Factors required in vitro for excision of the Bacteroides conjugative transposon, CTnDOT
Four genes have been found to be essential for excision of the Bacteroides conjugative transposon CTnDOT in vivo: intDOT, orf2c, orf2d, and exc. The intDOT gene encodes an integrase that is essential for integration and excision. The function of the other genes is still uncertain. Previously, we dev...
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| Veröffentlicht in: | Plasmid 2004-09, Vol.52 (2), p.119-130 |
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| creator | Sutanto, Yuri DiChiara, Jeanne M. Shoemaker, Nadja B. Gardner, Jeffrey F. Salyers, Abigail A. |
| description | Four genes have been found to be essential for excision of the
Bacteroides conjugative transposon CTnDOT in vivo:
intDOT,
orf2c,
orf2d, and
exc. The
intDOT gene encodes an integrase that is essential for integration and excision. The function of the other genes is still uncertain. Previously, we developed an in vitro system for the integration reaction. We have now developed an in vitro system for excision. In this system, the left and right junctions of CTnDOT,
attL, and
attR, are provided on separate plasmids. The excision reaction produced a cointegrate which contained the
attDOT (the joined ends of CTnDOT) and
attB (the chromosomal target site). Cointegrate formation was observed after electroporation of
Escherichia coli with the assay mixture and was also detected directly in the assay mixture by Southern hybridization. The highest reaction frequencies (10
−3) were obtained with a mixture that contained purified IntDOT and a cell extract from
Bacteroides thetaiotaomicron 4001, which contained the excision region of CTnDOT carried on a plasmid. An unexpected finding was that the addition of purified Exc, which is essential for excision in vivo, was not required for excision in vitro, nor did it increase the frequency of cointegrate formation. |
| doi_str_mv | 10.1016/j.plasmid.2004.06.003 |
| format | Article |
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Bacteroides conjugative transposon CTnDOT in vivo:
intDOT,
orf2c,
orf2d, and
exc. The
intDOT gene encodes an integrase that is essential for integration and excision. The function of the other genes is still uncertain. Previously, we developed an in vitro system for the integration reaction. We have now developed an in vitro system for excision. In this system, the left and right junctions of CTnDOT,
attL, and
attR, are provided on separate plasmids. The excision reaction produced a cointegrate which contained the
attDOT (the joined ends of CTnDOT) and
attB (the chromosomal target site). Cointegrate formation was observed after electroporation of
Escherichia coli with the assay mixture and was also detected directly in the assay mixture by Southern hybridization. The highest reaction frequencies (10
−3) were obtained with a mixture that contained purified IntDOT and a cell extract from
Bacteroides thetaiotaomicron 4001, which contained the excision region of CTnDOT carried on a plasmid. An unexpected finding was that the addition of purified Exc, which is essential for excision in vivo, was not required for excision in vitro, nor did it increase the frequency of cointegrate formation.</description><identifier>ISSN: 0147-619X</identifier><identifier>EISSN: 1095-9890</identifier><identifier>DOI: 10.1016/j.plasmid.2004.06.003</identifier><identifier>PMID: 15336489</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Attachment Sites, Microbiological - genetics ; Bacteroides - enzymology ; Bacteroides - genetics ; Bacteroides resistance gene transfer elements ; Bacteroides thetaiotaomicron ; Base Sequence ; Codon, Terminator ; CTnDOT ; DNA Transposable Elements - genetics ; DNA, Bacterial - chemistry ; DNA, Bacterial - genetics ; Electroporation ; Escherichia coli - genetics ; Excision factors ; Host factors ; In vitro excision system ; Integrases - genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Open Reading Frames ; Plasmids - genetics ; Recombination, Genetic ; Sequence Analysis, DNA</subject><ispartof>Plasmid, 2004-09, Vol.52 (2), p.119-130</ispartof><rights>2004 Elsevier Inc.