnight using 2 to 4 units of enzyme/pg of DNA and the digests (5 pg/slot) were subsequently separated by electrophoresis in O*S”h to l.Z”/b (w/v) agarose horizontal gels run in I x TBE buffer (89 m&i-Tris, 89 mu-boric acid, 2 mM-EDTA (pH 7.9)) at 2V/cm for approximately 16 h. Gels were transferred onto nitrocellulose in 18 x SSC (2.7 M-NaCl, 0.27 M-sodium citrate (pH 7-O)) and 1 M-ammonium acetate, but otherwise blotted according t’o the method of Southern (1975) with the modifications described by Wahl et al. (1979). The filters were baked for 2 h at 8O”C, prehybridized for 1 h and hybridized (Wahl et al., 1979) for 12 to 24 h with 1.5 x lo6 cts/min per ml of nicktranslated (Maniatis et al., 1975) ptk probe (spec. act. 10s to 3 x 10s cts/min per pg) at 65°C. The prehybridization was carried out in 4 x SET (0.6 M-NaCl, 8 mM-EDTA, 0.12 M-Tris (pH 8-O)), O-1 M-sodium phosphate (pH 7-O), 5% sodium pyrophosphate, 10 x Denhardt’s (0.2%, w/v, of bovine serum albumin/polyvinylpyrollidone and Ficoll)

and 2Opg denatured salmon sperm DNA/ml. For hybridization the same solution was used except 1 x Denhardt’s solution was used. After hybridization, 2 to 3 sequential 20-min washes were carried out at 55°C in a wash buffer containing 0.1 x SET, 0.1% sodium pyrophosphate, 0.1 M-phosphate buffer (pH 7), and 0.1% sodium dodecyl sulfate. Autoradiographic exposure was at, -80°C for 1 to 5 days using preflashed Kodak XAR-5 film with a DuPont intensifying screen. For dot-blot analyses, purified DNA samples were heated for 15 min at 95”C, quickly cooled, adjusted to a final concentration of 10 X SSC, 1 M-ammonium acetate, in a volume of 200 ~1, then applied to wetted nitrocellulose using a. minifold apparatus (Schleicher and Schuell, Inc.). The blotted nutrocellulose was washed for 5 min in 0.1 x SSC, 1 M-ammonium acetate, then processed as a Southern blot with the exception that the length of hybridization was shortened to 5 h. (c) Calcium phosphate transformation

with genomic DNA

DNA transformation with high molecular weight genomic DNA was carried out using the procedure of Wigler et al. (1978) with the modifications described by Lewis et al. (1980). Briefly, 80 to 100 pg of calcium phosphate-precipitated DNA were added to each plate of 2 x lo6 cells. After 4 h, the cells were shocked with dimethylsulfoxide for 30 min, placed in non-selective medium for 20 h, then fed with HAT medium. After 10 to 14 days, HAT-resistant colonies were isolated using cloning cylinders. (d) Metaphase preparation, in situ hybridization chromosome banding

and

To obtain metaphase spreads, cells were incubated for 1 h at 37°C in 100 ng of colcemid/ml (Gibco). harvested by trypsinization, hypotonically shocked in 0.075 M-KC1 (37°C) for 15 min, fixed with methanol/acetic acid (3 : 1. v/v) and spread by splashing onto glass slides (Wahl et al., 1982). The in situ hybridizations were carried out according to the method of Harper & Saunders (1981). Thus RNase-treated metaphase spreads were denatured at 70°C in 507; formamide, then hybridized with a nicktranslated tritiated ptk probe (spec. act. 2 x IO’ to 5 x 10’ cts/min per pg). After 16 h of hybridization, the slides were washed. and then coated with Kodak NTB-2 emulsion. Four to seven days later, the coated slides were developed and stained. For trypsin G-banding of metaphase spreads, the method of Francke & Oliver (1978) was used and for C-banding, aged (4 weeks old) fixed spreads were treated for 1 min with a 526 barium hydroxide sohrtion at 50°C (Sumner, 1972). Banded metaphase spreads were photographed at a magnification of 100 x under oil-immersion using a Zeiss photomicmscope and Kodak 2415 Technical Pan film. (e) CZoning To clone the 8.8 x lo3 base BglII restriction fragment, we first fractionated the genomic DNA of LC2-3E17-5 on a Hoechst/cesium chloride gradient, and then isolated the fraction that was maximally enriched in ptk sequences. For this gradient fractionation, approximately IO’ cells were gently lysed, incubated with the dye Hoechst 33258, and then centrifuged in a final volume of 4.5 ml containing 1.68 g C&l/ml (Manuelidis, 1977). Centrifugation was carried out to equilibrium in a type 50 Ti rotor spun at 42,000 revs/min for 24 h. The gradient was

