Interference of the Simian Virus 40 Origin of Replication by the Cytomegalovirus Immediate Early Gene Enhancer: Evidence for Competition of Active Regulatory Chromatin Conformation in a Single Domain

Abstract

Replication origins are often found closely associated with transcription regulatory elements in both prokaryotic and eukaryotic cells. To examine the relationship between these two elements, we studied the effect of a strong promoter-enhancer on simian virus 40 (SV40) DNA replication. The human cytomegalovirus (CMV) immediate early gene enhancer-promoter was found to exert a strong inhibitory effect on SV40 origin-based plasmid replication in Cos-1 cells in a position- and dose-dependent manner. Deletion analysis indicated that the effect was exerted by sequences located in the enhancer portion of the CMV sequence, thus excluding the mechanism of origin occlusion by transcription. Insertion of extra copies of the SV40 origin only partially alleviated the inhibition. Analysis of nuclease-sensitive cleavage sites of chromatin containing the transfected plasmids indicate that the chromatin was cleaved at one of the regulatory sites in the plasmids containing more than one regulatory site, suggesting that only one nuclease-hypersensitive site existed per chromatin. A positive correlation was found between the degree of inhibition of DNA replication and the decrease of P1 cleavage frequency at the SV40 origin. The CMV enhancer was also found to exhibit an inhibitory effect on the CMV enhancer-promoter driving chloramphenicol acetyltransferase expression in a dose-dependent manner. Together these results suggest that inhibition of SV40 origin-based DNA replication by the CMV enhancer is due to intramolecular competition for the formation of active chromatin structure.

A majority of DNA replication origins in both prokaryotic (3, 11, 35, 42, 53, 56, 59) and eukaryotic (1, 5, 12, 17–19, 24, 25, 36, 38, 41, 43, 48, 60, 62–66, 68, 71, 75, 78, 81, 83, 84, 93–95) cells are closely associated with a transcription unit. Recent characterization of origins of replication in mammalian cells has shown that the origins are located very close to a transcription unit or even within a transcription unit (see review in reference 20). The tight association between transcription and replication units has raised the question of whether or not these two processes are coupled (32, 36, 42, 64, 91).

Mutual influences between replication and transcription processes have been demonstrated. Activation of DNA replication by transcription has been found in both prokaryotic (3, 11, 35, 56, 70) and eukaryotic (48, 60) cells. On the other hand, transcription has been found to suppress replication in bacterial plasmid DNA (47, 53) and in yeast (77, 82), tetrahymena (61), and human (33) cells. Conversely, DNA replication can enhance transcription (28, 32, 64, 90, 91). Evidence for coupling or a correlation between transcription and replication processes has also been observed in Bacillus subtilis (42), in Physarum (64), in temporal regulation of transcription in active genes in early S phase (26, 37), and in several mammalian genes (5, 41, 71, 94). Transcription factors have also been implicated in regulating replication origins (7, 13, 18, 19, 30, 31, 38, 52).

To understand the role of transcription factor regulatory elements and the transcription process in the regulation of eukaryotic DNA replication, we studied the effect of strong promoter-enhancer on the simian virus 40 (SV40) replication origin. Our results indicate that the human cytomegalovirus (HCMV) immediate early (IE) gene enhancer inhibits SV40 origin-dependent replication in a position- and dose-dependent manner. A similar inhibitory effect was observed in a plasmid containing a Rous sarcoma virus (RSV)-mouse mammary tumor virus (MMTV) enhancer-promoter. Our results based on probing chromatin structure at regulatory sequences by P1 nuclease suggest that inhibition of the SV40 origin by the CMV IE enhancer is likely to be the result of competition between the SV40 origin and CMV enhancer for the formation of active chromatin conformation. P1 nuclease analysis provided strong evidence that only one of the regulatory elements in a DNA containing multiple regulatory elements can form active chromatin structure.

MATERIALS AND METHODS

Plasmid constructions.

Table 1 describes the plasmids used in this study. Plasmid pCMVIE, a gift from Felicia Wu, contains a CMV IE enhancer-promoter in front of a chloramphenicol acetyltransferase (CAT) reporter gene. For the construction of pSCM1 plasmids (Fig. 1a), the CMV sequence in pCMVIE, containing the HCMV IE gene enhancer and promoter, was excised by digestion with restriction endonucleases ClaI and HindIII, extended with HindIII linker, and then inserted at the HindIII site (nucleotide [nt] 5001/1 in pSV2cat) of pSV2cat or pSV2neo to generate plasmid pSCM-1cat or pSCM-1neo. Insertion of the CMV IE gene enhancer-promoter at other sites of pSV2cat was achieved by adding appropriate restriction endonuclease linkers to the CMV fragment or by blunt-end ligation using T4 DNA polymerase or Klenow fragment. Plasmids were named pSCM-x(y), where x is the nucleotide number of pSV2cat at which the CMV enhancer-promoter was inserted and y represents the inserted orientation relative to the direction of transcription from the SV40 early promoter, either the same [(+)] or opposite [(−)] direction.

TABLE 1.

