Department of Genetics, University of Cambridge, Downing Site, Cambridge CB2 3EH, UK1
Author for correspondence: David K. Summers. Tel: +44 1223 333991. Fax: +44 1223 333992. e-mail: dks11{at}cam.ac.uk
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ABSTRACT |
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Keywords: dimer resolution, cerXer recombination system, cell-cycle checkpoint, Rcd, plasmid stability
Abbreviations: GalK, galactokinase
a Present address: Laboratory of Receptor Signalling, The Babraham Institute, Babraham Hall, Cambridge CB2 4AT, UK.
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INTRODUCTION |
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cer-mediated dimer resolution is necessary, but not sufficient, for the stable maintenance of ColE1 and related plasmids. A mutation which inactivates the promoter, Pcer, within the cer site reduces plasmid stability despite having no detectable effect on plasmid dimer resolution (Summers & Sherratt, 1988 ; Patient & Summers, 1993
). Pcer directs the synthesis of an approximately 75 nt transcript, Rcd, whose predicted secondary structure is reminiscent of small antisense RNAs involved in the control of plasmid replication and transposition (Sharpe et al., 1999
). The level of Rcd is elevated in multimer-containing cells and it has been proposed that the transcript is part of a cell-cycle checkpoint which blocks division until multimer resolution is complete (Patient & Summers, 1993
). An unusual feature of Rcd action is that growth and division appear to be stopped in a co-ordinated manner since cells recovered from solid medium after Rcd overexpression show cell-cycle arrest just before septation but do not filament (Patient & Summers, 1993
). In broth culture septation is blocked by Rcd induction and moderate filamentation is apparent, with cells increasing to approximately three times their normal length (Rowe & Summers, 1999
).
One of the most intriguing aspects of the Rcd checkpoint hypothesis is the activation of Pcer by plasmid multimerization. How might this be achieved? The separation between the -10 and -35 hexamers in Pcer is only 15 bp, compared to the consensus spacing for an Escherichia coli promoter of 17±1 bp (Hawley & McClure, 1983 ; Harley & Reynolds, 1987
). We have speculated that distortion of the promoter in a cercer synaptic complex might align the -10 and -35 regions for recognition by RNA polymerase (Summers, 1998
). We refer to this as the local twisting hypothesis, although the distortion may conceivably include elements of both twist and writhe in the strict topological sense. Since recombination (and therefore synaptic complex formation) is limited to cer sites on the same molecule, this would ensure that Rcd was only made in multimer-containing cells. Support for the idea that Pcer can be activated by realignment of its -10 and -35 sequences came from the analysis of
, an up-promoter derivative of Pcer in which the -35 hexamer is replaced by a new sequence at a more conventional spacing from the -10 (Patient & Summers, 1993
; Fig. 1
). This derivative promoter is active even in the absence of multimers, suggesting that the short spacer is crucial to proper monomerdimer control at Pcer.
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METHODS |
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Media and bacterial transformation.
For the routine growth of bacteria L-broth (Kennedy, 1971 ) and Oxoid iso-sensitest agar were used (37 °C). Where appropriate, 50 µg ampicillin ml-1 (Sigma) was added to the media. Plasmid transformation of calcium chloride-treated cells was by the method of Cohen et al. (1972)
. TG1 was maintained on minimal M9 agar (Sambrook et al., 1989
). For transformation TG1 cells were grown in 2YT broth (Sambrook et al., 1989
) and transfected cells were spread in soft agar overlays on L-agar plates. Enzymes were used according to the manufacturers recommendations. All enzymes were obtained from Promega except ThaI, which was obtained from Gibco-BRL.
Site-directed mutagenesis.
Single-stranded M13mp19cer phage DNA was prepared by phenol extraction of a polyethylene glycol phage precipitate (Sambrook et al., 1989 ) and used as a template for mutagenesis. Site-directed mutagenesis reactions were performed using the Sculptor in vitro mutagenesis system (Amersham). Oligonucleotides for mutagenesis were synthesized by the Protein and Nucleic Acid Chemistry Facility, Department of Biochemistry, University of Cambridge. Oligonucleotides used to introduce extra bases into the promoter (Fig. 1
) were degenerate within the 14 base insert region. The oligonucleotide to generate a 14 bp intervening spacing (i.e. to remove 1 base) was synthesized with a T missing from the wild-type sequence (Fig. 1
). Plaques were picked at random and sequenced to identify mutants. DNA sequences were determined using the Sequenase DNA sequencing kit (version 2.0, US Biochemicals) and an automated DNA sequencer (model 377; Applied Biosystems). Approximately 75% of the plaques obtained by this method were mutant.