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c458t-bd616e22402ef9853a1a6832e0259289f0ab1dc0e32ec1633491b63b3351618b3</citedby><cites>FETCH-LOGICAL-c458t-bd616e22402ef9853a1a6832e0259289f0ab1dc0e32ec1633491b63b3351618b3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/j.plasmid.2004.06.003$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,778,782,3539,27907,27908,45978</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/15336489$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Sutanto, Yuri</creatorcontrib><creatorcontrib>DiChiara, Jeanne M.</creatorcontrib><creatorcontrib>Shoemaker, Nadja B.</creatorcontrib><creatorcontrib>Gardner, Jeffrey F.</creatorcontrib><creatorcontrib>Salyers, Abigail A.</creatorcontrib><title>Factors required in vitro for excision of the Bacteroides conjugative transposon, CTnDOT</title><title>Plasmid</title><addtitle>Plasmid</addtitle><description>Four genes have been found to be essential for excision of the
Bacteroides conjugative transposon CTnDOT in vivo:
intDOT,
orf2c,
orf2d, and
exc. The
intDOT gene encodes an integrase that is essential for integration and excision. The function of the other genes is still uncertain. Previously, we developed an in vitro system for the integration reaction. We have now developed an in vitro system for excision. In this system, the left and right junctions of CTnDOT,
attL, and
attR, are provided on separate plasmids. The excision reaction produced a cointegrate which contained the
attDOT (the joined ends of CTnDOT) and
attB (the chromosomal target site). Cointegrate formation was observed after electroporation of
Escherichia coli with the assay mixture and was also detected directly in the assay mixture by Southern hybridization. The highest reaction frequencies (10
−3) were obtained with a mixture that contained purified IntDOT and a cell extract from
Bacteroides thetaiotaomicron 4001, which contained the excision region of CTnDOT carried on a plasmid. An unexpected finding was that the addition of purified Exc, which is essential for excision in vivo, was not required for excision in vitro, nor did it increase the frequency of cointegrate formation.</description><subject>Attachment Sites, Microbiological - genetics</subject><subject>Bacteroides - enzymology</subject><subject>Bacteroides - genetics</subject><subject>Bacteroides resistance gene transfer elements</subject><subject>Bacteroides thetaiotaomicron</subject><subject>Base Sequence</subject><subject>Codon, Terminator</subject><subject>CTnDOT</subject><subject>DNA Transposable Elements - genetics</subject><subject>DNA, Bacterial - chemistry</subject><subject>DNA, Bacterial - genetics</subject><subject>Electroporation</subject><subject>Escherichia coli - genetics</subject><subject>Excision factors</subject><subject>Host factors</subject><subject>In vitro excision system</subject><subject>Integrases - genetics</subject><subject>Molecular Sequence Data</subject><subject>Mutagenesis, Site-Directed</subject><subject>Open Reading Frames</subject><subject>Plasmids - genetics</subject><subject>Recombination, Genetic</subject><subject>Sequence Analysis, DNA</subject><issn>0147-619X</issn><issn>1095-9890</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkE2L2zAQhkXZ0s1u-xNadNpT7c5YtmKdSjfdLwjkksLehCyPd2USK5HssP33VUigxz0NDM87L_Mw9hUhR0D5o893GxO3rs0LgDIHmQOID2yGoKpM1Qou2AywnGcS1fMlu4qxBwBZoPzELrESQpa1mrHne2NHHyIPtJ9coJa7gR_cGDzvfOD0Zl10fuC-4-Mr8dtEU_CupcitH_rpxYzuQHwMZog7H_3wnS_Ww-_V-jP72JlNpC_nec3-3N-tF4_ZcvXwtPi1zGxZ1WPWtBIlFUUJBXWqroRBI2tREBSVKmrVgWmwtUBpZVEKUSpspGiEqFBi3YhrdnO6uwt-P1Ec9dZFS5uNGchPUeNcqiqRCaxOoA0-xkCd3gW3NeGvRtBHpbrXZ6X6qFSD1Elpyn07F0zNltr_qbPDBPw8AZTePDgKOlpHg6U2-bSjbr17p-IfdsCKFg</recordid><startdate>20040901</startdate><enddate>20040901</enddate><creator>Sutanto, Yuri</creator><creator>DiChiara, Jeanne M.</creator><creator>Shoemaker, Nadja B.</creator><creator>Gardner, Jeffrey F.</creator><creator>Salyers, Abigail A.</creator><general>Elsevier Inc</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>RC3</scope></search><sort><creationdate>20040901</creationdate><title>Factors required in vitro for excision of the Bacteroides conjugative transposon, CTnDOT</title><author>Sutanto, Yuri ; DiChiara, Jeanne M. ; Shoemaker, Nadja B. ; Gardner, Jeffrey F. ; Salyers, Abigail A.