Plasmid constructs

Clone name	Insertion
Parental pSV2cat derivatives
pSCM-1(−)	HCMV P/Ea at nt 1/5001 of pSV2cat, orientation (−)
pSCM-1(+)	HCMV P/E inserted position as in pSCM-1(−), where inserted orientation is (+)
pSCM-4755(−)	HCMV P/E at nt 4755 of pSV2cat, orientation (−)
pSCM-4755(+)	HCMV P/E inserted position as in pSCM-4755(−), where inserted orientation is (+)
pSCM-4486(−)	HCMV P/E at nt 4486 of pSV2cat, orientation (−)
pSCM-3502(−)	HCMV P/E at nt 3502 of pSV2cat, orientation (−)
pSCM-3502(+)	HCMV P/E insertion position as in pSCM-3502(−), where inserted orientation is (+)
pSCM-3369(−)	HCMV P/E at nt 3369 of pSV2cat, orientation (−)
pSCM-3369(+)	HCMV P/E insertion position as in pSCM-3369(−), where inserted orientation is (+)
pSCM-2824(+)	HCMV P/E at nt 2824 of pSV2cat, orientation (+)
pSCM-2543(−)	HCMV P/E at nt 2543 of pSV2cat, orientation (−)
pSCM-2543(+)	HCMV P/E inserted position as in pSCM-2543(−), where inserted orientation is (+)
pSCM-554(−)	HCMV P/E at nt 554 of pSV2cat, orientation (−)
pSCM-554(+)	HCMV P/E inserted position as in pSCM-554(−), where inserted orientation is (+)
pS2CM-1(+)/ori-2618(−)	1 HCMV P/E at nt 1/5001 of pSV2cat, orientation (+); 1 SV40 origin at nt 2618 of pSV2cat, orientation (−)
pS2CM-1(−)/ori-2618(−)	Each HCMV P/E and SV40 origin inserted position as in plasmid pS2CM-1(+)/ori-2618(−), where inserted orientations are (−) and (−), respectively
pS2CM-1(+)/ori-2618(+)	Each HCMV P/E and SV40 origin inserted position is as in plasmid pS2CM-1(+)/ori-2618(−), where inserted orientations are (+) and (+), respectively
pS2CM-1(−)/ori-2618(+)	Each HCMV P/E and SV40 origin inserted position as in plasmid pS2CM-1(+)/ori-2618(−), where inserted orientations are (−) and (+), respectively
pS3CM-1(−)/ori(2X)-4751(−)	One HCMV P/E at nt 1/5001 of pSV2cat, orientation (−); tandem repeats of origin at nt 4751 of pSV2cat, orientation (−)
pSCM2-2618(−)	Tandem repeats of HCMV P/E at nt 2618 of pSV2cat, orientation (−)
pSCM2-554(−)/3502(−)	Two copies of HCMV P/E separately inserted at nt 554 and 3502 of pSV2cat, where inserted orientations are (−)
pSCM2-3502(−)/2618(−)	Two copies of HCMV P/E separately inserted at nt 3502 and 2618 of pSV2cat, where inserted orientations are (−)
pSCM2-3502(+)/2618(−)	Two copies of HCMV P/E inserted as in pSCM2-3502(−)/2618(−), where inserted orientations are (+) and (−), respectively
pSVA-1	3 copies of 171-bp fragment alpha satellite DNA from monkey kidney cells inserted at nt 1/5001 of pSV2cat
pSCAGr	20 copies of human triplet CAG repeats inserted at nt 1/5001 of pSV2cat
pSVL-1	564 bp from HindIII digest of lambda phage genomic DNA inserted at nt 1/5001 of pSV2cat
pSVLs-1	125 bp from HindIII digest of lambda phage genomic DNA inserted at nt 1/5001 of pSV2cat
pSVB-1	Bent DNA from parasite Leishmania minicircle with 690 bp inserted at nt 1/5001 of pSV2cat
pSVT-1	Human telomere DNA with 35 bp inserted at nt 1/5001 of pSV2cat
pSVP-554	Fragment from phage 174 with 603 bp inserted at nt 1/5001 of pSV2cat
Parental pCATBasic derivatives
pSC-2266(−)	SV40 origin at nt 2266 of PCATBasic, orientation (−)
pSC-2266(+)	SV40 origin inserted position is as in pSC-2266(−), where orientation is (+)
pS2C-2266(−)	Tandem repeats of SV40 origin at nt 2266 of pCATBasic, orientation (−)
pS2C-2266(+)	Tandem repeats of SV40 origin inserted position as in pS2C-2266(−), where orientation is (+)
pS3C-2266(−)	3 tandem repeats of SV40 origin at nt 2266 of pCATBasic, orientation (−)
pSC-2266(−)/CMV-2242(+)	1 SV40 origin at nt 2266 of pCATBasic, orientation (−); 1 HCMV P/E at nt 2242, orientation (+)
pSC-2266(+)/CMV-2242(+)	Each SV40 origin and HCMV P/E inserted position as in pSC-2266(−)/CMV-2242(+), where orientations are (+) and (+), respectively
pSC-2266(−)/CMV-2242(−)	Each SV40 origin and HCMV P/E inserted position as in pSC-2266(−)/CMV-2242(+), where orientations are (−) and (−), respectively
pSC-2266(+)/CMV-2242(−)	Each SV40 origin and HCMV P/E inserted position as in pSC-2266(−)/CMV-2242(+), where orientations are (+) and (−), respectively
pS2C-2266(−)/CMV-2242(+)	As in pSC-2266(−)/CMV-2242(+), but SV40 origin replaced with 2 copies
pS2C-2266(+)/CMV-2242(+)	As in pSC-2266(+)/CMV-2242(+), but SV40 origin replaced with 2 copies
pS2C-2266(−)/CMV-2242(−)	As in pSC-2266(−)/CMV-2242(−), but SV40 origin replaced with two copies
pS2C-2266(+)/CMV-2242(−)	As in pSC-2266(+)/CMV-2242(−), but SV40 origin replaced with 2 copies
pS3C-2266(−)/CMV-2242(+)	As in pSC-2266(−)/CMV-2242(+), but SV40 origin replaced with 3 copies
pS3C-2266(−)/CMV-2242(−)	As in pS3C-2266(−)/CMV-2242(+), but orientation of HCMV P/E is (−)
pCM	HCMV P/E at nt 2266 of pCATBasic, orientation (+)
pCM-en1	As in pCM, insertion of additional HCMV enhancer at nt 3910 of pCATBasic, orientation (+)
pCM-en2	As in pCM, insertion of 2 additional of HCMV enhancer at nt 3910 and 4134 of pCATBasic, orientations (−) and (+), respectively
pCM-en4	As in pCM, insertion of tandem repeats of HCMV enhancer at nt 3910 of pCATBasic, orientation (−); each with 1 copy of enhancer at nt 4134 and 16, orientations (+) and (−), respectively

^aP/E, promoter-enhancer. 

FIG. 1.

(a) Map and replication efficiency of pSCM plasmids. The circular map represents the parental plasmid, pSV2cat. Positions of restriction sites at which the HCMV IE gene enhancer-promoter is inserted are indicated on the map, together with the replication efficiency of that plasmid relative to the parental plasmid. + and − represent the orientation of the CMV promoter relative to the SV40 early promoter. (b) Map of pCATBasic vector. AmpR, ampicillin resistance.

Insertion of restriction fragments of CMV enhancer-promoter sequence into the HindIII site of pSV2cat was performed similarly. BAL31 deletion mutations of the CMV enhancer or promoter sequence were constructed by deletion from a restriction site as indicated in the text.

Plasmid pL, used as an internal control of replication efficiency, was constructed by replacing the HindIII-SmaI fragment of pSV2neo with a 564-bp HindIII fragment of lambda phage DNA.

Plasmid clones containing a human triplet CAG repeat, bent DNA sequence from Leishmania minicircle (49), and monkey cell alpha satellite repeat sequence of monomer, dimer, and trimer sizes (171, 342, and 513 bp, respectively) were constructed by inserting the appropriate DNA fragment into the HindIII site of plasmid pSV2cat or pSV2neo. Inserted sequences were checked by DNA sequencing.