Sequencing of Pcer derivative constructs.
Plasmid templates for sequencing were isolated using a Qiagen plasmid mini kit (Qiagen tip-20). DNA sequences were determined using the ABI PRISM Dye Terminator cycle sequencing ready reaction kit with AmpliTaq DNA Polymerase FS (Perkin Elmer), and a model 377 DNA sequencer.
Assay of galactokinase activity.
Transformant colonies were inoculated into L-broth containing 50 µg ampicillin ml-1 and incubated in a 37 °C shaking water bath overnight. They were then subcultured into fresh medium and incubated at 37 °C until an OD650 0·20·3 was reached. GalK activity was assayed by the method of McKenney et al. (1981) . Enzyme activity is expressed as the mean±SD of a minimum of six independent assays.
Preparation of dimer plasmids and recombination analysis.
Plasmid DNA was prepared from JC8679 by alkaline lysis (Sambrook et al., 1989 ). DNA was electrophoresed on a 0·85% (w/v) agarose gel and dimer bands were cut out from the gel and eluted into TE buffer (pH 8·0) using the Geneclean kit (Bio 101). DS941 xerC was then transformed with dimer plasmid DNA and transformant colonies containing dimers were identified after preparation of plasmid DNA by standard techniques. Analysis of cer-mediated recombination was as described by Summers & Sherratt (1984
, 1988
).
Assay of host cell growth.
A single colony of the host E. coli strain harbouring the test plasmid was picked from a selective plate and inoculated into 10 ml L-broth containing 100 µg ampicillin ml-1 and incubated overnight at 30 °C in a shaking water bath. Cultures were diluted in L-broth to OD600 0·0050·01; a change in density of the culture at 37 °C was followed by measurement of OD600. To prevent the accumulation of plasmid-free cells, antibiotic selection was maintained throughout. The overnight incubation was carried out at 30 °C to reduce Rcd growth inhibition of cells carrying the +CA Pcer derivative. If the overnight incubation was at 37 °C, cells carrying the +CA Pcer derivative became effectively non-viable.
Oligonucleotide probe preparation.
For Northern blot and dot blot analysis oligonucleotide probes were used. Probes were end-labelled using T4 polynucleotide kinase and [-32P]ATP (Amersham). The phosphorylation reaction contained Promega T4 polynucleotide kinase buffer, 200 ng probe, and 10 units polynucleotide kinase in 20 µl. After 30 min incubation at 37 °C, the mixture was heated to 90 °C to inactivate the kinase. The labelled probe was then purified using a S200-HR spin column (Pharmacia). To probe for Rcd an oligonucleotide complementary to a region just downstream of Pcer was used: 5'-GGTAAAAATGGCAACAAACC-3'. To probe for ß-lactamase, an oligonucleotide probe with the sequence 5'-CGGGATAATACCGCGCC-3' was used.
Isolation of RNA.
E. coli cells harbouring plasmids were cultured in L-broth containing 100 µg ampicillin ml-1, overnight in a shaking water bath at 30 °C. The cells were then diluted into fresh medium to an OD650 0·010·02 and incubated at 37 °C until an OD650 0·3 was reached. The cells were harvested and resuspended in 200 µl of solution 1 (0·3 M sucrose, 0·01 M sodium acetate, pH 4·8). They were lysed by the addition of 200 µl of solution 2 (2% SDS, 0·01 M sodium acetate, pH 4·8) preheated to 65 °C. RNA was isolated by three extractions with acid phenol (Bioline) at 65 °C. The initial phenol extraction was followed by rapid freezing in liquid nitrogen. The RNA was then extracted with phenol/chloroform at room temperature, followed by extraction with chloroform/isoamyl alcohol. Then 10% (v/v) 3 M sodium acetate was added and the RNA was precipitated by adding 2 vols of ethanol (at -20 °C, 90 min). The RNA pellet was washed with 70% ethanol and dissolved in water. DNA was removed by treatment with DNase I (Sigma); the RNA was extracted with phenol/chloroform, re-precipitated and resuspended in water. RNA concentrations were determined by measurement of A260.
Northern blot analysis.
