Replication control of a small cryptic plasmid of Escherichia coli
Identifieur interne : 001F89 ( Istex/Corpus ); précédent : 001F88; suivant : 001F90Replication control of a small cryptic plasmid of Escherichia coli
Auteurs : Ján Burian ; Stanislav Stuchl K ; William W. KaySource :
- Journal of Molecular Biology [ 0022-2836 ] ; 1999.
English descriptors
Abstract
The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n′-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.
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DOI: 10.1006/jmbi.1999.3266
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title xml:lang="en">Replication control of a small cryptic plasmid of Escherichia coli</title><author><name sortKey="Burian, Jan" sort="Burian, Jan" uniqKey="Burian J" first="Ján" last="Burian">Ján Burian</name><affiliation><mods:affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Stuchl K, Stanislav" sort="Stuchl K, Stanislav" uniqKey="Stuchl K S" first="Stanislav" last="Stuchl K">Stanislav Stuchl K</name><affiliation><mods:affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Kay, William W" sort="Kay, William W" uniqKey="Kay W" first="William W" last="Kay">William W. Kay</name><affiliation><mods:affiliation>E-mail: wkay@uvic.ca</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:32B0C69EF610A6D0BB325DC605889AF830E82B8F</idno><date when="1999" year="1999">1999</date><idno type="doi">10.1006/jmbi.1999.3266</idno><idno type="url">https://api-v5.istex.fr/document/32B0C69EF610A6D0BB325DC605889AF830E82B8F/fulltext/pdf</idno><idno type="wicri:Area/Istex/Corpus">001F89</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001F89</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main" xml:lang="en">Replication control of a small cryptic plasmid of Escherichia coli</title><author><name sortKey="Burian, Jan" sort="Burian, Jan" uniqKey="Burian J" first="Ján" last="Burian">Ján Burian</name><affiliation><mods:affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Stuchl K, Stanislav" sort="Stuchl K, Stanislav" uniqKey="Stuchl K S" first="Stanislav" last="Stuchl K">Stanislav Stuchl K</name><affiliation><mods:affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</mods:affiliation></affiliation></author><author><name sortKey="Kay, William W" sort="Kay, William W" uniqKey="Kay W" first="William W" last="Kay">William W. Kay</name><affiliation><mods:affiliation>E-mail: wkay@uvic.ca</mods:affiliation></affiliation><affiliation><mods:affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="ISSN">0022-2836</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999">1999</date><biblScope unit="volume">294</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="49">49</biblScope><biblScope unit="page" to="65">65</biblScope></imprint><idno type="ISSN">0022-2836</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0022-2836</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>BD-1</term><term>BD-2</term><term>DNA replication</term><term>IHF</term><term>ORF</term><term>SCP</term><term>SSB</term><term>autoregulation</term><term>plasmid</term><term>repA</term><term>ssDNA</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n′-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.</div></front></TEI><istex><corpusName>elsevier</corpusName><author><json:item><name>Ján Burian</name><affiliations><json:string>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</json:string></affiliations></json:item><json:item><name>Stanislav Stuchlı́k</name><affiliations><json:string>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</json:string></affiliations></json:item><json:item><name>William W Kay</name><affiliations><json:string>E-mail: wkay@uvic.ca</json:string><json:string>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Regular article</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>plasmid</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>autoregulation</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>DNA replication</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>IHF</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>repA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ssDNA : single-stranded DNA</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SSB : ssDNA-binding protein</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>SCP : small cryptic plasmids</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>ORF : open reading frame</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>IHF : integration host factor</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>BD-1 : binding domain I</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>BD-2 : binding domain II</value></json:item></subject><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n′-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.