Oligonucleotides corresponding to SV40 21-bp repeats, human telomere repeat (5 to 15 repeats of TTAGGG), and NFκB sequence (ATCAACGGGACTTTCCAA repeated twice) with HindIII linker sequence at the ends were synthesized using an oligonucleotide synthesizer. These oligonucleotides were cloned into pSV2cat at the HindIII site. The presence of the target insert was confirmed by DNA sequencing.

pSC series plasmids were constructed from the parental plasmid, pCATBasic (Promega), by the insertion of an SV40 origin fragment or its polymer (SV40 nt 5197 to 325) at the HindIII site (nt 2266 in the parental plasmid) to obtain plasmids pSC-2266, pS2C-2266 (containing two copies of the SV40 origin), and pS3C-2266 (containing three copies of the SV40 origin). The CMV enhancer/promoter was then inserted at the HindIII site of these plasmids to obtain the corresponding plasmids pSC-2266/CMV, pS2C-2266/CMV, and pS3C-2266/CMV. Plasmid pCM was constructed by inserting one copy of the CMV promoter-enhancer (NcoI-HindIII fragment) at the XbaI (nt 2266) site of pCATBasic (Promega). Plasmids pCM-en1, pCM-en2, and pCM-en4 were derived from pCM by the insertion of an additional copy of the CMV enhancer fragment, ClaI to NcoI (−598 to −223), at the positions indicated in Table 1.

Plasmid pMAMneo-CAT was obtained from Clontech. pMneo-CAT was constructed from pMAMneo-CAT by removing the MluI-to-BspEI fragments, deleting the RSV-MMTV sequences.

Transient DNA replication assay.

Cos-1 cells at 50 to 60% confluence were cotransfected by the DEAE-dextran method (79) with 1 μg of each plasmid DNA to be tested and pL. This cell line expresses SV40 T antigen constitutively, thus allowing the replication of plasmids containing the SV40 DNA replication origin. Transfection efficiency was around 1 to 2% as determined by transfection with plasmid containing β-galactosidase reporter gene. At 48 h after transfection, plasmid DNA was recovered from the transfected cells by the Hirt method (39), digested with restriction nuclease DpnI to remove parental unreplicated DNA and then with appropriate restriction endonucleases as indicated in the figure legends, and analyzed by agarose gel electrophoresis and Southern blotting. Replication efficiency of a test plasmid relative to the parental pSV2cat plasmid was determined using the cotransfecting pL as the internal standard. The radioactivity of each DNA band was measured with an Instantimager (Packard). The ratio of the replicated test plasmid relative to pL was corrected for the input amount in the same Southern blot.

Two-dimensional gel electrophoresis analysis of DNA replication intermediates.

Plasmid DNA recovered from transfected cells was cleaved with restriction endonucleases StuI and FspI and analyzed by a two-dimensional electrophoresis technique described previously (87).

CAT assay.

CAT activity was measured by the method of Gorman et al. (27). Each set of measurements included a mock control and a pSV2cat positive control.

P1 nuclease digestion of intracellular nucleoprotein complexes.

Cos-1 cells transfected with plasmids were lysed at 48 h after transfection with lysis buffer (0.5% NP-40, 10 mM Tris-HCl [pH 7.5], 0.2 mM ZnCl₂), and the nuclei were digested with P1 nuclease as described previously (15). After P1 nuclease digestion, plasmid DNA was extracted by the Hirt method and purified by phenol and chloroform extractions. The purified plasmid DNA was digested with restriction endonucleases as described in the text and figure legends and then analyzed by agarose gel electrophoresis and Southern blot analysis using appropriate probes that abut one end of the cutting sites (indirect end labeling).

Transformation of Cos-1 cells.

Cos-1 cells were transfected with plasmid pSCM-1neo, and the transfected cells were selected with G418 (300 μg/ml). Surviving cell colonies were picked using Pasteur pipettes and grown in the presence of G418 (300 μg/ml).

RESULTS

Position-dependent inhibition of the SV40 origin activity by the CMV IE gene enhancer-promoter.

To study the effect of a neighboring strong transcription unit on the activity of the SV40 origin of replication, we inserted the CMV IE gene enhancer-promoter at the HindIII site (nt 1/5001) located immediately on the early promoter side of the SV40 replication origin in plasmid pSV2cat (Fig. 1a). The resulting plasmid, pSCM-1(−), with the CMV promoter in the opposite direction of the SV40 early promoter, was found to accumulate about 1 to 3% of the amount of DNA of the parental plasmid, pSV2cat, using the cotransfected plasmid pL as a quantitative internal standard (Fig. 2a; compare the region of test plasmid DNA in lanes 2 and 8). Similar results were obtained when the CMV sequence was inserted in the opposite direction (data not shown). Control plasmids containing insertions of various DNA fragments similar in size to the CMV enhancer-promoter at the HindIII site were also analyzed (Fig. 2b). Insertion of lambda phage DNA fragments, alpha satellite, telomeric, and CAG repeats, and Leishmania bent DNA had no significant effect on replication of plasmid DNA. Thus, the inhibition of replication of plasmid pSCM-1 is specifically mediated by the CMV IE sequence.

FIG. 2.

Gel electrophoretic and Southern analysis of replication of test plasmids transfected into Cos-1 cells. Lane sets A and B represent input plasmid DNA samples used for transfection and plasmid DNA extracted from transfected cells 48 h posttransfection and treated with restriction endonuclease DpnI to remove unreplicated and methylated input DNA, respectively. Plasmid pL cotransfected with the test plasmids is the internal standard used to standardize transfection efficiency. (a) Analysis of pSCM plasmids. Lanes 1 and 7, pSV2cat parental plasmid; lanes 2 and 8, pSCM-1(−); lanes 3 and 9, pSCM-4755(−); lanes 4 and 10, pSCM-3369(−); lanes 5 and 11, pSCM-3502(−); lanes 6 and 12, pSCM-2618(−). All DNA samples had been digested with endonuclease EcoRI and probed with pSCM-2618(−) plasmid DNA. The amount of test plasmid DNA before and after transfection relative to the internal standard was determined by the radioactivity in the Southern blot as described in Materials and Methods. (b) Analysis of replication of control plasmids with insertion of a 513-bp monkey alpha satellite repeat (lanes 1 and 3) and 564-bp lambda phage HindIII fragment (lanes 2 and 4) at the HindIII site of pSV2cat. (c) Analysis of transient DNA replication of pMAMneo-CAT and its derivative pMAneo-CAT, with the RSV-MMTV transcription enhancer-promoter deleted. Lanes 1 and 4, pSV2cat; lanes 2 and 5, pMAMneo-CAT; lanes 3 and 6, pMAneo-CAT. With pL as an internal control, pMAMneo-CAT replication efficiency is about 25% of that of pSV2cat, whereas that of the deletion mutation is about 75%. Sizes of markers (M) are indicated in nucleotides.