Twenty micrograms of total RNA was electrophoresed on polyacrylamide gels containing 7 M urea (Sambrook et al., 1989 ). RNA was blotted onto Genescreen Plus nylon membrane (DuPont) with a Bio-Rad electroblot apparatus (5 h at 75 V). The transfer buffer used was 0·5x TBE (0·045 M Tris/borate, 0·001 M EDTA, pH 8·0). Pre-hybridization was at 37 °C for 1 h in a solution containing 6x standard saline citrate (SSC), 0·5% SDS, 7·5x Denhardts solution and sonicated salmon sperm DNA (200 µg ml-1). Hybridization was at 37 °C for 24 h in pre-hybridization solution containing the labelled probe. Hybridized membranes were then washed for 4x10 min at 37 °C in 6x SSC containing 0·1% SDS, followed by 2x10 min at 40 °C in 6x SSC containing 0·1% SDS.
Dot blot analysis.
Serial dilutions of RNA samples were prepared and dot blotted onto Genescreen Plus nylon membrane (DuPont) by the method of Sambrook et al. (1989) . Filters were probed for Rcd as described above. Filters were probed for ß-lactamase in the same pre-hybridization solution but the pre-hybridization and hybridization steps were at 40 °C and the washes were at 40 °C and 43 °C.
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RESULTS |
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An isolated Pcer promoter does not show monomerdimer regulation
We next investigated the monomerdimer control of Pcer and its derivatives. A key question was whether monomerdimer regulation is exhibited by isolated promoters or whether the rest of the cer site plays an essential part. It is possible to formulate a plausible hypothesis in which isolated promoters would respond to plasmid dimerization. ArgR and PepA are involved in interactions between pairs of cer sites (Guhathakurta & Summers, 1995 ) and in the model of Hodgman et al. (1998)
sites are brought together when PepA forms a bridge between ArgR hexamers bound to their respective ARG boxes. The ARG box overlaps Pcer so that the PepA/ArgR bridge provides a means of communication between promoters in a dimer: this might be necessary, or even sufficient, for their activation.
To investigate monomerdimer control of isolated promoters we used the PcergalK fusion plasmids whose construction was described in the previous section. These plasmids contain the cer ARG box and Pcer, but the cer sequences downstream of the Rcd transcription start (including the XerCD binding site) have been deleted. Transcription from Pcer and its derivatives was compared in strains containing monomers or dimers of the PcergalK constructs. Plasmids used in the assay contained either wild-type Pcer, the T deletion or the +C, +CA, +CAT and +CATT insertions. These four insertion derivatives were chosen because they reconstitute the ARG box. Dimers of all the plasmids showed a small (1339%) increase in transcription compared to the monomers (Table 2
). The fact that this increase was small and that similar increases were seen for the wild-type promoter and its up-regulated derivatives suggests that the effect is unlikely to represent a biologically significant activation of Pcer. It seems more likely to result from differences in the galK dosage in monomers and dimers. This interpretation was supported by the observation that even in an argR mutant strain (where a PepA/ArgR bridge would be impossible) the wild-type PcergalK fusion plasmid showed an increased GalK activity of 15% between monomers and dimers (55±10 and 63±13 GalK units, respectively). We noticed that the monomer plasmids all showed slightly lower GalK activity in the xerC strain than in the wild-type (compare Tables 1
and 2
). This may be due to the reduced viability of xerC cells because XerC is required for the resolution of chromosome dimers formed during replication.
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DISCUSSION |
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Although the activity of Pcer derivatives was determined primarily by spacer length there was some variation within each group, reflecting the fact that the sequence of the spacer region also plays a role. The interaction between RNA polymerase and the spacer is largely non-specific, but if the sequence is intrinsically bent, or has the necessary flexibility to bend in the appropriate orientation, this may assist promoter activation (Collis et al., 1989 ; Lozinski et al., 1991
). Equally, bends in the spacer that mis-orientate the DNA and disrupt proteinDNA interactions can decrease promoter activity. The Pcer mutant promoter with an 18 bp spacer created by insertion of TTT shows considerably lower promoter activity than the other promoter mutants with an 18 bp spacer. The insertion of TTT at the insertion site generates a run of five Ts. Such a T-tract is a rigid structure and will distort the DNA (Koo et al., 1986
; Hagerman, 1990
). Presumably this T-tract bending misplaces the proteinDNA interfaces, thereby reducing promoter activation.