Rearrangements in Satellite visualized under long-wavelength ultraviolet illumination and approximately 100 ~1 of the interband region (between the sDNA and main-band DNA) was carefully eluted. This fraction contained approximately 10 pg of DNA and was enriched approximately 15-fold in ptk DN$ sequences as compared to total genomic DNA analyzed in Southern blots. This DNA was purified by precipitation with ethanol, digested with BgZII and ligated directly into the BgZII site of the positive selection plasmid vector pKGW (P. Green & H. Boyer, personal communication). The ligation products were transformed into competent D1210 host cells prepared by the CaCl, method of Morrison (1977). The cells were then plated onto L-plates supplemented with 1 mM-isopropyl-beta-Dthiogalactoside and 50 pg kanamycin/ml. Under these conditions, only colonies carrying a recombinant plasmid will grow. When the surviving colonies had reached a diameter of 0.1 mm, they were transferred to nitrocellulose. amplified with chloramphenicol, and hybridized with a nick-translated 3.6~ lo3 base ptk BumHI fragment according to standard methods (Maniatis et al.. 1982). Approximately 15 recombinant colonies were found to contain the 8.8 kb BgZII tk-mouse junction fragment. One of these clones was chosen at random and its plasmid was designated as pl 1. (f) Suhhcloning and sequencing of the mouse repeat The 235 bp unit repeat of pll was subcloned into pUCl9. The subcloning was carried out by digesting pll DNA with BstNI and isolating the 235 bp DNA fragment by electroeluting it onto DEAE paper (Winberg & Hammarskjold, 1980). This fragment was end-filled, ligated with BamHI linkers (New England Biolabs), and inserted into the alkaline phosphatase-treated (Maniatis et al.. 1982) BamHI site of pUCl9 DNA (according to st’andard procedures; Maniatis et al., 1982). The ligation products were transformed into competent JM 83 cells (Morrison, 1977) and the positive transformants were ident,ified as white colonies on LB agar plates containing 80 pg ampicillin/ml and 0603% 5-bromo-4-chloro-3indolyl-beta-n-galactopyranoside (XGAL). One of the positive clones obtained was found to contain the monomeric repeat and was designated as PSAT. DNA sequences were obtained for both strands of the PSAT insert according to the following strategy. First. Hind111 and EcoRT, single cutters of PSAT that cleave on opposite sides of t’he BamHI insert, were each used to linearize a separate group of PSAT molecules. These linearized molecules were end-labeled using polynucleot’ide kinase (Berkner & Folk, 1977), and the labeled inserts were excised with EcoRI for those molecules labeled at the HindIII site and with Hind111 for those molecules labeled at, the EcoRI site. The excised and endlabeled inserts were then separated from the vector DNA on a soy; (w/v) agarose gel, electroeluted (Winberg & Hammarskjold, 1980). and sequenced according to the method of Maxam & Gilbert (1980). The redundancy provided by sequencing both strands of the insert in PSAT made it possible to cross-check the accuracy of over 8596 of t,he DNA sequence.

3. Results (a) DNA

rearrangements

in LCZ progeny

In this study we carried out experiments to determine the molecular basis for the ptk restriction