When the CMV sequence was moved away from the SV40 origin, the suppression of SV40 origin-dependent DNA replication was reduced, but the effect depended on the position of the CMV IE enhancer-promoter sequence relative to the SV40 origin (Fig. 2a, with one orientation of CMV; results were similar with the other orientation [Fig. 1]). There is no simple relationship between the distance of the two regulatory elements and the degree of inhibition (see Discussion). For example, insertion of the CMV sequence at the PvuII site (nt 323) immediately on the late promoter side of origin reduced the accumulated DNA amount to about 1 to 5% of that for pSV2cat, but CMV sequence located at the NdeI site at nt 554 reduced DNA replication to only about 45% of the control. On the other hand, the CMV enhancer-promoter at the EcoRI site about 2.6 kb away from the SV40 origin reduced the accumulated DNA amount to about 20% of the control (Fig. 2a, lanes 6 and 12). These results suggest a position-dependent effect of the CMV IE gene enhancer-promoter on SV40 origin-based DNA replication. The position-dependent inhibitory effect is highly reproducible, since all experiments were performed at least three times and each set of data was carefully analyzed by counting the radioactivity in each DNA band with an Instantimager. Similar results were obtained when plasmid pSV2neo was used as the starting vector.

To examine whether the suppression effect is specific to the CMV sequence, we carried out replication analysis of a commercially available plasmid, pMAMneo-CAT, that contains an RSV-MMTV transcription regulatory element and an SV40 origin. This plasmid replicated poorly compared to pSV2cat. Deletion of the RSV-MMTV transcription regulatory element induced replication activity about threefold, indicating that this transcription regulatory element also exerts a negative effect on the SV40 origin (Fig. 2c).

Dosage effect of the CMV enhancer-promoter on SV40 DNA replication.

The results above indicate a position-dependent effect of CMV sequence on SV40 origin activity. To further analyze the mechanism of inhibition of the SV40 origin by the CMV sequence, we inserted multiple copies of the CMV sequence, either in tandem or separately, in pSV2cat. Insertion of extra copies of the CMV sequence either in tandem or at different sites further inhibited SV40 origin-dependent replication (Fig. 3).

FIG. 3.

Dosage effect of CMV IE enhancer-promoter on SV40 origin activity. Replication efficiency relative to that of the parental plasmid pSV2cat was determined for plasmids containing one copy (1×) or two copies (2×) of the HCMV IE enhancer-promoter (P/E) inserted at the positions indicated. The orientation of the CMV promoter relative to the SV40 early promoter is indicated as (+) or (−). Plasmids containing two copies of the CMV enhancer-promoter replicate much less efficiently than plasmids with only one.

Inhibition of SV40 origin-dependent replication is located in the enhancer portion of the CMV regulatory element; exclusion of an origin occlusion mechanism.

To determine whether inhibition of the SV40 origin is due to interference of initiation at the origin by transcription from the CMV promoter, we removed the CMV promoter by BAL31 nuclease deletion from the 3′ end of the CMV enhancer-promoter. As shown in Fig. 4, deletion up to nt −91 including the TATA and CAAAT promoter elements did not diminish the ability of the CMV sequence to suppress SV40 origin-dependent replication when the CMV sequence was inserted at the HindIII site in either orientation. Furthermore, the SV40 origin was not suppressed by the insertion of the minimal CMV promoter from nt −42 to +107 at either HindIII (early side of origin) or NdeI (late side of origin) in either orientation. These results indicate that transcription toward or away from the origin from either the early or late side does not affect SV40 origin-dependent DNA replication and that the CMV promoter is dispensable for the inhibitory effect.

FIG. 4.

Effect of deletion of the HCMV IE enhancer-promoter on SV40 origin activity. The sequence organization of various repeats of the IE enhancer (filled bars) and promoter elements (open bars) is shown at the top. The portion retained after deletion is shown as solid lines. The deleted CMV sequence was inserted at the HindIII site (nt 1/5001) of pSV2cat, and the replication efficiency of these plasmids relative to that of the parental plasmid was determined by using the cotransfected pL internal standard.

The fact that promoter deletion is still fully capable of inhibiting SV40 DNA replication indicates that the suppressing sequences of CMV are located within the enhancer sequence. To further locate the suppression elements, we inserted portions of the CMV enhancer at the HindIII site of pSV2cat and studied the transient replication of these plasmids in Cos-1 cells. The 5′ portion of the enhancer represented by the restriction fragments ClaI-NdeI (nt −598 to −348 relative to CMV IE transcription start site) and ClaI-NcoI (nt −598 to −223) reduced the plasmid DNA amount to about 11 to 18% and 6%, respectively, of the control plasmid (Fig. 4). Thus, the 5′ portion of the enhancer still contained repressor elements, but the effect was less than that of the whole enhancer. Similarly, the 3′ portion of the enhancer, represented by the restriction fragments NdeI-HindIII and NcoI-HindIII, also suppressed plasmid replication to about 6% of the control (Fig. 4). These data indicate that SV40 origin-suppressing elements are located throughout the enhancer.

To map the suppressing elements further, we studied the effect of deletion in the 3′ portions of the CMV enhancer. As summarized in Fig. 4, BAL31 deletion analysis suggests a suppressing element between nt −135 and −76. This region includes an NF-κB and two SP1 transcription factor binding sites. Similarly, deletion of sequence in the 5′ enhancer portion from −598 to −507 raised the replicated DNA amount from 18 to 75% of the level for pSV2cat. This result suggests a suppressing element between nt −598 and −507.

Since the SV40 enhancer also contains NF-κB and SP1 sites, we sought to determine if these transcription factor binding sequences in the 3′ portion of the CMV enhancer could inhibit the SV40 origin through some kind of competition with the SV40 enhancer. To this end, we constructed plasmids containing NF-κB binding site oligonucleotides corresponding to the 18-bp repeats in the CMV IE enhancer or its oligomers and inserted them at the HindIII site of pSV2cat. No suppression of plasmid replication was observed (data not shown). Similarly, insertion of 21-bp repeats which contained multiple SP1 binding sites from SV40 DNA at the HindIII site of pSV2cat also did not appreciably affect the replication of plasmid DNA in Cos-1 cells (data not shown). Thus, neither the SP1 nor NF-κB binding site alone seems capable of inhibiting SV40 DNA replication when inserted at the HindIII site immediate to the early side of the SV40 origin.