</abstract><qualityIndicators><score>8</score><pdfWordCount>10722</pdfWordCount><pdfCharCount>64582</pdfCharCount><pdfVersion>1.3</pdfVersion><pdfPageCount>17</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>273</abstractWordCount><abstractCharCount>1768</abstractCharCount><keywordCount>13</keywordCount></qualityIndicators><title>Replication control of a small cryptic plasmid of Escherichia coli</title><pii><json:string>S0022-2836(99)93266-7</json:string></pii><genre><json:string>research-article</json:string></genre><host><title>Journal of Molecular Biology</title><language><json:string>unknown</json:string></language><publicationDate>1999</publicationDate><issn><json:string>0022-2836</json:string></issn><pii><json:string>S0022-2836(00)X0285-9</json:string></pii><volume>294</volume><issue>1</issue><pages><first>49</first><last>65</last></pages><genre><json:string>journal</json:string></genre></host><categories><wos><json:string>science</json:string><json:string>biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>health sciences</json:string><json:string>biomedical research</json:string><json:string>biochemistry & molecular biology</json:string></scienceMetrix><inist><json:string>sciences appliquees, technologies et medecines</json:string><json:string>sciences biologiques et medicales</json:string><json:string>sciences biologiques fondamentales et appliquees. psychologie</json:string><json:string>phytopathologie. zoologie agricole. protection des cultures et des forets</json:string></inist></categories><publicationDate>1999</publicationDate><copyrightDate>1999</copyrightDate><doi><json:string>10.1006/jmbi.1999.3266</json:string></doi><id>32B0C69EF610A6D0BB325DC605889AF830E82B8F</id><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api-v5.istex.fr/document/32B0C69EF610A6D0BB325DC605889AF830E82B8F/fulltext/pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api-v5.istex.fr/document/32B0C69EF610A6D0BB325DC605889AF830E82B8F/fulltext/zip</uri></json:item><istex:fulltextTEI uri="https://api-v5.istex.fr/document/32B0C69EF610A6D0BB325DC605889AF830E82B8F/fulltext/tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main" xml:lang="en">Replication control of a small cryptic plasmid of Escherichia coli</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>ELSEVIER</publisher><availability><p>©1999 Academic Press</p></availability><date>1999</date></publicationStmt><notesStmt><note>Edited by M. Gottesman</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Effect of RepA on pKL1 replication. Strains producing different amounts of RepA were analyzed by Western blotting using anti-RepA antiserum (a) and resulting amplification of pKL1 DNA by agarose electrophoresis and EtBr staining (b). Lane 1, shows RepA produced from pKL1. Lanes 1–5 show Western blots of RepA and electrophoresis of plasmids isolated from E. coli DH11 harboring pKL1 (lane 1), pKL1 plus pTZ19R grown at 37 °C (lane 2), pKL1 plus pBGL-R grown at 37 °C (lane 3), pKL1 pBGL-R and pGP1-2 cultivated at 30 °C (lane 4) and pKL1 plus pBGL-R and pGP1-2 cultivated at 30 °C and then shifted to 42 °C for two hours (lane 5). Plasmid pTZ19R is the vector used for construction of pBGL-R. The arrows to the right indicate the various plasmids and plasmid forms: pGP1-2 (G), pBGL-R (B), presumably pKL1 dimer (Kd) and pKL1 open (Ko) and supercoiled (Ks) forms.</note><note type="content">Figure 2: Modulated replication of pKL1. Different strains based on E. coli DH11 harboring pKL1 and DH11F harboring pKL1 and F′lacIq are presented; each strain containing a different RepA-producing or RepA-binding plasmid respectively. The strains were grown in the absence or presence of IPTG (inducer of repA expression). Shown is the relative amplification of the pKL1 copy number calculated as the ratio of relative copy number of pKL1 in the presence of the parent plasmid of the relevant helper plasmid to the relative copy number of pKL1 in the presence of particular helper plasmid. Parent plasmids were pTZ19R, pUK21, pK194, pPD1 and pACYC184. Relevant helper plasmids were pREP1, pURREP21, pKREP194, pPDREP1 and pACori1. Also shown is the high amplification of pKL1 in the presence of pREP1, moderate amplification in the presence of pUKREP21, low amplification in the presence of pKREP194, practically no amplification in the presence of pPDREP1 and lastly suppression of pKL1 copy number in the presence of pACori1.