pattern changes detected in the LC2 cell line. One

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possibility is that the restriction pattern changes result from DNA rearrangements at the site(s) of ptk insertion. Alternatively, if the LC2 cells actually consisted of a collection of independent transformants, the detection of restriction pattern changes could result merely from the normal population dynamics of a heterogeneous collection of tk+ transformants. This latter possibility seems unlikely, however, since the LC2 cell line represents the only tk+ clone to emerge from a dish of ptkcells. Nevertheless, to resolve this injected ambiguity, we isolated single cell subclones from the LC2 culture and characterized their restriction pattern. From 24 subclones characterized, 11 sets of restriction patterns were observed, 10 of which are digest). This shown in Figure 1 (for HinfI restriction analysis revealed that all the subclones share some restriction fragments in common, including some that are not endogenous to the ptk DNA and are derived from novel junction fragments at the site(s) of ptk insertion (see Fig. 1; the novel restriction fragments are denoted by dots). This result clearly demonstrates that all the subclones are indeed descendants of a single transformed ceil. Tn light of the above results, it appears likely that the heterogeneity in the LC2 subclones is a consequence of DNA rearrangements near the site(s) of ptk insertion. This possibility was confirmed when the subclone LC2-3, which was obtained in the subclone analysis above, was observed to give rise to daughter cells that exhibited changes in their restriction pattern. Thus, three of the four single cells isolated from an LC2-3 HAT culture each have a unique and non-parental tk restriction pattern (as observed with RgZII, BarnHI, HinfI and PvuII digests; see Fig. 2 for RgZII digests). In addition, Southern blot,s of these three subclones suggested that they may have undergone amplification and deletion events; this is indicated by the hybridization intensity differences of restriction fragments shared in common between the parental LC2-3 cell line and the three subclones (see Fig. 2). It is important to note that the LC2-3 subclone used in the above analysis represents a predominant member of the LC2 culture; that is. nine out of the 24 subclones originally isolated from the LC2 culture have the same restriction pattern as LC2-3. This suggests that the type of instability detected in the LC%3 culture is probably common to a large proportion of the LC2 cells. Thus, the high frequency of DNA rearrangements in LC2-3 could account for all the restriction pattern changes previously detected in the LC2 cell line. As DNA tranformation is not typically associated with a high frequency of spontaneous DNA rearrangements (Perucho & Wigler, 1980), it is likely that the LC2 instability is brought about by either the unique properties of the mouse DNA flanking the site(s) of ptk integration or the unusual arrangement of the ptk inserts themselves. To examine these possibilities, we first characterized

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Figure 2. Southern blot’ of single cell subclones of LCZ-3. DNAs from the LC2-3 parental line (lane 3) and from 3 single cell LC2-3 subclones (3H, 3G, 3F) were digested with BgZII, separated in a 0.8% agarose gel. which was then blotted and hybridized with a ptk probe. Note that all these subclones have a restriction pattern different from that of the LC2-3 parental cell line. Standard sizes are shown at the right (in kb).

Figure 1. (a) Hid restriction map of ptk linearized at the HindIII site. Note that only the molecular weights of fragments larger than 0.5 kb are given. (b) Southern blot of 10 single cell subclones isolated from LC2. Twenty-four subclones were isolated from LC2 cells that had been propagated in HAT medium for approximately 17 months. Among these, 11 different classes of ptk-specific restriction patterns were observed. Ten of these are illustrated above for Hinff digests. The digested DNA

samples (5 pg each) were separated by electrophoresis in a 1’3% agarose gell, blotted onto nitrocellulose and hybridized with a ptk probe. The ptk junction fragments are indicated with dots t,o the left of the Figure. Sizes are shown at the right (in kb).

the organization of the ptk inserts and defined the nature of the flanking mouse genomic DNA by restriction mapping. For these and the subsequent studies to be described below, we used the subclone LC2-3. (b) Organization

of tk sequences in LC2-3

For this mapping study, we used double-digest and partial-probe analyses in conjunction with a novel approach for establishing the linkage relationships of restriction fragments in genomic DNA.

This latter method, described in more detail elsewhere (Lo, 1985) involved using DNA transformation to introduce LC2-3 genomic DNA into tk- mouse L cells, and then examining the tk’ transformants for the cotransfection of tk restriction fragments from the donor genomic DNA. The feasibility of this approach is based on the fact that, DNA transfected by this method is usually randomly degraded to pieces of 5 to 15 kb in size before integration (Wake et al., 1984). As a result, only closely apposed parental restriction fragments are expected to be cotransfected. Using this approach, 12 calcium phosphate transfectants were isoiated, one of which (LC2-3d2) contained all the tk restriction fragments found in LC2-3. This result indicates that all the ptk DNA in LC2-3 must be integrated in close proximity within a single chromosomal integration site. The remaining 11 transfectants were each found to have different subsets of the parental ptk restriction fragments. An analysis of how these restriction fragments were coinherited allowed us to establish the relative positions of all the ptk restrict’ion fragment’s in LC2-3. Using the linkage data obtained from these secondary transfectants in conjunction with data derived from conventional Southern restriction analyses (Lo, 1985), we succeeded in constructing a