Suppression of replication of plasmids containing two or three copies of origin of replication by the CMV enhancer-promoter.

The severe inhibition of the SV40 origin by the CMV enhancer-promoter inserted at the EcoRI site 2.6 kb from the SV40 origin suggests a long-range effect of the CMV enhancer-promoter. To examine whether this effect is due to long-range interactions between SV40 and CMV regulatory elements or due to global effects of DNA topology or nucleoprotein organization by the presence of CMV regulatory elements, we inserted one or more extra copies of the SV40 origin in pSCM-1(−). We reasoned that if the inhibition is through direct one-to-one protein-mediated interactions between CMV and SV40 regulatory elements, then one of the SV40 origins should always escape the inhibitory effect and replication proficiency should be restored by the insertion of extra copies of the SV40 origin. As shown in Fig. 5a, insertion of extra origins in this plasmid did not result in recovery of full replication activity. A plasmid with insertion of an extra copy of the origin at nt 2618 in pSCM-1(−) was still severely inhibited by a single copy of CMV [plasmid pS2CM-1(−)/ori-2618(+) in Fig. 5a]. Even for the plasmid containing three copies of the SV40 origin, one at the normal position and tandem copies at nt 4751, replication was still repressed by the CMV sequence to about 36% of the level for the control plasmid pSV2cat. Similar inhibition efficiency by the CMV promoter-enhancer was observed for plasmid constructs containing one, two, or three copies of the SV40 origin in tandem but with the CMV enhancer inserted on the late (Fig. 5b) or early (data not shown) side of the origin. These results suggest that the CMV enhancer-promoter exerts a global inhibitory effect that is not due to a direct one-to-one interaction between SV40 and CMV regulatory sequences.

FIG. 5.

Inhibition of replication of plasmids containing multiple copies of the SV40 origin by a single copy of the HCMV enhancer-promoter. (a) Additional copies of the SV40 origin (ori) were inserted in plasmid pSCM-1 at the positions indicated in the plasmid names. Average replication efficiency of parental plasmid pSV2cat is taken as 100%. (b) pSC constructs (see Materials and Methods) containing one, two, or three tandem copies of the SV40 origin or their derivatives with a single HCMV enhancer-promoter insertion were analyzed for replication efficiency. The minus sign following the number 2266 indicates that the SV40 late promoter was used to drive CAT gene expression. Replication efficiency was determined using pL as internal standard. Replication efficiency of the pSC plasmids (pSC, pS2C, and pS3C) without CMV insertion is taken as 100%.

Competition between SV40 origin and CMV IE enhancer for the establishment of active chromatin conformation; evidence for the presence of only one nuclease-hypersensitive site per chromatin domain.

The results described above indicate that the inhibition of SV40 origin-dependent replication by the CMV enhancer is not due to one-to-one interaction between SV40 and CMV regulatory sequences. To further elucidate the mechanisms of inhibition of SV40 origin activity by the CMV enhancer, we examined the possibility that the CMV enhancer alters the chromatin conformation at the SV40 origin.

Active regulatory sequences either as origin of replication or as promoter-enhancer have been shown to exhibit nucleosome-free conformation and are hypersensitive to nuclease digestion (7, 29). The special nucleoprotein organization at the regulatory sequences has also been shown to establish a nucleosome phase in chromatin (52, 73, 76, 92). We first considered the possibility that the organization of transcription factors in the CMV enhancer altered the nucleosome phase, causing occlusion of the SV40 origin. Analysis of nucleosome phases at the SV40 origin using micrococcal nuclease digestion mapping did not reveal gross alteration of nucleosome phases at the SV40 origin between plasmids pSV2cat and pSCM-1(−) in the transfected cells (data not shown). We then examined the possibility that competition for the formation of active chromatin conformation as defined by nuclease hypersensitivity may cause the reduced frequency of forming active chromatin at the SV40 origin and thus the reduction of replication activity. Evidence for such competition has been inferred from a previous study of SV40 genomes containing two separate copies of origin. These variant SV40 genomes replicate using only one origin at a time, and only one origin is nuclease hypersensitive in the DNA (74, 89). This origin interference mechanism has also been observed in yeast (8, 9, 22, 45, 54). If this mutual exclusion also occurs between the SV40 origin and CMV enhancer, then the frequency of initiation at the SV40 origin would be reduced relative to the parental pSV2cat and the effect would be manifested as inhibition of DNA replication as described above.

To test this hypothesis, we assayed active chromatin conformation at the CMV enhancer and SV40 origin using nuclease hypersensitivity analysis. It has been well established that nuclease hypersensitivity is a hallmark of active chromatin at the regulatory region (29). Since we are interested in the relative accessibility between different regulatory sites in a single DNA molecule, a steady-state digestion pattern would be needed. The traditional DNase I digestion, however, is unsuitable for such an analysis because all chromatin DNA is eventually digested into small fragments. A detailed kinetic analysis of different sites digested would be required to obtain relative accessibility that may be changed during DNase I digestion. In the present work, we used the unique property of P1 nuclease to determine the relative nuclease accessibility in different regions of chromatin. Our lab has shown that P1 nuclease does not attack nucleosomal DNA under low-salt conditions even in prolonged digestion and with an excess amount of the enzyme, whereas naked DNA or DNA in the nucleosome-free region is readily cleaved. Furthermore, P1 cleaved SV40 chromatin only once at the regulatory sites without further degrading viral DNA even in prolonged digestion with an excess amount of enzyme (15). This unique property allowed us to assay the accessibility of various regions of a chromatin to P1 nuclease without worrying about conditions for limited digestion as in the case of DNase I digestion.