</note><note type="content">Figure 3: Circular map of plasmid pKL1. The map shows the plasmid replication regulatory elements. Fragments ori 1–4 contain a functional or partial minimal replication region when RepA is provided in trans. repA encodes the replication initiator protein RepA; BD-1, BD-2 are two distinct RepA binding sites; Prep is the promoter of repA; the IHF box is an integration host factor protein (IHF) binding region; oriT is a putative origin of conjugative transfer in the presence of a mobilization plasmid; n′-pasH is a functional ssi site; a putative stem-loop structure is indicated; A,B,C and E are Sau3AI- generated fragments of pKL1; Tn1737Km marks the location of transposon Tn1737Km inserted in pKL1 (pKL1∷Tn1737Km; Burian et al., 1997) and the location of new EcoRI restriction sites located close to both ends of Tn1737Km such that when excised from pKL1∷Tn1737Km, the linear pKL1 with EcoRI ends was used to create plasmids pTZKL1, pKL1Km and pKL1Cm (Table 1).</note><note type="content">Figure 4: The repA transcription initiation site. The primer 5′-TTGCTTCTTCGAGATCCTCGGT located 54 bp downstream of the repA start codon was used for the sequencing reaction of repA encoded by pKL1 and to direct the primer extension reaction on total E. coli DH11 (lane 1) and DH1 (lane 2) RNA. An asterix marks the transcription start site located 35 bp upstream of the repA start codon.</note><note type="content">Figure 5: RepA-DNA binding sites, BD-1 and BD-2. Reactions containing the 32P end-labelled Sau3AI fragment C of pKL1 and RepA were prepared and analyzed as described in Material and Methods. (a) The major part of the DNA sequence of Sau3AI fragment C. The IHF box is a putative IHF binding region; direct repeats, DR, are underlined; the RepA binding motif 5′-CAACGTT is highlighted; Prep is the +1 region of the repA promoter; RBS is the ribosome binding site (underlined); repA marks the translation of the first eight codons of repA; arrows mark “hot spots” of DNase I activity in the presence of RepA; rectangles A-E mark RepA protected regions of DNA against DNase I activity. (b) A typical DNase I protection assay; lanes 1–3, different loadings of the reaction in the presence of RepA; lanes 4 and 5, different loadings of the reaction in the absence of RepA; and lane 6, a reaction where DNA was hydrolyzed with 1 M piperidine at high temperature. Arrows mark “hot spots” of DNase I activity in the presence of RepA; rectangles A-E mark RepA protected regions of DNA against DNase I activity.</note><note type="content">Figure 6: Gel retardation assays. 32P end-labelled Sau3AI DNA fragment C of pKL1 was incubated with RepA or RepA and IHF, separated by polyacrylamide gel electrophoresis and examined by autoradiography. (a) RepA-DNA binding: lane 1, no protein; lanes 2–6, 12, 14, 16, 18 and 20 pmol of RepA respectively. (b) Effect of IHF on RepA-DNA binding: lane 1, no protein; lane 2, 1 pmol of IHF; lane 3, 1 pmol of IHF plus 4 pmol of RepA; lane 4, 1 pmol of IHF and 8 pmol of RepA; lane 5, 1 pmol of IHF and 12 pmol of RepA; lane 6, 1 pmol of IHF and 16 pmol of RepA.</note><note type="content">Figure 7: Effect of RepA oligomerization on DNA binding. Samples of RepA crosslinked with various levels of BS3 were separated by SDS-PAGE and blotted onto nitrocellulose which were then incubated with different [32P]DNA probes (Southwestern blot) containing BD-1 and/or BD-2 RepA binding domains. (a) A SDS-PAGE of BS3 crosslinked RepA: lane 0, Mr standards (14.85, 21.63, 29.35, 47.11, 72.960, 111.2 and 200.42 kDa); lane 1, RepA; lanes 2–6, RepA crosslinked with 0.6, 0.8, 1, 2 and 4 mM BS3, respectively. Southwestern blots of crosslinked RepA using [32P]DNA probes containing RepA binding sites (b) BD-1 and BD-2, (c) BD1 or (d) BD-2.