Rearrangements in Satellite DNA

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Figure 3. Restriction maps of tk inserts in LC2-3. In the upper part of t’he Figure there is a restriction map of the ptk plasmid linearized with HindIII. This form of ptk was used to generate the LC2 cell line. Beneath the ptk map is a bar diagram, which helps to illustrate the relative orientation of the ptk inserts in the LC2-3 genomic DNA. In the lower part of the Figure there is a complete restriction map for all the ptk inserts in LC2-3. The arrows above each map indicate the boundaries of the tk mRNA (McKnight, 1980) and point in the direction of transcription. B. BarnHI: Bg, RglII: H. NincIT; Hd, HindHI; P, P&I; Pv. PvuII; R, EcoRI; S, S&I.

restriction map for all the tk inserts in LC2-3 (Fig. 3). This map revealed that, in LC2-3, there are four partial copies of the injected ptk sequences, two of which contain the intact tk structural gene and promoter (as indicated by the presence of the 2.08 kb PvuII fragment of ptk; Colbere-Garapin et al., 1979; McKnight, 1980; McKnight & Gavis, 1980: McKnight et al., 1981). These sequences are arranged as direct and inverted tandem repeats and, surprisingly, one of the inverted repeats is palindromic (the one on the right side of Fig. 3). Interspersed between these ptk inserts are unidentified sequences that may be of mouse origin. A very striking feature of this restriction map is that the flanking mouse DNA is highly devoid of recognition sites for each of 12 restriction enzymes used in the mapping studies (data not shown). We observed, at the most, four restriction sites in a stretch of DNA that is at least 50 to 100 kb in size (Lo, 1985). These results strongly suggest that the flanking DNA is composed of simple sequence repeats.

(c) Instability

is not heritable by DNA transfection

Given the unusual structure of the tk inserts and the apparently repetitive nature of the adjacent mouse DNA in LC2-3, it seemed possible that either of these properties could be responsible for the instability observed in this cell line. To examine this further, we used the calcium phosphate transfectant LC2-3d2, which was generated in the above mapping studies, to determine if the instability in LC2-3 is heritable via its ptkcontaining genomic DNA. The LC2-3d2 cells contain the entire cluster of ptk inserts of LC2-3, including at least 6.4 kb of the mouse DNA flanking the right end of this array (unpublished results and

also see below). Restriction analysis (using seven enzymes, data not shown) demonstrated that the ptk array in this cell line is identical with that of LC2-3 but, as expected, the host environment surrounding these ptk inserts was found to be quite different. This was indicated by the fact that the ptk-containing high molecular weight DNA of LC2-3 and LC2-3d2 banded in very different positions in Hoechst/cesium chloride density gradients (Manuelidis, 1977; also see below). In light of these observations, we used the LC2-3d2 cell line to determine if the ptk cluster underwent rearrangement within the context of a new chromosomal environment. We expected that if the palindromic structure of the ptk sequences or the short stretch of repetitive mouse DNA immediately flanking the ptk cluster were responsible for the LC2-3 instability, the LC2-3d2 cells would exhibit instability at a level comparable with that observed with the LC2-3 cells. To examine this possibility, we isolated 12 single-cell subclones from LC2-3d2 and characterized their ptk restriction patterns. We found that each of the LC2-3d2 subclones had ptk restriction patterns that were identical with the parent (data not shown). This result contrasts with the previous finding of DNA rearrangements in three of four subclones isolated from the LC2-3 culture (Fig. 2) and suggests that the 1X2-3 instability is not heritable via the transfected ptk array. These observations indicate that the high degree of instability in LC2-3 is not related to the repeat structure of the ptk sequences or to the presence of the immediately flanking mouse DNA. Rather, it seems likely that the instability is brought about by DNA sequences outside of the immediate area of ptk integration. To characterize the chromosomal environment around the ptk insertions, we carried out in situ hybridization of metaphase chromosomes spreads. restriction

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K. A. Butner and C. W. Lo .