P1 nuclease digestion of nuclei transfected by plasmids containing different combinations of SV40 and CMV enhancer-promoter elements showed that plasmids were mainly cleaved once to generate full-length linear DNA (Fig. 6a and b, lanes 1 to 4). These results indicate that only one site in the DNA is accessible to P1 nuclease irrespective of how many regulatory elements are present; no products corresponding to simultaneous double or multiple cleavages at regulatory elements were observed even in plasmids containing four regulatory elements. For example, plasmid pS3CM-1(−)/ori(2X)-4751(−) contains three copies of the SV40 origin, one at nt 1/5001, tandem copies at nt 4751, and a CMV element at nt 1/5001. P1 digestion generated only full-length linear DNA (lanes 4); no significant amount of products corresponding to double, or triple cleavages at regulatory sequences were observed. Mapping of P1 cleaved sites in this plasmid by indirect end labeling showed that P1 cleaved only at one of the four sites containing the regulatory sequences in a single molecule but not two, three, or four sites simultaneously (lanes 8). The fact that four cleavage products corresponding to the sites of the regulatory elements were observed using by indirect end labeling indicates unambiguously that each site is independently cleaved. Had they been cleaved simultaneously, one would see only the band closest to the probe. Similar results were obtained for plasmids containing two copies of the CMV enhancer-promoter in addition to the SV40 origin and for viral DNA containing two copies of the SV40 origin, or one SV40 origin and an MMTV promoter (data not shown). These results suggest that only one regulatory region could form a nuclease-hypersensitive, active chromatin conformation in a single chromatin domain containing more than one regulatory sequence.

FIG. 6.

Mapping of P1 nuclease cleavage sites of transfected plasmid DNA in transfected cell nuclei. Cells at 48 h after transfection were treated with lysis buffer, and the nuclei were digested with P1 nuclease as described in Materials and Methods. (a) Mapping of P1 cleavage sites in plasmids containing multiple regulatory sequences. Lanes 1 to 4, plasmid DNA extracted after P1 digestion. The main product is full linear DNA (L). The positions of forms I, II, and III of pSV2cat DNA are indicated. Lanes 5 to 8, FspI restriction enzyme digestion products of DNA from lanes 1 to 4. The blot was probed with the FspI-EcoRI (nt 1845 to 2618) fragment of pSV2cat (indirect end labeling). This mapped the P1 cleavage sites from the clockwise direction in the pSV2cat map in Fig. 1. Lanes 1 and 5, pSV2cat parental plasmid. A cluster of cleavage sites at the SV40 regulatory sequence not well resolved in this gel electrophoresis condition is labeled “S.” Lanes 2 and 6, pSCM-554(−), with insertion of the CMV sequence at nt 554. Two major clusters of cleavage sites corresponding to SV40 origin (S) and CMV (H) enhancer were observed. Lanes 3 and 7, plasmid pSCM2-3502(+)/2618(−), with one SV40 origin and two copies of CMV at nt 2618 and 3502, respectively. Three major cleavage clusters were seen, one at the SV40 origin (S) and one each at the two copies of the CMV enhancer (H). Lanes 4 and 8, plasmid pS3CM-1(−)/ori(2x)-4751(−), containing three SV40 origins, one at nt 1 and tandem copies at 4751 in addition to the CMV sequence at nt 1. Four major cleavage clusters corresponding to the four regulatory sites were seen. (b) The same blot as in panel a but with an FspI-HaeII (1845 to 976) fragment. This maps the cleavage sites from the counterclockwise direction in the pSV2cat map. Sizes of markers (M) are indicated in nucleotides.

The single cleavage of plasmid DNA in nuclei was not due to incomplete P1 digestion since P1 nuclease was added in excess with prolonged digestion (3 h). Exogenous plasmid DNA added to nuclei before P1 digestion was rapidly cleaved into tiny fragments within 20 min. Digestion of in vitro-assembled chromatin by P1 nuclease indicated that partially assembled linear plasmid DNA was cleaved into small fragments whereas fully assembled chromatin from linear plasmid DNA was fully resistant to P1 cleavage (data not shown). If chromatin DNA in nuclei was first cleaved with endonuclease BspE1, which cleaved the plasmid once, and then digested with P1, we observed again P1 cleavage at either the SV40 origin or CMV enhancer (data not shown). These results indicate that (i) P1 could cut linear DNA containing partially assembled nucleosomes in vitro and (ii) linearization of plasmid in vivo with a restriction endonuclease did not prevent P1 from cutting at either of the regulatory sites in the plasmid. Therefore, we believe that limited cleavage of transfected plasmid DNA is indeed due to existence of only one nuclease-hypersensitive site per plasmid DNA.

If competition between regulatory sites in a single molecule for the formation of active chromatin conformation is the mechanism for suppressing SV40 replication, then one would expect a direct correlation between the relative frequency of P1 cleavage at the SV40 origin and the degree of inhibition of SV40 origin-based DNA replication. As shown in Fig. 7, this expectation is indeed borne out; a positive correlation between relative replication activity and percentage of P1 cleavage at the SV40 origin was observed (average of three independent experiments), indicating that the higher the sensitivity of SV40 origin to P1 nuclease, the better its replication activity. The large effect on the accumulated plasmid DNA by a relatively small change in P1 cleavage is due to the fact that DNA replication is an amplification process. The analysis shown in Fig. 7 is a linear regression plot and does not imply any specific mechanism for the correlation observed. This result, together with the observations described above, suggests that reduction of SV40 origin-based DNA replication in plasmids containing a strong enhancer element is the result of competition for the formation of active chromatin conformation at the origin of SV40 DNA replication.

FIG. 7.

Correlation between percentage of P1 cleavage at SV40 origin and DNA replication efficiency in pSCM plasmids. The ration of intensity of DNA bands representing cleavage at the SV40 origin to the total intensity of DNA bands from the cleavage of SV40 and CMV regulatory sequence is plotted against the replication efficiency of the plasmid relative to the parental plasmid pSV2cat.

Using higher-resolving gels, we found that there were six P1 cleavage sites in SV40 regulatory sequences that map to SV40 enhancers, 21-bp repeat, and SV40 core origin (data not shown). There were two major P1 cleavage sites in the CMV enhancer that map to the 5′ and 3′ portions of the enhancer. These sites were unchanged in different plasmids, but there were subtle changes in the relative intensity in different plasmids, suggesting a potential modulating effect of local sequence microenvironment on the fine organization of chromatin structure at the regulatory sites.

Interference of CMV enhancer-promoter-dependent transcription by the CMV enhancer.

To further test the competition model described above, we analyzed the effect of inserting a CMV IE enhancer fragment on CAT reporter gene activity driven by the CMV enhancer-promoter. We reasoned that competition between the two enhancers in the establishment of active chromatin conformation would reduce the transcription initiated from the CMV enhancer-promoter in a manner dependent on the copy number of the competing enhancers. This was indeed observed (Fig. 8). Relative to the control without enhancer insertion, CAT activity decreased in a manner relative to the number of the competing enhancer. This result supports the competitive model described above.

FIG. 8.