</note><note type="content">Figure 8: DNA sequence comparisons of selected regions of pKL1. (a) Plasmids and parts thereof with particular Genbank accession numbers are: dnaBCG is the consensus sequence of dnaB, dnaC and dnaG protein-dependent initiation signal; n′ PRS is putative n′ protein (PriA) recognition site; pF-ssiA is E. coli F plasmid ssiA (D90179); pColE2-ssiA is E. coli plasmid ColE2 ssiA. (D90186); pF-ssiF is the E. coli F plasmid ssiF (D90181); p15A-ori is an E. coli plasmid p15A fragment specifying the origin of replication (J01748); pColE1-ori is the plasmid ColE1 origin of replication (M25196); p15A-ori is the plasmid p15A primer precursor DNA 3′ to the origin of replication (M24166); pR485-ori is the plasmid R485 replication origin region (M11688); pRSF1030-ori is the plasmid RSF1030 replication origin region with RNA I and primer precursor transcript (J101784); pSF is the sequence of Salmonella flexneri 2 MDa plasmid DNA associated with reactive arthritis (M25995); pR100 is the plasmid R100 replication-associated protein, repA, gene (M26840); pBR322-plasmid pBR322 (J01749). (b) pColD-ori is the plasmid ColD replication origin region (M12575); pColA-ori is the plasmid ColA replication origin region (M12574); pBR322 is plasmid pBR322 (J01749). (c) Inverted repeats located on oriT and pKL1 are indicated by arrows. oriT is the oriT region of the Salmonella typhimurium plasmid R64. Short but significant homologies were found between pKL1 (334–395 bp) and the single-strand initiation signals ssiA and ssiF of plasmid F ssiA as well as to plasmids ColE2, p15A, ColE1, pACYC184 and pas-BL of pBR322 (Bahk et al., 1988; Marians et al., 1982; Murotsu et al., 1984; Nomura et al., 1982, 1991). The same part of the pKL1 sequence was also homologous to parts of the replication origins of plasmids RSF1030 (Som & Tomizawa, 1982) and R485 (Stalker & Helinski, 1985), part of the sequence of the S. flexneri 2 MDa plasmid (Stieglitz et al., 1989) and a region of the plasmid R100 repA gene (Ohtsubo et al., 1986). There is a short region of homology between (b) pKL1 (620–680 bp) and parts of the replication origins of plasmids ColA, ColD (Zverev & Khmel, 1984) and pBR322 as well as a clear homology of (c) pKL1 (432–531 bp) and the oriT region of plasmid R64 (Komano et al., 1988).</note><note type="content">Table 1: Bacterial strains, plasmids and phages used in this study</note><note type="content">Table 2: Plaque morphology assay</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a" type="main" xml:lang="en">Replication control of a small cryptic plasmid of Escherichia coli</title><author xml:id="author-0000"><persName><forename type="first">Ján</forename><surname>Burian</surname></persName><affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Stanislav</forename><surname>Stuchlı́k</surname></persName><affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</affiliation></author><author xml:id="author-0002"><persName><forename type="first">William W</forename><surname>Kay</surname></persName><email>wkay@uvic.ca</email><note type="correspondence"><p>Corresponding author</p></note><affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</affiliation></author><idno type="istex">32B0C69EF610A6D0BB325DC605889AF830E82B8F</idno><idno type="DOI">10.1006/jmbi.1999.3266</idno><idno type="PII">S0022-2836(99)93266-7</idno></analytic><monogr><title level="j">Journal of Molecular Biology</title><title level="j" type="abbrev">YJMBI</title><idno type="pISSN">0022-2836</idno><idno type="PII">S0022-2836(00)X0285-9</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1999"></date><biblScope unit="volume">294</biblScope><biblScope unit="issue">1</biblScope><biblScope unit="page" from="49">49</biblScope><biblScope unit="page" to="65">65</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1999</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n′-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.