Figure 4. In situ hybridization of a metaphase spread of LC2-3F. Hybridization was carried out w&h 0.2 pg of 3Hlabeled ptk probe/ml and exposure was for 5 days at 4°C. Of 71 metaphase spreads examined. the only significant hybridization (51/160 grains) occurred within the pericentromeric region of a single metacentric chromosome (referred to as M7). In particular, grains were clustered between the 2 centromeric constrictions of this chromosome (see arrows). Magnification: 1000 x (d) LocaEization of tk inserts in the pericentromeric heterochromatin In situ hybridization of metaphase chromosome spreads was carried out for the subclone LC2-3 and its rearranged daughter LC2-3F. For this analysis, a nick-translated tritiated ptk probe was used. In both cell lines, the only significant, hybridization was localized to a region bounded by the two centromeric constrictions of a single bi-armed or metacentric chromosome (which we will refer to as M7) (Fig. 4; see arrow). This is based on the examination of 29 LC2-3 and 71 LC2-3F metaphase spreads. In LC2-3, 25% (24/99) and in LC2-3F, 32% (51/160) of the total grains were clustered in this region. Also consistent with this localization is the observation that this particular region has been expanded (lengthened) in an LC2-3 derived cell line (LC2-3E17-5) that contains a 25. to 50-fold amplification of the ptk insertions. Note that, as the standard mouse karyotype includes only acrocentric chromosomes (Nesbitt & Francke, 1973), the M7 chromosome is probably the product’ of a chromosomal fusion event (Rushton, 1973). The morphological details of this chromosome can be seen more clearly in the G-banded karyotype illustrated in Figure 5(a). Given that the sites of ptk hybridizat’ion in LC2-3 and LCZ-3F are identical, it is likely that the DNA rearrangement(s) that led to the formation of the subclone LC2-3F occurred intrachromosomally. Also in the course of these metaphase analyses, we noted that. the karyotype of LC2-3 and LC2-3F very httle heterogeneity. This demonstrated observation indicates that the DNA rearrangements detected in the tk restriction pattern cannot be the result of gross chromosomal instability in the LC2-3

cell lineage. This result, is also consistent with the previous Southern analyses that revealed no restriction pattern changes at three other random regions of the mouse genome, e.g. at the alphafetoprotein, globin or alpha-act’in loci. As the centromeres of mouse chromosomes are typically associated with constitutive heterochromatin, the observation of ptk inserts in the pericentromeric region suggests that the ptk DNA may be embedded in constitutive heterochromat,in. To examine this possibility, we used t’he C-banding technique to stain selectively t,he constitutive heterochromatin of the LC2-3 chromosomes (Arrighi & Hsu, 1974). The results of this analysis revealed that the region between the two centromerit constrictions of the M7 chromosome is indeed contained within a large C band (Fig. 5(b), see arrow). This suggests that the ptk inserts are integrated in constitutive heterochromatin. (e) Flanking

mouse DNA consists of mouse satellite DNA

As mouse centromeres and, in particular, the pericentromeric C-banded regions of mouse chromosomes have been shown to contain large amounts of sDNA (Pardue & Gall, 1970), the above observations suggest that the chromosomal environment around the ptk insert may also consist of sDNA. This possibility is consistent with the results of our restriction analysis, which indica.te that the mouse DNA flanking the ptk inserts consists of long (>50 kb) stretches of highly repetitive sequences. To determine the molecular nature of the host sequences surrounding the ptk insertions, we fractionated high molecular weight

Rearrangements in Satellite DNA

Figure 5. Cytogenetic analvsis. (a) Trypsin G-banded metaphase of LC2-3. Arrow kdicates chromosome M7. In particular, notice the location of the 2 centromeric constrictions. (b) C-banded metaphase spread of Lc2-3. Arrow (I-banded wntromeric

denotes chromosome M7. Pu’otice that region covers const,rictions.