In isolation, our observation that a +2 bp derivative of Pcer shows high, constitutive activity was unremarkable. It did, however, suggest a mechanism by which the wild-type promoter might be activated: instead of inserting extra bases, the effect of a non-optimal spacer length might be counteracted by twisting or bending the promoter. Inserting 2 bp into the spacer is equivalent to a 69° relative rotation of the -10 and -35 sequences; something which could perhaps be achieved by appropriate proteinDNA interactions. A precedent for promoter activation by protein-mediated twisting is provided by the mer operon of Tn501, which encodes the mercuric ion detoxification system. The promoter of the mer operon has a 19 bp spacer region which is essential for induction of the operon in the presence of mercuric ions and for repression in their absence. Induction is controlled by binding of MerR at the mer operator sequence, which is a 7 bp perfect dyad sequence between the -35 and -10 hexamers (Parkhill & Brown, 1990 ). The MerR protein binds to this sequence in the presence or absence of mercuric ions. In the absence of mercuric ions MerR acts as a repressor, but in the presence of mercuric ions the MerR protein is allosterically modified. This modified form of MerR causes underwinding of the DNA at the operator site, which realigns the -35 and -10 hexamers into the orientation found in a promoter with an 18 bp spacer (OHalloran et al., 1989
; Ansari et al., 1992
).
The MerR system is relatively simple, with a single protein activating the promoter by inducing a change in its structure. By analogy it seemed possible that Pcer activation in a plasmid dimer might involve only the two promoters and ArgR hexamers bound to their ARG boxes (the isolated promoter model). Alternatively it might require the formation of full nucleoprotein complexes at cer. A previous observation that dimerization of a plasmid containing a PcergalK fusion resulted in increased GalK activity (H. Withers & D. Summers, unpublished data) seemed to support the isolated promoter model but the more detailed study reported here suggests that this is not the case. The small increase in GalK activity observed for fusion plasmid dimers in this work appears likely to reflect a copy-number effect (i.e. dimers of the fusion plasmid had slightly more than half the copy number of monomers) and in any case was not ArgR-dependent. A slight increase in galK gene dosage for dimers is not unexpected because the copy-number control of ColE1-like plasmids exhibits exponential kinetics (Summers, 1998 ).
The lack of support for the isolated promoter model implies that monomerdimer regulation of Pcer is likely to require the full nucleoprotein complexes which form at cer during dimer resolution. We have published a structural hypothesis for cerXer recombination (Hodgman et al., 1998 ) which proposes that nucleoprotein complexes form initially at individual cer sites in plasmid monomers. These single-site complexes are able to pair via an ArgR/PepA bridge which can form between two sites in a dimer or in separate monomers. However, only in the dimer is the paired structure sufficiently long-lived to isomerize into a recombination-competent synaptic complex. Some of the basic concepts of the Hodgman model were included in a model proposed by Sträter et al. (1999)
. However, the latter does not discuss structures for single-site complexes but envisages that the synaptic complex forms de novo on a plasmid dimer. In either case, the synaptic complex is the only nucleoprotein assembly unique to a plasmid dimer so we presume that it is within this that Pcer activation must take place.
If Pcer is activated by distortion within the synaptic complex, the +CA derivative is likely to be subjected to similar forces, but they may have different consequences for promoter regulation. For the +CA derivative the -10 and -35 sequences in an isolated promoter are positioned appropriately for recognition by RNA polymerase, and the local twisting model predicts that in the synaptic complex they should be moved out of alignment, deactivating the promoter (Fig. 5). To test this hypothesis we needed to generate a high level of synaptic complexes in the host cells. One possible approach would be to use recombination-deficient mutants of the XerC to trap the synaptic complex. However, there may be subtle differences in structure between a frozen synaptic complex and its recombination-proficient counterpart; in any case we have no idea whether Pcer is activated before or after the XerC-mediated strand exchange. Given our poor understanding of the system, we decided it was more appropriate to increase the frequency of synaptic complexes by stimulating recombination. We used the sbcA strain JC8679, in which plasmid multimers are generated continuously by homologous recombination, thus providing a constant supply of substrate for Xercer recombination. The demonstration by both Northern blot analysis and growth rate experiments that the +CA promoter derivative is less active in JC8679 than in DS941 (where the plasmids are mostly monomeric) is consistent with the predictions of the local twisting hypothesis. We would not expect complete de-activation of the +CA promoter since only about 50% of the plasmids are multimeric at any time. Furthermore, the synaptic complex modelled by Hodgman et al. (1998)
is asymmetric, with only one of the recombining sites subject to distortion during synaptic complex formation (the other site retaining the structure seen in the single-site complex). This would mean that only one wild-type promoter in a synaptic complex would fire and only one +CA derivative promoter would be down-regulated.