</p></abstract><textClass><keywords scheme="keyword"><list><head>article-category</head><item><term>Regular article</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Keywords</head><item><term>plasmid</term></item><item><term>autoregulation</term></item><item><term>DNA replication</term></item><item><term>IHF</term></item><item><term>repA</term></item></list></keywords></textClass><textClass xml:lang="en"><keywords scheme="keyword"><list><head>Abbreviations</head><item><term>ssDNA</term><term>single-stranded DNA</term></item><item><term>SSB</term><term>ssDNA-binding protein</term></item><item><term>SCP</term><term>small cryptic plasmids</term></item><item><term>ORF</term><term>open reading frame</term></item><item><term>IHF</term><term>integration host factor</term></item><item><term>BD-1</term><term>binding domain I</term></item><item><term>BD-2</term><term>binding domain II</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1999-09-29">Modified</change><change when="1999">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api-v5.istex.fr/document/32B0C69EF610A6D0BB325DC605889AF830E82B8F/fulltext/txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: ce:floats; body; tail"><istex:xmlDeclaration>version="1.0" encoding="utf-8"</istex:xmlDeclaration><istex:docType PUBLIC="-//ES//DTD journal article DTD version 4.5.2//EN//XML" URI="art452.dtd" name="istex:docType"><istex:entity SYSTEM="gr1" NDATA="IMAGE" name="GR1"></istex:entity><istex:entity SYSTEM="gr2" NDATA="IMAGE" name="GR2"></istex:entity><istex:entity SYSTEM="gr3" NDATA="IMAGE" name="GR3"></istex:entity><istex:entity SYSTEM="gr4" NDATA="IMAGE" name="GR4"></istex:entity><istex:entity SYSTEM="gr5a" NDATA="IMAGE" name="GR5a"></istex:entity><istex:entity SYSTEM="gr5b" NDATA="IMAGE" name="GR5b"></istex:entity><istex:entity SYSTEM="gr6" NDATA="IMAGE" name="GR6"></istex:entity><istex:entity SYSTEM="gr7" NDATA="IMAGE" name="GR7"></istex:entity><istex:entity SYSTEM="gr8" NDATA="IMAGE" name="GR8"></istex:entity></istex:docType><istex:document><converted-article version="4.5.2" docsubtype="fla" xml:lang="en"><item-info><jid>YJMBI</jid><aid>93266</aid><ce:pii>S0022-2836(99)93266-7</ce:pii><ce:doi>10.1006/jmbi.1999.3266</ce:doi><ce:copyright type="full-transfer" year="1999">Academic Press</ce:copyright><ce:doctopics><ce:doctopic><ce:text>Regular article</ce:text></ce:doctopic></ce:doctopics></item-info><head><ce:dochead><ce:textfn>Regular article</ce:textfn></ce:dochead><ce:title>Replication control of a small cryptic plasmid of <ce:italic>Escherichia coli</ce:italic><ce:cross-ref refid="FN1"><ce:sup>1</ce:sup></ce:cross-ref><ce:footnote id="FN1"><ce:label>1</ce:label><ce:note-para><ce:bold><ce:italic>Edited by M. Gottesman</ce:italic></ce:bold></ce:note-para></ce:footnote></ce:title><ce:author-group><ce:author><ce:given-name>Ján</ce:given-name><ce:surname>Burian</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF2"><ce:sup>2</ce:sup></ce:cross-ref></ce:author><ce:author><ce:indexed-name>Stuchlik</ce:indexed-name><ce:given-name>Stanislav</ce:given-name><ce:surname>Stuchlı́k</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref><ce:cross-ref refid="AFF3"><ce:sup>3</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>William W</ce:given-name><ce:surname>Kay</ce:surname><ce:cross-ref refid="AFF1"><ce:sup>1</ce:sup></ce:cross-ref><ce:cross-ref refid="COR1">*</ce:cross-ref><ce:e-address>wkay@uvic.ca</ce:e-address></ce:author><ce:affiliation id="AFF1"><ce:label>1</ce:label><ce:textfn>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</ce:textfn></ce:affiliation><ce:affiliation id="AFF2"><ce:label>2</ce:label><ce:textfn>Microtek International (1998) Ltd., 6761 Kirkpatrick Cres. Saanichton, British Columbia V8M 1Z8, Canada</ce:textfn></ce:affiliation><ce:affiliation id="AFF3"><ce:label>3</ce:label><ce:textfn>Department of Molecular Biology, Faculty of Natural Science, Comenius University Mlynská Dolina, 842 15 Bratislava, Slovakia</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>*</ce:label><ce:text>Corresponding author</ce:text></ce:correspondence></ce:author-group><ce:date-received day="15" month="7" year="1999"></ce:date-received><ce:date-revised day="29" month="9" year="1999"></ce:date-revised><ce:date-accepted day="30" month="9" year="1999"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of <ce:italic>Escherichia coli</ce:italic>, was elucidated. The identified <ce:italic>ori</ce:italic> region encompasses a copy-number control element (<ce:italic>cop</ce:italic>) and an active single-strand initiation signal (<ce:italic>ssi</ce:italic>), n′-pasH, which were essential for efficient plasmid replication. The <ce:italic>cop</ce:italic> region also harbors a region of plasmid incompatibility, <ce:italic>inc</ce:italic>, encompassing a stem-loop structure, the <ce:italic>repA</ce:italic> promoter, P<ce:inf><ce:italic>rep</ce:italic></ce:inf>, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate <ce:italic>repA</ce:italic> transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the <ce:italic>cop</ce:italic> region and plasmid replication was shown to be dependent on host IHF encoding genes <ce:italic>himA</ce:italic> and <ce:italic>himD</ce:italic>. Low concentrations of IHF predisposed the <ce:italic>cop</ce:italic> region to RepA binding, although when highly expressed <ce:italic>in trans</ce:italic> RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the <ce:italic>cop</ce:italic> locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>plasmid</ce:text></ce:keyword><ce:keyword><ce:text>autoregulation</ce:text></ce:keyword><ce:keyword><ce:text>DNA replication</ce:text></ce:keyword><ce:keyword><ce:text>IHF</ce:text></ce:keyword><ce:keyword><ce:text><ce:italic>repA</ce:italic></ce:text></ce:keyword></ce:keywords><ce:keywords class="abr"><ce:section-title>Abbreviations</ce:section-title><ce:keyword><ce:text>ssDNA</ce:text><ce:keyword><ce:text>single-stranded DNA</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>SSB</ce:text><ce:keyword><ce:text>ssDNA-binding protein</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>SCP</ce:text><ce:keyword><ce:text>small cryptic plasmids</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>ORF</ce:text><ce:keyword><ce:text>open reading frame</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>IHF</ce:text><ce:keyword><ce:text>integration host factor</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>BD-1</ce:text><ce:keyword><ce:text>binding domain I</ce:text></ce:keyword></ce:keyword><ce:keyword><ce:text>BD-2</ce:text><ce:keyword><ce:text>binding domain II</ce:text></ce:keyword></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo lang="en"><title>Replication control of a small cryptic plasmid of Escherichia coli</title></titleInfo><titleInfo type="alternative" lang="en" contentType="CDATA"><title>Replication control of a small cryptic plasmid of</title></titleInfo><name type="personal"><namePart type="given">Ján</namePart><namePart type="family">Burian</namePart><affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Stanislav</namePart><namePart type="family">Stuchlı́k</namePart><affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">William W</namePart><namePart type="family">Kay</namePart><affiliation>E-mail: wkay@uvic.ca</affiliation><affiliation>Department of Biochemistry and Microbiology and the Canadian Bacterial Diseases Network, University of Victoria, Petch Building, P. O. Box 3055, Victoria, British Columbia V8W 3P6, Canada</affiliation><description>Corresponding author</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article"></genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1999</dateIssued><dateModified encoding="w3cdtf">1999-09-29</dateModified><copyrightDate encoding="w3cdtf">1999</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><physicalDescription><internetMediaType>text/html</internetMediaType></physicalDescription><abstract lang="en">The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n′-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.</abstract><note type="footnote">Edited by M. Gottesman</note><note type="content">Section title: Regular article</note><note type="content">Figure 1: Effect of RepA on pKL1 replication. Strains producing different amounts of RepA were analyzed by Western blotting using anti-RepA antiserum (a) and resulting amplification of pKL1 DNA by agarose electrophoresis and EtBr staining (b). Lane 1, shows RepA produced from pKL1. Lanes 1–5 show Western blots of RepA and electrophoresis of plasmids isolated from E. coli DH11 harboring pKL1 (lane 1), pKL1 plus pTZ19R grown at 37 °C (lane 2), pKL1 plus pBGL-R grown at 37 °C (lane 3), pKL1 pBGL-R and pGP1-2 cultivated at 30 °C (lane 4) and pKL1 plus pBGL-R and pGP1-2 cultivated at 30 °C and then shifted to 42 °C for two hours (lane 5). Plasmid pTZ19R is the vector used for construction of pBGL-R. The arrows to the right indicate the various plasmids and plasmid forms: pGP1-2 (G), pBGL-R (B), presumably pKL1 dimer (Kd) and pKL1 open (Ko) and supercoiled (Ks) forms.