Suppression of transcription of CAT gene by the HCMV enhancer-promoter by the HCMV enhancer fragment. pCM is a plasmid with a CAT gene driven by the HCMV enhancer-promoter. CAT activity of this plasmid is taken as 100%. pCM-en1, pCM-en2, and pCM-en4 contain one, two, and four copies of the HCMV 5′ enhancer (ClaI-NcoI fragment) inserted at the positions indicated in Table 1. Extra copies of the enhancer inhibit CAT activity driven by the HCMV enhancer-promoter.

Enhancement of CMV promoter-initiated CAT activity by the combination of SV40 origin and either CMV or SV40 enhancer.

In contrast to the severe inhibition of SV40 origin-dependent DNA replication by the CMV enhancer-promoter in pSCM-1(+), CAT activity of this plasmid was actually 6-fold higher than that of the parental plasmid pSV2cat and 200-fold higher than that of plasmid pCM without the SV40 origin when assayed at 48 h posttransfection in Cos-1 cells (Fig. 9). Since the pSCM-1(+) DNA amount is about 1 to 2% of the pSV2cat amount, the CAT activity per DNA template was actually 200- to 400-fold higher than that of pSV2cat at 48 h posttransfection. These results showed that although DNA replication was suppressed by the CMV sequence, transcription was stimulated by the presence of the SV40 origin.

FIG. 9.

Enhancement of transcription activity by CMV. CAT activity in cells transfected with pSV2cat, pSCM-1(+), pSCM-1(−), pSCM-1(+)-1, and pSCM-1(+)-2 was analyzed at 48 h posttransfection as described in Materials and Methods. CAT activity was sixfold higher in pSCM-1(+) than in pSV2cat. Only a background level was observed for pSCM-1(−). pSCM-1(+)-1, a derivative of pSCM-1(+), contains the portion of the CMV enhancer-promoter from nt −135 to +107 including the promoter element plus a small segment of the CMV enhancer. pSCM-1(+)-2 contains nt −598 to −223 of the enhancer.

The increased transcription is the result of enhancement of the CMV promoter by the SV40 regulatory sequence, since reversing the direction of the CMV promoter or deletion of the CMV promoter resulted in only a background level of CAT activity (Fig. 9). Removal of the SV40 promoter reduced the enhancement level only about threefold. Removal of both SV40 and CMV enhancer-promoters (21- and 72-bp repeats) resulted in only background CAT activity. These results indicate that the high level of transcription observed is initiated by the CMV promoter and that stimulation of CAT activity from the CMV promoter does not depend on the SV40 promoter. Enhanced CAT activity depended on the presence of either the SV40 or CMV enhancer. High-level CAT activity was still observed in plasmids devoid of either the SV40 or CMV enhancer. However, deletion of both enhancers resulted in poor CAT activity from the minimum CMV promoter (positions −42 to +107). The enhancement, however, depended on the presence of the SV40 origin of replication. These results suggest an up-regulation of the CMV promoter by the combined effect of an enhancer and SV40 core origin.

DISCUSSION

Mechanisms of inhibition of SV40 origin-dependent replication by the CMV IE gene enhancer.

We considered several mechanisms for the effect of the CMV IE gene enhancer on SV40 origin-dependent replication. First, we can exclude the transcription-mediated origin occlusion mechanism (33, 47, 53, 61, 82) since the promoter is dispensable (Fig. 4). Second, the possibility that the inhibition is due to protein-mediated one-to-one interaction between the SV40 origin and CMV enhancer using a looping mechanism (57, 67, 78) seems unlikely because insertion of extra copies of the SV40 origin did not reverse the effect. Third, we found no alteration in the bulk nucleosome phase relative to the SV40 origin after insertion of the CMV enhancer. However, since we looked at only the overall bulk nucleosome phase, we could not exclude the possibility that a subpopulation of plasmid existed with an altered nucleosome phase. Until there is a method to separate different chromatin populations, this possibility remains to be tested.

It is also possible that the CMV sequence blocks replication fork passage. However, we observed no significant accumulation of replication intermediates in the agarose gels even in plasmids that were severely inhibited by the CMV enhancer. Analysis of replication intermediates by two-dimensional gel electrophoresis showed that there is no significant delay or block of replication fork movement in the region containing the CMV sequence. Furthermore, since SV40 DNA replication is bidirectional, it is unlikely that a block of one of the replication forks would cause significant inhibition of DNA replication. Because we were comparing the replication between two DNA molecules differing in the insertion of a 700-bp CMV sequence, a change in the elongation rate must be due to the slowdown of elongation in the CMV sequence. Since elongation occupies only about 10% of the time of overall replication, a 20% reduction in replication would mean a 1,400% (20% × 10 × 5/0.7) decrease in elongation rate through the CMV enhancer. This was not observed in the two-dimensional gel analysis of replication intermediates containing the CMV sequence. Thus, we believe that the inhibition of DNA replication by the CMV sequence is not due to retardation of replication fork movement but must be due to inhibition of DNA replication initiation.

Finally, based on previous examples of origin dominance and interference in SV40 and in yeast, we examined the possibility that inhibition of SV40 origin-based DNA replication by the CMV enhancer could be due to competition for the formation of active chromatin conformation at the regulatory sites. Our P1 nuclease mapping data indeed are consistent with this interpretation. First, the fact that only one nuclease-sensitive site is present per DNA molecule irrespective of how many regulatory sites are present supports a mechanism of competition for the formation of active chromatin at SV40 origin. Second, the correlation between percentage of origin existing as nuclease-sensitive conformation and DNA replication efficiency is consistent with this interpretation. The relatively small change in P1 cleavage compared to the large reduction in replication activity is mainly due to the fact that replication is an amplified process. Thus, a reduction of 20% in replication initiation efficiency would result in a 10-fold reduction of accumulated DNA after 10 rounds of replication. However, the specific relationship between P1 sensitivity and initiation efficiency is not known, and the quantitative relationship between chromatin structure and DNA replication initiation remains to be examined.

An interesting observation in this report is the position-dependent effect of the CMV enhancer on SV40 origin activity. Although enhancer action is generally considered to be position independent, Fromm and Berg showed that the SV40 enhancer at a certain position of the plasmid failed to activate promoters (23). Activity of the yeast origin of replication has also been found to depend on the position in a plasmid (8, 9, 16, 54) and on chromosomal location (21, 86). In yeast, hierarchy of origins is influenced by the origin-specific replication enhancer (22, 45). These observations suggest that local sequence microenvironment may influence the activity of a cis-regulatory element. How local sequences affect the activity of regulatory elements remains to be determined.

