An Indicator Gene to Demonstrate Intracellular Transposition of Defective Retroviruses
An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo- MLV(neo)RT]. NeoRT contains th...
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| Veröffentlicht in: | Proceedings of the National Academy of Sciences - PNAS 1988-04, Vol.85 (7), p.2219-2223 |
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| creator | Heidmann, Thierry Heidmann, Odile Nicolas, Jean-François |
| description | An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo- MLV(neo)RT]. NeoRT contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)RT provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo- MLV(neo)RT provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 107 cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected. |
| doi_str_mv | 10.1073/pnas.85.7.2219 |
| format | Article |
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It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo- MLV(neo)RT]. NeoRT contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)RT provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo- MLV(neo)RT provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 107 cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected.</description><identifier>ISSN: 0027-8424</identifier><identifier>EISSN: 1091-6490</identifier><identifier>DOI: 10.1073/pnas.85.7.2219</identifier><identifier>PMID: 2832848</identifier><language>eng</language><publisher>United States: National Academy of Sciences of the United States of America</publisher><subject>3T3 cells ; Cancer ; Cell lines ; Cellular Biology ; Defective Viruses - genetics ; DNA ; DNA Transposable Elements ; DNA, Recombinant ; Drug Resistance ; Genes ; Genes, Synthetic ; Genes, Viral ; Genetic transposition ; Genetics ; Gentamicins - pharmacology ; Introns ; Life Sciences ; Moloney murine leukemia virus - genetics ; NIH 3T3 cells ; Poly A - genetics ; Proviruses ; Proviruses - genetics ; Retroviridae Proteins - genetics ; Reverse transcription ; RNA ; RNA Splicing ; RNA, Viral - genetics ; Viral Envelope Proteins - genetics ; Viruses</subject><ispartof>Proceedings of the National Academy of Sciences - PNAS, 1988-04, Vol.85 (7), p.2219-2223</ispartof><rights>Distributed under a Creative Commons Attribution 4.0 International License</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c533t-37aa706966a3e2574f57b8f54403e4a5451cf3c89cfb365e61a36d73b2035c813</citedby><orcidid>0000-0003-4204-803X</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Uhttp://www.pnas.org/content/85/7.cover.gif</thumbnail><linktopdf>$$Uhttps://www.jstor.org/stable/pdf/31101$$EPDF$$P50$$Gjstor$$H</linktopdf><linktohtml>$$Uhttps://www.jstor.org/stable/31101$$EHTML$$P50$$Gjstor$$H</linktohtml><link.rule.ids>230,314,776,780,799,881,27901,27902,57992,58225</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2832848$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink><backlink>$$Uhttps://hal.science/hal-02012578$$DView record in HAL$$Hfree_for_read</backlink></links><search><creatorcontrib>Heidmann, Thierry</creatorcontrib><creatorcontrib>Heidmann, Odile</creatorcontrib><creatorcontrib>Nicolas, Jean-François</creatorcontrib><title>An Indicator Gene to Demonstrate Intracellular Transposition of Defective Retroviruses</title><title>Proceedings of the National Academy of Sciences - PNAS</title><addtitle>Proc Natl Acad Sci U S A</addtitle><description>An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo- MLV(neo)RT]. NeoRT contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)RT provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo- MLV(neo)RT provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 107 cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected.</description><subject>3T3 cells</subject><subject>Cancer</subject><subject>Cell lines</subject><subject>Cellular Biology</subject><subject>Defective Viruses - genetics</subject><subject>DNA</subject><subject>DNA Transposable Elements</subject><subject>DNA, Recombinant</subject><subject>Drug Resistance</subject><subject>Genes</subject><subject>Genes, Synthetic</subject><subject>Genes, Viral</subject><subject>Genetic transposition</subject><subject>Genetics</subject><subject>Gentamicins - pharmacology</subject><subject>Introns</subject><subject>Life Sciences</subject><subject>Moloney murine leukemia virus - genetics</subject><subject>NIH 3T3 cells</subject><subject>Poly A - genetics</subject><subject>Proviruses</subject><subject>Proviruses - genetics</subject><subject>Retroviridae Proteins - genetics</subject><subject>Reverse transcription</subject><subject>RNA</subject><subject>RNA Splicing</subject><subject>RNA, Viral - genetics</subject><subject>Viral Envelope Proteins - genetics</subject><subject>Viruses</subject><issn>0027-8424</issn><issn>1091-6490</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1988</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkc1r3DAQxUVJSbZprz0EAj4FerA7-rLk45K2SWChUNJehVY7Ig5eayPJS_Lf1663S07taWDeb96M9Aj5SKGioPjnXW9TpWWlKsZo84YsKDS0rEUDJ2QBwFSpBRNn5F1KjwDQSA2n5JRpzrTQC_Jr2Rd3_aZ1NodY3GCPRQ7FF9yGPuVoM47qWB123dDZWNxH26ddSG1uQ18EP6IeXW73WPzAHMO-jUPC9J689bZL-OFQz8nPb1_vr2_L1febu-vlqnSS81xyZa2Cuqlry5FJJbxUa-2lEMBRWCkkdZ473Ti_5rXEmlpebxRfM-DSacrPyafZ98F2ZhfbrY0vJtjW3C5XZuoBAzoa6_3EXs3sLoanAVM22zZND7M9hiEZpWmtxz_8L0hFw4H_2V7NoIshpYj-eAIFM6VjpnSMlkYZNjtfHpyH9RY3R_wQxyt9mvurvp6_-pdu_NB1GZ_zCF7M4GMacz2SnFKg_DebQqpU</recordid><startdate>19880401</startdate><enddate>19880401</enddate><creator>Heidmann, Thierry</creator><creator>Heidmann, Odile</creator><creator>Nicolas, Jean-François</creator><general>National