Finally it is interesting to contrast our analysis of Pcer with a study of proU promoter regulation. The proU promoter responds to changes in extracellular osmolarity and, like Pcer, has a suboptimal (16 bp) spacer (Jordi et al., 1995 ). Jordi et al. (1995)
predicted that if the proU promoter is activated by twisting, increasing the spacer length from 16 to 18 bp would reverse the effect of increased osmolarity on its activity. However, a proU promoter derivative with an 18 bp spacer showed essentially normal osmoregulation. Jordi et al. (1995)
discounted the hypothesis that changes in DNA twist activate the proU promoter and proposed instead that changes in promoter flexibility, facilitating writhe, were responsible. In contrast to the proU results, we have found that a Pcer derivative with an 18 bp spacer does indeed show a reversed response to plasmid multimerization, consistent with promoter activation involving twist. Monomerdimer control of Pcer therefore appears to represent a distinct and novel mechanism of transcriptional control.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Ansari, A. Z., Chael, M. L. & OHalloran, T. V. (1992). Allosteric underwinding of DNA is a critical step in positive control of transcription by Hg-MerR. Nature 355, 87-89.[Medline]
Aoyama, T., Takanami, M., Ohtsuka, E., Taniyama, Y, Marumoto, R., Sato, H. & Ikehara, M. (1983). Essential structure of E. coli promoter: effect of spacer length between the two consensus sequences on promoter function. Nucleic Acids Res 17, 5855-5864.[Medline]
Bachmann, B. J. (1972). Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev 36, 525-557.[Medline]
Berman, M. L. & Landy, A (1979). Promoter mutations in the transfer RNA gene tyrT of Escherichia coli. Proc Natl Acad Sci USA 76, 4303-4307.[Abstract]
Blakely, G., May, G., McCulloch, R., Arciszewska, L. K., Burke, M., Lovett, S. T. & Sherratt, D. J. (1993). Two related recombinases are required for site-specific recombination at dif and cer in E. coli K12. Cell 75, 351-361.[Medline]
Cohen, S. N., Chang, A. C. Y. & Hsu, L. (1972). Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci USA 69, 2110-2114.[Abstract]
Collis, C. M., Molloy, P. L., Both, G. W. & Drew, H. R. (1989). Influence of the sequence-dependent flexure of DNA on transcription in E. coli. Nucleic Acids Res 17, 9447-9468.[Abstract]
Colloms, S. D., Sykora, P., Szatmari, G. & Sherratt, D. J. (1990). Recombination at ColE1 cer requires the Escherichia coli xerC gene product, a member of the lambda integrase family of site-specific recombinases. J Bacteriol 172, 6973-6980.[Medline]
Guhathakurta, A. & Summers, D. K. (1995). Involvement of ArgR and PepA in the pairing of ColE1 dimer resolution sites. Microbiology 141, 1163-1171.[Abstract]
Guhathakurta, A., Viney, I. & Summers, D. K. (1996). Accessory proteins impose site selectivity during ColE1 dimer resolution. Mol Microbiol 20, 613-620.[Medline]
Hagerman, P. J. (1990). Sequence-directed curvature of DNA. Annu Rev Biochem 59, 755-781.[Medline]
Harley, C. B. & Reynolds, R. P. (1987). Analysis of E. coli promoter sequences. Nucleic Acids Res 15, 2343-2361.[Abstract]
Hawley, D. K. & McClure, W. R. (1983). Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res 11, 2237-2255.[Abstract]
Hodgman, T. C., Griffiths, H. & Summers, D. K. (1998). Nucleoprotein architecture and ColE1 dimer resolution: a hypothesis. Mol Microbiol 29, 545-558.[Medline]
Jordi, B. J. A. M., Owen-Hughes, T. A., Hulton, C. S. J. & Higgins, C. F. (1995). DNA twist, flexibility and transcription of the osmoregulated proU promoter of Salmonella typhimurium. EMBO J 14, 5690-5700.[Abstract]
Kennedy, C. K. (1971). Induction of colicin production by high temperature or inhibition of protein synthesis. J Bacteriol 108, 10-19.[Medline]
Koo, H.-S., Wu, H.-M. & Crothers, D. M. (1986). DNA bending at adenine thymine tracts. Nature 320, 501-506.[Medline]
Lozinski, T., Adrych-Rozek, K., Markiewicz, W. T. & Wierzchowski, K. L. (1991). Effect of DNA bending in various regions of a consensus-like Escherichia coli promoter on its strength in vivo and structure of the open complex in vitro. Nucleic Acids Res 19, 2947-2953.[Abstract]
McKenney, K., Shimatake, H., Court, D., Schmeisser, U., Brady, C. & Rosenberg, M. (1981). A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase. In Gene Amplification and Analysis , pp. 383-415. Edited by J. G. Chirikjian & T. S. Papa. Amsterdam:Elsevier.