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(> 100 kb) LC2-3 genomic DNA on a Hoechst/ equilibrium cesium chloride density gradient (Manuelidis, 1977). Hoescht’s dye preferentially binds to A +T-rich DNA and exaggerates the difference in density between mouse sDNA and main-band sequences. Using this fractionation scheme, we found that the LC2-3 ptk sequences banded at a position between main-band and sDNA. Similar results were obtained with two other LC2-3 derivatives (LC2-3E17-5, LC2-3B) (Fig. 6), both of which have retained the ptk inserts within the same cytogenetic location as the ptk inserts in LC2-3. These findings suggest that the ptk cluster in LC2-3 (and its derivatives) is embedded in a large block of sDNA. This conclusion is based on the fact that the ptk DNA is G+C-rich (McKnight, 1980) and, consequently, ptk associated with the sDNA would be expected to fractionate in a region between main-band and sDNA. To examine directly whether the tk inserts are flanked by mouse sDNA, we cloned and characterized the 8.8 kb BgZII mouse-tk junction fragment of LC2-3 (see Figs 3 and 7(a)). The cloning was carried out in a RecA- host using a positive selection plasmid vector (pKGW). This approach was undertaken to avoid potential problems with the cloning of highly repetitive DNA in lambda cloning vectors. To facilitate the cloning, we used genomic DNA from LC2-3E17-5, a subclone of LC2-3 that has amplified tk inserts including this 8.8 kb BglII fragment (approx. 25-fold; unpublished results). The strategy we undertook consisted of first enriching for the ptk-containing sequences by fractionating the genomic DNA from this cell line on a Hoechst/cesium chloride density gradient and then using the ptk-enriched fractions to generate a genomic library. This library was screened with a tk-specific probe and of 80 positive clones obtained approximately 20% were found to have the 8.8 kb mouse genomic insert. One of the latter clones designated at pll was chosen for further analysis. The insert within pll has been mapped with PvuII, EcoRI, SstI and BgZII. The result of this analysis indicated that the pll insert is identical with the 8.8 kb BgZII junction fragment of LC2-3 (Fig. 3). Consistent with this conclusion is the fact that the two restriction fragments released by EcoRI digestion of the pll insert were observed to either hybridize only with the ptk probe (the 2.4 kb fragment) or almost entirely with mouse DNA (the 6.4 kb fragment), as would be expected based on the LC2-3 restriction map. To characterize furt.her t,he mouse sequences within pl 1, we nick-t,ranslated pll and used it to probe dot blots containing various amounts of mouse (Ltk-) genomic DNA. From the intensity of hybridization. it was clear that the mouse sequences within pll are highly repeated to t,he order of 10’ to IO6 copies per haploid genome (data not shown). Furthermore, hybridization of pll to dot blots of LCZ-3 DNA fractionated on a Hoechst/cesium chloride density gradient revealed very strong hybridization to the lighter A+T-rich fractions and, in particular. to

K. A. Butner

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Figure 6. Localization of ptk and pll sequences in LC2-3E17-5 DNA fractionated in a cesium chloride density gradient. (a) Hoechst/CsCl density-gradient fractionation of LC2-3E17-5 genomic DNA. Approximately 10’ LC2-3El7-5 cells were lysed, incubated with Hoechst dye, adjusted to 8 ml containing 1.68 g CsCl/ml and centrifuged in a 50 Ti rotor at 32,000 revs/min for 63 h followed by 40,000 revs/min for 32 h (Manuelidis, 1977). Fractions of 150 ~1 were collected and 5% of every 3rd fraction was separated by electrophoresis in a O.Sy’ 1 x TBE agarose gel. The photograph of the gel was taken under ultraviolet transillumination. The positions of the satellite and main band DNAs were approximated by their positions in the intact gradient as visualized with long-wavelength ultraviolet illumination. L, top of gradient; H, bottom of gradient; S, satellite DNA; I. interband region; M: main-band DNA; R, RNA pellet. (b) Dot blots of LC2b-3E17-5 gradient fractions. The DNA remaining in each fraction analyzed in the above gel was purified and dot blotted onto nit’rocellulose. The blot was hybridized with a ptk probe, washed and exposed at -80°C for 16 h with a DuPont intensifying screen. The resulting autoradiographic image is illustrated in the upper row of dots. Sfter the dot blot had decayed to background radioactivity levels. it was reprobed with a pll probe, washed and exposed for 1 h without, a screen. The result is illustrated in the lower row of dots. The brackets in both series of dots indicate the fractions containing the strongest hybridization signal.

those fractions containing sDNA (Fig. 6). Together, these results suggest that the pll insert is likely to contain mouse satellite DNA. Also consistent with that a 235 bp this possibility is the finding restriction fragment is released in large molar excess when pll is cut with restriction enzymes (MnZI or BstNI) that cleave once within the 235 bp repeat of mouse sDNA (data not shown). To determine how the sDNA repeats are organized in pll, we carried out an end-labeling and partial digestion experiments using the 6.4 kb EcoRI/BgZII fragment labeled at the BgZII end (Fig. 7(a)).