</note><note type="content">Figure 2: Modulated replication of pKL1. Different strains based on E. coli DH11 harboring pKL1 and DH11F harboring pKL1 and F′lacIq are presented; each strain containing a different RepA-producing or RepA-binding plasmid respectively. The strains were grown in the absence or presence of IPTG (inducer of repA expression). Shown is the relative amplification of the pKL1 copy number calculated as the ratio of relative copy number of pKL1 in the presence of the parent plasmid of the relevant helper plasmid to the relative copy number of pKL1 in the presence of particular helper plasmid. Parent plasmids were pTZ19R, pUK21, pK194, pPD1 and pACYC184. Relevant helper plasmids were pREP1, pURREP21, pKREP194, pPDREP1 and pACori1. Also shown is the high amplification of pKL1 in the presence of pREP1, moderate amplification in the presence of pUKREP21, low amplification in the presence of pKREP194, practically no amplification in the presence of pPDREP1 and lastly suppression of pKL1 copy number in the presence of pACori1.</note><note type="content">Figure 3: Circular map of plasmid pKL1. The map shows the plasmid replication regulatory elements. Fragments ori 1–4 contain a functional or partial minimal replication region when RepA is provided in trans. repA encodes the replication initiator protein RepA; BD-1, BD-2 are two distinct RepA binding sites; Prep is the promoter of repA; the IHF box is an integration host factor protein (IHF) binding region; oriT is a putative origin of conjugative transfer in the presence of a mobilization plasmid; n′-pasH is a functional ssi site; a putative stem-loop structure is indicated; A,B,C and E are Sau3AI- generated fragments of pKL1; Tn1737Km marks the location of transposon Tn1737Km inserted in pKL1 (pKL1∷Tn1737Km; Burian et al., 1997) and the location of new EcoRI restriction sites located close to both ends of Tn1737Km such that when excised from pKL1∷Tn1737Km, the linear pKL1 with EcoRI ends was used to create plasmids pTZKL1, pKL1Km and pKL1Cm (Table 1).</note><note type="content">Figure 4: The repA transcription initiation site. The primer 5′-TTGCTTCTTCGAGATCCTCGGT located 54 bp downstream of the repA start codon was used for the sequencing reaction of repA encoded by pKL1 and to direct the primer extension reaction on total E. coli DH11 (lane 1) and DH1 (lane 2) RNA. An asterix marks the transcription start site located 35 bp upstream of the repA start codon.</note><note type="content">Figure 5: RepA-DNA binding sites, BD-1 and BD-2. Reactions containing the 32P end-labelled Sau3AI fragment C of pKL1 and RepA were prepared and analyzed as described in Material and Methods. (a) The major part of the DNA sequence of Sau3AI fragment C. The IHF box is a putative IHF binding region; direct repeats, DR, are underlined; the RepA binding motif 5′-CAACGTT is highlighted; Prep is the +1 region of the repA promoter; RBS is the ribosome binding site (underlined); repA marks the translation of the first eight codons of repA; arrows mark “hot spots” of DNase I activity in the presence of RepA; rectangles A-E mark RepA protected regions of DNA against DNase I activity. (b) A typical DNase I protection assay; lanes 1–3, different loadings of the reaction in the presence of RepA; lanes 4 and 5, different loadings of the reaction in the absence of RepA; and lane 6, a reaction where DNA was hydrolyzed with 1 M piperidine at high temperature. Arrows mark “hot spots” of DNase I activity in the presence of RepA; rectangles A-E mark RepA protected regions of DNA against DNase I activity.</note><note type="content">Figure 6: Gel retardation assays. 32P end-labelled Sau3AI DNA fragment C of pKL1 was incubated with RepA or RepA and IHF, separated by polyacrylamide gel electrophoresis and examined by autoradiography. (a) RepA-DNA binding: lane 1, no protein; lanes 2–6, 12, 14, 16, 18 and 20 pmol of RepA respectively. (b) Effect of IHF on RepA-DNA binding: lane 1, no protein; lane 2, 1 pmol of IHF; lane 3, 1 pmol of IHF plus 4 pmol of RepA; lane 4, 1 pmol of IHF and 8 pmol of RepA; lane 5, 1 pmol of IHF and 12 pmol of RepA; lane 6, 1 pmol of IHF and 16 pmol of RepA.</note><note type="content">Figure 7: Effect of RepA oligomerization on DNA binding. Samples of RepA crosslinked with various levels of BS3 were separated by SDS-PAGE and blotted onto nitrocellulose which were then incubated with different [32P]DNA probes (Southwestern blot) containing BD-1 and/or BD-2 RepA binding domains. (a) A SDS-PAGE of BS3 crosslinked RepA: lane 0, Mr standards (14.85, 21.63, 29.35, 47.11, 72.960, 111.2 and 200.42 kDa); lane 1, RepA; lanes 2–6, RepA crosslinked with 0.6, 0.8, 1, 2 and 4 mM BS3, respectively. Southwestern blots of crosslinked RepA using [32P]DNA probes containing RepA binding sites (b) BD-1 and BD-2, (c) BD1 or (d) BD-2.