Academy of Sciences of the United States of America</general><general>National Acad Sciences</general><general>National Academy of Sciences</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>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>1XC</scope><orcidid>https://orcid.org/0000-0003-4204-803X</orcidid></search><sort><creationdate>19880401</creationdate><title>An Indicator Gene to Demonstrate Intracellular Transposition of Defective Retroviruses</title><author>Heidmann, Thierry ; Heidmann, Odile ; Nicolas, Jean-François</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c533t-37aa706966a3e2574f57b8f54403e4a5451cf3c89cfb365e61a36d73b2035c813</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1988</creationdate><topic>3T3 cells</topic><topic>Cancer</topic><topic>Cell lines</topic><topic>Cellular Biology</topic><topic>Defective Viruses - genetics</topic><topic>DNA</topic><topic>DNA Transposable Elements</topic><topic>DNA, Recombinant</topic><topic>Drug Resistance</topic><topic>Genes</topic><topic>Genes, Synthetic</topic><topic>Genes, Viral</topic><topic>Genetic transposition</topic><topic>Genetics</topic><topic>Gentamicins - pharmacology</topic><topic>Introns</topic><topic>Life Sciences</topic><topic>Moloney murine leukemia virus - genetics</topic><topic>NIH 3T3 cells</topic><topic>Poly A - genetics</topic><topic>Proviruses</topic><topic>Proviruses - genetics</topic><topic>Retroviridae Proteins - genetics</topic><topic>Reverse transcription</topic><topic>RNA</topic><topic>RNA Splicing</topic><topic>RNA, Viral - genetics</topic><topic>Viral Envelope Proteins - genetics</topic><topic>Viruses</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Heidmann, Thierry</creatorcontrib><creatorcontrib>Heidmann, Odile</creatorcontrib><creatorcontrib>Nicolas, Jean-François</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>Hyper Article en Ligne (HAL)</collection><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Heidmann, Thierry</au><au>Heidmann, Odile</au><au>Nicolas, Jean-François</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>An Indicator Gene to Demonstrate Intracellular Transposition of Defective Retroviruses</atitle><jtitle>Proceedings of the National Academy of Sciences - PNAS</jtitle><addtitle>Proc Natl Acad Sci U S A</addtitle><date>1988-04-01</date><risdate>1988</risdate><volume>85</volume><issue>7</issue><spage>2219</spage><epage>2223</epage><pages>2219-2223</pages><issn>0027-8424</issn><eissn>1091-6490</eissn><abstract>An indicator gene for detection and quantitation of RNA-mediated transposition was constructed (neoRT). It was inserted into a Moloney murine leukemia provirus (Mo-MLV) deleted for the envelope gene to test for intracellular transposition of defective retroviruses [Mo- MLV(neo)RT]. NeoRT contains the selectable neo gene (which confers resistance to the drug G418), inactivated by a polyadenylylation sequence inserted between the neo promotor and coding sequence. The polyadenylylation sequence is flanked (on the antisense strand of the DNA) by a donor and an acceptor splice site so as to be removed upon passage of the provirus through an RNA intermediate. 3T3 cells transfected with the defective Mo-MLV(neo)RT provirus are sensitive to G418. After trans-complementation with Mo-MLV, viral transcripts confer resistance to G418 upon infection of test cells. In the resistant cells, the polyadenylylation sequence has been removed, as a result in most cases of precise splicing of the intronic domain. Retrotransposition of the defective Mo- MLV(neo)RT provirus was demonstrated by submitting transfected G418-sensitive clones to selection. Between 1 and 10 G418-resistant clones were obtained per 107 cells. Several possess additional copies, with evidence for precise removal of the intronic domain. By using target test cells in coculture experiments, extracellular intermediates of retrotransposition could not be detected.</abstract><cop>United States</cop><pub>National Academy of Sciences of the United States of America</pub><pmid>2832848</pmid><doi>10.1073/pnas.85.7.2219</doi><tpages>5</tpages><orcidid>https://orcid.org/0000-0003-4204-803X</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | 3T3 cells Cancer Cell lines Cellular Biology Defective Viruses - genetics DNA DNA Transposable Elements DNA, Recombinant Drug Resistance Genes Genes, Synthetic Genes, Viral Genetic transposition Genetics Gentamicins - pharmacology Introns Life Sciences Moloney murine leukemia virus - genetics NIH 3T3 cells Poly A - genetics Proviruses Proviruses - genetics Retroviridae Proteins - genetics Reverse transcription RNA RNA Splicing RNA, Viral - genetics Viral Envelope Proteins - genetics Viruses |
| title | An Indicator Gene to Demonstrate Intracellular Transposition of Defective Retroviruses |
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