Figure 7. Organization of mouse repeats flanking the site of tk insertion. (a) Restriction map. A BgZII restriction map of the tk inserts in LC2-3 is illustrated (see also Fig. 3). For orientation, the 4 ptk inserts have been shown as 4 open arrows above the restriction map and the 8.8 kb tk-mouse junction fragment cloned in pl 1 is delineated by a bracket. Also shown is the 6.4 kb EcoRI/BgZII restriction fragment used for the endlabeling/partial digest experiment illustrated in (b). ITsing the data obtained from the experiment illustrated in (b), the MnZI restrict,ion sites at the right end of the 8.8 kb BgZII fragment were mapped. The arrangement of these sites suggests that this region of mouse DNA is composed of a tandem array of sDNA repeats. (b) Partial digestion of the 6.4 kb EcoRI/BgZII fragment of pll. pll was digested with BgZII, end-labeled at the BgZII site using a Klenow reaction, then digested with EcoRI and separated in a 0.8% agarose gel. The 6.4 kb EcoRI/BgZII fragment was purified from the gel by electroelution and used as a substrate for partial digestion with BstNI. Enzymatic reactions were carried out at 37°C for 1 h in the presence of variable amounts of enzyme. The digests were separated on a 2”/0 agarose gel and the autoradiograms obtained is illustrated above. Notice the even spacing of the ladder released by BstNI. The amounts of enzyme (units/pg) used in each digest are as follows: lane a, 2: b, 1: c. 0.5; d. 0.25: e. 0.12; f, 0.2; g, 0.1; h. 0.05: i. 0.025; j, 0.012.

Rearrangements in Satellite

PSAT s DNA

BstNI 1o 20 30 40 50 60 v -lGGAATATGE EGAGAAAACT GAMATCACG GAAAATGAGA AATACACACT TTAGGACGTG X%AATATGG

SGAGAAAACT GAAMTCACG MnII

AA&TTGGTG70 PSAT

555

DNA

--

-

GAAAATGAGA AATACACACT TTAGGACGTG

80 90 100 110 120 AGGAAAACTG AAAAAGGTGG AAAATTTAGA AATGTCCACT GTAGGACGTG

s DNA

AGGAAAACTG AAAAAGGTGG MAATTTAGA AAATATGGSG --

p SAT

130 140 150 160 170 180 GAATATGGCA AGAAAACTGA AAATEATGGA AAATGAGAAA CATCCACTTG ACGACTTGAA

sDNA

GAATATGGCA AGAAAACTGA AAATCATGGA MATGAGAAA

PSAT

AAATGACM

s DNA

AAATGACGAA ATCACTA@A

190

200 ATCACTAIAA

210 AACGTGAAiU

220

AATGTCCACT GTAGGACGTG

CATCCACTTG ACGACTTGAA

230

ATGAGAAATG CACACTGAAG Gmm

AACGTGAAAA ATGAGAAATG CACACTGAAG GACCT

Figure 8. The DNA sequence of the repeat unit in PSAT compared with the consensus sequence of the major mouse satellite repeat (Horz & Altenburger, 1981). The underlined nucleotides are single base-pair differences. Kate that the blocked gap corresponds to a single nucleotide deletion in PSAT. The bold-face nucleotides at positions 70 to 73 correspond to the MnZI restriction site and those at 1 to 3 and 233 to 235 correspond to the 2 halves of the restriction site recognized by BstNI. Cleavage sites for B&N1 and MnZI are indicated by filled and open triangles. respectively.