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c458t-bd616e22402ef9853a1a6832e0259289f0ab1dc0e32ec1633491b63b3351618b3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2004</creationdate><topic>Attachment Sites, Microbiological - genetics</topic><topic>Bacteroides - enzymology</topic><topic>Bacteroides - genetics</topic><topic>Bacteroides resistance gene transfer elements</topic><topic>Bacteroides thetaiotaomicron</topic><topic>Base Sequence</topic><topic>Codon, Terminator</topic><topic>CTnDOT</topic><topic>DNA Transposable Elements - genetics</topic><topic>DNA, Bacterial - chemistry</topic><topic>DNA, Bacterial - genetics</topic><topic>Electroporation</topic><topic>Escherichia coli - genetics</topic><topic>Excision factors</topic><topic>Host factors</topic><topic>In vitro excision system</topic><topic>Integrases - genetics</topic><topic>Molecular Sequence Data</topic><topic>Mutagenesis, Site-Directed</topic><topic>Open Reading Frames</topic><topic>Plasmids - genetics</topic><topic>Recombination, Genetic</topic><topic>Sequence Analysis, DNA</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Sutanto, Yuri</creatorcontrib><creatorcontrib>DiChiara, Jeanne M.</creatorcontrib><creatorcontrib>Shoemaker, Nadja B.</creatorcontrib><creatorcontrib>Gardner, Jeffrey F.</creatorcontrib><creatorcontrib>Salyers, Abigail A.</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><jtitle>Plasmid</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Sutanto, Yuri</au><au>DiChiara, Jeanne M.</au><au>Shoemaker, Nadja B.</au><au>Gardner, Jeffrey F.</au><au>Salyers, Abigail A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Factors required in vitro for excision of the Bacteroides conjugative transposon, CTnDOT</atitle><jtitle>Plasmid</jtitle><addtitle>Plasmid</addtitle><date>2004-09-01</date><risdate>2004</risdate><volume>52</volume><issue>2</issue><spage>119</spage><epage>130</epage><pages>119-130</pages><issn>0147-619X</issn><eissn>1095-9890</eissn><abstract>Four genes have been found to be essential for excision of the
Bacteroides conjugative transposon CTnDOT in vivo:
intDOT,
orf2c,
orf2d, and
exc. The
intDOT gene encodes an integrase that is essential for integration and excision. The function of the other genes is still uncertain. Previously, we developed an in vitro system for the integration reaction. We have now developed an in vitro system for excision. In this system, the left and right junctions of CTnDOT,
attL, and
attR, are provided on separate plasmids. The excision reaction produced a cointegrate which contained the
attDOT (the joined ends of CTnDOT) and
attB (the chromosomal target site). Cointegrate formation was observed after electroporation of
Escherichia coli with the assay mixture and was also detected directly in the assay mixture by Southern hybridization. The highest reaction frequencies (10
−3) were obtained with a mixture that contained purified IntDOT and a cell extract from
Bacteroides thetaiotaomicron 4001, which contained the excision region of CTnDOT carried on a plasmid. An unexpected finding was that the addition of purified Exc, which is essential for excision in vivo, was not required for excision in vitro, nor did it increase the frequency of cointegrate formation.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>15336489</pmid><doi>10.1016/j.plasmid.2004.06.003</doi><tpages>12</tpages></addata></record> |
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| subjects | Attachment Sites, Microbiological - genetics Bacteroides - enzymology Bacteroides - genetics Bacteroides resistance gene transfer elements Bacteroides thetaiotaomicron Base Sequence Codon, Terminator CTnDOT DNA Transposable Elements - genetics DNA, Bacterial - chemistry DNA, Bacterial - genetics Electroporation Escherichia coli - genetics Excision factors Host factors In vitro excision system Integrases - genetics Molecular Sequence Data Mutagenesis, Site-Directed Open Reading Frames Plasmids - genetics Recombination, Genetic Sequence Analysis, DNA |
| title | Factors required in vitro for excision of the Bacteroides conjugative transposon, CTnDOT |
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