A prediction from the competition model is that replacement of the SV40 enhancer by the CMV enhancer should not inhibit SV40 origin-dependent replication since there would be no competition. To test this prediction, we replaced the SV40 enhancer with the CMV 5′ enhancer (ClaI to NcoI site), with the direction of the CMV enhancer in the orientation toward the early SV40 promoter. This construction not only did not cause inhibition of SV40 origin-dependent replication but actually increased replication threefold relative to that of plasmid pSV2cat. Thus, the CMV 5′ enhancer was actually superior to the endogenous enhancer in stimulating SV40 origin activity, and the prediction was borne out. However, the SV40 origin was inhibited when the CMV 5′ enhancer was inserted in the opposite orientation. In contrast, the 3′ portion of the enhancer had only a slight effect on SV40 origin activity used to replace the SV40 enhancer. These results suggest that specific protein-protein interactions are required for stimulation of the SV40 origin by the enhancer as described previously (20).

Single P1 nuclease cleavage site in plasmids containing more than one regulatory sequence.

In this study we used P1 nuclease to probe the chromatin conformation at the regulatory sequences in the various plasmids in vivo. Previously we showed that P1 made only a single cut at the SV40 regulatory sequence while leaving the rest of DNA intact even after overdigestion with an excess amount of the enzyme (15). In vitro nucleosome reconstitution experiments using pBR322 plasmid DNA confirmed the resistance of nucleosomal DNA to P1 nuclease digestion under low-salt conditions, whereas naked DNA is degraded into acid-soluble fragments (data not shown). This unique property of P1 nuclease allows us to probe the relative accessibility of different regulatory sequences in a single molecule to P1 digestion in limiting digestion conditions. This type of analysis would be much more difficult with the traditional DNase I method because the sensitivity to DNase I is only relative and eventually all DNA sequences are degraded into small fragments.

Using P1 nuclease as a probe, we were able to clearly show that in the plasmid containing more than one regulatory sequence, only one is accessible to P1 nuclease digestion. This was observed in different plasmid constructs containing either homologous or heterologous regulatory sequences in two to three copies in various locations and therefore is unlikely to be due to some specific sequence effect. To our knowledge, this is the first demonstration of mutual exclusion of regulatory sequences to form active conformation in a single chromatin domain. The mutual exclusion may result from simple competition of the binding of regulatory factors, with the binding of the first site setting up a nucleosome array that precludes the binding of transcription factors to the other sites. Alternatively, competitive binding of regulatory sequences to nuclear matrix may determine the biological activity of regulatory elements and exclude the other regulatory elements activated at the same time. It is also possible that the formation of nuclease-hypersensitive sites require topological tension and the tension can support the formation of only one active chromatin conformation. In fact, Leonard and Patient (51) have provided evidence that P1 nuclease cleaves the AT sequence upstream of the Xenopus beta-globin gene under torsional stress. Torsional stress has also been implicated in the generation of DNase I sensitivity of active genes (85).

Several published observations are consistent with the mutual exclusion model. Shenk showed that in viral DNA containing two origins of replication, only one is active at one time (74). Replicator interference or dominance similar to that of SV40 has also been found in yeast (8, 16, 22, 45, 54, 94). In plasmids that contain multiple copies of a given yeast origin, only one origin is activated per DNA molecule. Furthermore, the activity of the origin depends on the location of in the DNA, indicating a local sequence effect. Our result of dose-dependent suppression of CMV enhancer-promoter-driven CAT activity by the CMV enhancer is also consistent with the model of mutual exclusion for the formation of active regulatory sites within a given chromatin domain. Suppression of DNase I-hypersensitive sites by a distant exogenous hypersensitive site has been documented (10, 34, 73). These observations are consistent with the competition model described above. However, origin interference could also be resulted from replication fork interference by a nearby dominant origin (86). Such a possibility needs to be considered in further study of origin interference in order to distinguish between initiation and elongation as the source of interference.

Modulation of SV40 replication origin by transcription regulatory elements.

Modulation of SV40 origin activity by other transcription binding sites has also been previously observed. Arizumi et al. showed that SV40 origin activity could be modulated by immunoglobulin heavy-chain enhancer in a manner that depended on the SV40 enhancer-promoter and the relative orientation of the two regulatory sequences (2). Their observation that replacement of the SV40 enhancer-promoter with the immunoglobulin enhancer either enhanced or inhibited SV40 origin activity is similar to our data for the CMV IE gene enhancer. In the case of the immunoglobulin enhancer, the 5′ enhancer element activates SV40 origin activity when placed on the early side but suppresses replication when placed on the late side of the core origin without the SV40 enhancer-promoter. This result is the reverse of what we observed for the CMV enhancer. Unfortunately, Arizumi et al. did not study the effect of inverting the immunoglobulin enhancer. In our case, the CMV enhancer activates the SV40 core origin in one orientation but inhibits in the other orientation when placed on the late side of the core origin (data not shown).

In contrast to the CMV IE gene enhancer, other transcription factor binding sites such as the c-Myc enhancer (46), BK virus NFI site, multimers of MMTV NFI or ATF transcription factor binding site (30, 31) and a 69-bp monkey cell DNA containing AP1 sites (80), and multiple copies of promoter elements (40) have been shown to activate SV40 minimum or enhancerless origin-dependent replication. In the case of monkey cell DNA containing an AP1 site, SV40 origin activity is enhanced only when the sequence is inserted on the late side of the origin and the effect is abolished when it is inserted at a distance. Activation of the minimal SV40 origin by the transcription regulatory elements is similar to our observation of activation of enhancerless SV40 origin by the CMV 5′ enhancer. On the other hand, SV40 origin activity is suppressed by the RSV long terminal repeat enhancer (6) and by the bovine papillomavirus origin in a composite plasmid (69). These results could be understood from our competition model. However, further experimentation is required to confirm this hypothesis.

Stable maintenance of SV40 origin-based plasmids as extrachromosomal DNA in permissive cells is difficult, possibly due to cell killing by runaway DNA replication (14, 67). The finding that pSCM-1cat has five- to six-times-higher CAT activity compared to the parental plasmid but at the same time much less replication efficiency allows us to obtain stable transfectants containing plasmid pSCM-1(+)neo, using G418 as the selection agent. We were able to maintain eight such cell clones for at least 3 months, with each cell containing up to 500 copies of free plasmid DNA. These G418-resistant cell lines could still support the replication of plasmids containing the SV40 origin. The observation suggests that plasmid pSCM-1neo may be a useful vector for the expression of eukaryotic genes in mammalian cells.