Mandecki, W. & Reznikoff, W. S. (1982). A lac promoter with a changed distance between -10 and -35 regions. Nucleic Acids Res 10, 903-912.[Abstract]
Mandecki, W., Goldman, R. A., Powell, B. S. & Caruthers, M. H. (1985). lac up-promoter mutants with increased homology to the consensus promoter sequence. J Bacteriol 164, 1353-1355.[Medline]
Messing, J. (1983). New M13 vectors for cloning. Methods Enzymol 101, 20-78.[Medline]
Norrander, J., Kempe, T. & Messing, J. (1983). Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26, 101-106.[Medline]
OHalloran, T. V., Frantz, B., Shin, M. K., Ralston, D. M. & Wright, J. G. (1989). The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell 56, 119-129.[Medline]
Parkhill, J. & Brown, N. L. (1990). Site-specific insertion and deletion mutants in the mer promoter-operator region of Tn501; the nineteen base-pair spacer is essential for normal induction of the promoter by MerR. Nucleic Acids Res 18, 5157-5162.[Abstract]
Patient, M. E. & Summers, D. K. (1993). ColE1 multimer formation triggers inhibition of Escherichia coli cell division. Mol Microbiol 9, 1089-1095.[Medline]
Rowe, C. D. & Summers, D. K. (1999). The quiescent-cell expression system for protein synthesis in Escherichia coli. Appl Environ Microbiol 65, 2710-2715.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sharpe, M. E., Chatwin, H. M., Macpherson, C., Withers, H. L. & Summers, D. K. (1999). Analysis of the ColE1 stability determinant Rcd. Microbiology 145, 2135-2144.[Abstract]
Stefano, J. E. & Gralla, J. D. (1982). Spacer mutations in the lac ps promoter. Proc Natl Acad Sci USA 79, 1069-1072.[Abstract]
Stirling, C. J., Stewart, G. & Sherratt, D. J. (1988a). Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination. Mol Gen Genet 214, 80-84.[Medline]
Stirling, C. J., Szatmari, G., Stewart, G., Smith, M. C. M. & Sherratt, D. J. (1988b). The arginine repressor is essential for plasmid stabilizing site-specific recombination at the ColE1 cer locus. EMBO J 7, 4389-4395.[Abstract]
Stirling, C. J., Colloms, S. D., Collins, J. F., Szatmari, G. & Sherratt, D. J. (1989). xerB, an Escherichia coli gene required for plasmid ColE1 site-specific recombination, is identical to pepA, encoding aminopeptidase A, a protein with substantial similarity to bovine lens leucine aminopeptidase. EMBO J 8, 1623-1627.[Abstract]
Sträter, N., Sherratt, D. J. & Colloms, S. D. (1999). X-ray structure of aminopeptidase A from Escherichia coli and a model for the nucleoprotein complex in Xer site-specific recombination. EMBO J 18, 4513-4522.
Summers, D. K. (1991). The kinetics of plasmid loss. Trends Biotechnol 9, 273-278.[Medline]
Summers, D. K. (1998). Timing, self-control and a sense of direction are the secrets of multicopy plasmid stability. Mol Microbiol 29, 1137-1145.[Medline]
Summers, D. K. & Sherratt, D. J. (1984). Multimerization of high copy number plasmids causes instability: ColE1 encodes a determinant essential for plasmid monomerization and stability. Cell 36, 1097-1103.[Medline]
Summers, D. K. & Sherratt, D. J. (1988). Resolution of ColE1 dimers requires a DNA sequence implicated in the three-dimensional organization of the cer site. EMBO J 7, 851-858.[Abstract]
Summers, D. K., Beton, C. W. H. & Withers, H. L. (1993). Multicopy plasmid instability: the dimer catastrophe hypothesis. Mol Microbiol 8, 1031-1038.[Medline]
Vieira, J. & Messing, J. (1982). The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19, 259-268.[Medline]
Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119.[Medline]
Received 17 May 2001;
revised 19 July 2001;
accepted 26 July 2001.
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