Partial digestion with BstNI released a regular ladder of DNA fragments with a repeat length of approximately 235 bp (Fig. 7(b)). To confirm the satellite origin of the 235 bp repeat, we subcloned and sequenced the repeat unit of pll (Fig. 8). A comparison of the DNA sequence of the repeat unit with the consensus sequence of the major mouse sDNA repeat revealed that they are essentially identical, with the exception of eight nucleotide mismat’ches and one nucleotide deletion (Fig. 8).

4. Discussion Our results demonstrate that a high frequency of DNA rearrangements is associated with a cluster of tk inserts in a ptk-transformed mouse L cell line. These ptk inserts were localized to the pericentromerit region of a mouse chromosome. In addition, the mouse DNA flanking the site of ptk integration was demonstrated to contain classical mouse sDNA. Surprisingly, our restriction mapping analyses revealed that some of the ptk inserts are arranged in palindromic arrays. These unusual structures are probably not responsible for the observed instability as no DNA rearrangement was detected in the subclones of LC2-3d2, a secondary transfectant that contains the entire LC2-3 ptk cluster along with several kb of flanking mouse sDNA. This finding is in agreement with the results of studies in prokaryotes that revealed that long perfect palindromes can become stabilized if their center of

symmetry is interrupted (Hagan & Warren, 1983), as is the case for the tk inserts in LC2-3. The above results indicate that neither t,he of the ptk inserts nor the imstructure sDNA is responsible for mediately adjacent propagating the instability in this cell line. On the basis of these considerations, we suggest that the instability may be related to the pericentromeric DNA environment of the ptk insertions. Consistent with this suggestion is the finding of another study in which DNA inserts localized to the centromere of a hamster chromosome were also observed to undergo DNA rearrangements (Wahl et al., 1984). If pericentromeric DNA is capable of generating instability, it is interesting to consider what mechanism might be involved in this process. One possibility might be the presence of specific nonsatellite DNA sequences in the pericentromeric region that can propagate instability. As no instability was observed in the ptk arrays of LC23d2, if such sequences exist in LC2-3 bhey must’ be situated at t’oo great a distance to be cotransfected with the ptk inserts. Note that at present we have no evidence for the existence of such DNA sequences. Another possible explanation is that instability may be propagated by the presence of very large blocks of repetit)ive DIVA. So, perhaps the overall structure of the satellite DNA can potentiate rearrangements by facilit’ating recombination, as has been suggest,ed to occur during the expansion and cont)raction of sDSA (Smith, 1976).

K. A. Butner and C. W. Lo Regardless of the mechanisms mediating pericentromeric DNA rearrangements, the question still remains as to whether the LC2 instability results from endogenous instability of mouse sDNA, or is a consequence of the ptk insertion event, Owing to the highly repetitive nature of sDNA, there is no way of directly addressing this question. However, at present, the former possibility seems more likely as it is consistent with the known fluidity of sDNA. Nevertheless, it is interesting to note that in nature non-satellite DNA have been observed to be inserted in the midst of sDNA (Carlson & Brutlag, 1978; McCutchan et al., 1982; Potter & Jones, 1983; Thayer & Singer, 1983), and if such spontaneous integration events can generate instability, then they may also play a significant role in the rapid evolution of sDNA. In the future we will use this cell line to determine whether intrachromosomal recombination, interchromosomal recombination (between homologous or non-homologous chromosomes), or saltatory replication play a role in these ptk-associated sDNA rearrangements. We hope that further studies with this cell line will provide important insights for understanding the fluidity associated with satellite DNA. We thank Andy Golden for excellent technical assistance, John Kaumeyer and Larry Yaeger for helpful discussions and suggestions on subcloning and DNA sequencing, Donna George for helpful suggestions on the preparation and banding of the metaphase spreads, Mary Harper for providing us with her in situ hybridization protocol, and Anna Chao for help in adapting this technique in our laboratory. In addition, we are grateful to Richard Axel, Philip Leder. Shirley Tilghman and Adrian Mint,y for providing us, respectively, with the herpes thymidine kinase, mouse globin, alpha-fetoprotein and actin probes. Also we thank Donna George and Kelly Tatchell for critical readings of the manuscript. This work was supported by NIH grants (CA 31042 and GM 30461) and a Basil O’Connor March of Dimes Grant 5-328. K.B. is supported by a NIH training grant (T32 GM 17517).

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by M. Gottesman