</note><note type="content">Figure 8: DNA sequence comparisons of selected regions of pKL1. (a) Plasmids and parts thereof with particular Genbank accession numbers are: dnaBCG is the consensus sequence of dnaB, dnaC and dnaG protein-dependent initiation signal; n′ PRS is putative n′ protein (PriA) recognition site; pF-ssiA is E. coli F plasmid ssiA (D90179); pColE2-ssiA is E. coli plasmid ColE2 ssiA. (D90186); pF-ssiF is the E. coli F plasmid ssiF (D90181); p15A-ori is an E. coli plasmid p15A fragment specifying the origin of replication (J01748); pColE1-ori is the plasmid ColE1 origin of replication (M25196); p15A-ori is the plasmid p15A primer precursor DNA 3′ to the origin of replication (M24166); pR485-ori is the plasmid R485 replication origin region (M11688); pRSF1030-ori is the plasmid RSF1030 replication origin region with RNA I and primer precursor transcript (J101784); pSF is the sequence of Salmonella flexneri 2 MDa plasmid DNA associated with reactive arthritis (M25995); pR100 is the plasmid R100 replication-associated protein, repA, gene (M26840); pBR322-plasmid pBR322 (J01749). (b) pColD-ori is the plasmid ColD replication origin region (M12575); pColA-ori is the plasmid ColA replication origin region (M12574); pBR322 is plasmid pBR322 (J01749). (c) Inverted repeats located on oriT and pKL1 are indicated by arrows. oriT is the oriT region of the Salmonella typhimurium plasmid R64. Short but significant homologies were found between pKL1 (334–395 bp) and the single-strand initiation signals ssiA and ssiF of plasmid F ssiA as well as to plasmids ColE2, p15A, ColE1, pACYC184 and pas-BL of pBR322 (Bahk et al., 1988; Marians et al., 1982; Murotsu et al., 1984; Nomura et al., 1982, 1991). The same part of the pKL1 sequence was also homologous to parts of the replication origins of plasmids RSF1030 (Som & Tomizawa, 1982) and R485 (Stalker & Helinski, 1985), part of the sequence of the S. flexneri 2 MDa plasmid (Stieglitz et al., 1989) and a region of the plasmid R100 repA gene (Ohtsubo et al., 1986). There is a short region of homology between (b) pKL1 (620–680 bp) and parts of the replication origins of plasmids ColA, ColD (Zverev & Khmel, 1984) and pBR322 as well as a clear homology of (c) pKL1 (432–531 bp) and the oriT region of plasmid R64 (Komano et al., 1988).</note><note type="content">Table 1: Bacterial strains, plasmids and phages used in this study</note><note type="content">Table 2: Plaque morphology assay</note><subject><genre>article-category</genre><topic>Regular article</topic></subject><subject lang="en"><genre>Keywords</genre><topic>plasmid</topic><topic>autoregulation</topic><topic>DNA replication</topic><topic>IHF</topic><topic>repA</topic></subject><subject lang="en"><genre>Abbreviations</genre><topic>ssDNA : single-stranded DNA</topic><topic>SSB : ssDNA-binding protein</topic><topic>SCP : small cryptic plasmids</topic><topic>ORF : open reading frame</topic><topic>IHF : integration host factor</topic><topic>BD-1 : binding domain I</topic><topic>BD-2 : binding domain II</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Molecular Biology</title></titleInfo><titleInfo type="abbreviated"><title>YJMBI</title></titleInfo><genre type="journal">journal</genre><originInfo><dateIssued encoding="w3cdtf">19991119</dateIssued></originInfo><identifier type="ISSN">0022-2836</identifier><identifier type="PII">S0022-2836(00)X0285-9</identifier><part><date>19991119</date><detail type="volume"><number>294</number><caption>vol.</caption></detail><detail type="issue"><number>1</number><caption>no.</caption></detail><extent unit="issue pages"><start>1</start><end>288</end></extent><extent unit="pages"><start>49</start><end>65</end></extent></part></relatedItem><identifier type="istex">32B0C69EF610A6D0BB325DC605889AF830E82B8F</identifier><identifier type="DOI">10.1006/jmbi.1999.3266</identifier><identifier type="PII">S0022-2836(99)93266-7</identifier><accessCondition type="use and reproduction" contentType="copyright">©1999 Academic Press</accessCondition><recordInfo><recordContentSource>ELSEVIER</recordContentSource><recordOrigin>Academic Press, ©1999</recordOrigin></recordInfo></mods></metadata><serie></serie></istex></record>Pour manipuler ce document sous Unix (Dilib)
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