Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter
RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from...
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| Veröffentlicht in: | Nucleic acids research 1995-06, Vol.23 (12), p.2259-2268 |
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| creator | Thompson, James D. Ayers, David F. Malmstrom, Terra A. McKenzie, Timothy L. Ganousis, Louis Chowrira, Bharat M. Couture, Larry Stinchcomb, Dan T. |
| description | RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human met1 tRNA-derived pol III promoter >100-fold by modifying the 3′ terminus of the transcripts to hybridize to the 5′ terminus. This terminal duplex Includes the 8 nt leader sequence present in the primary wild-type met) tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed ‘TRZ’, was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs. |
| doi_str_mv | 10.1093/nar/23.12.2259 |
| format | Article |
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Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human met1 tRNA-derived pol III promoter >100-fold by modifying the 3′ terminus of the transcripts to hybridize to the 5′ terminus. This terminal duplex Includes the 8 nt leader sequence present in the primary wild-type met) tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed ‘TRZ’, was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/23.12.2259</identifier><identifier>PMID: 7610054</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>AIDS/HIV ; Base Sequence ; Blotting, Northern ; Cell Line ; Gene Expression ; Human immunodeficiency virus ; Humans ; Molecular Sequence Data ; Nucleic Acid Conformation ; Plasmids ; Promoter Regions, Genetic ; Retroviridae - genetics ; RNA Polymerase III - genetics ; RNA Probes ; RNA, Catalytic - genetics ; RNA, Catalytic - metabolism ; RNA, Messenger - chemistry ; RNA, Messenger - metabolism ; RNA, Transfer, Met - genetics</subject><ispartof>Nucleic acids research, 1995-06, Vol.23 (12), p.2259-2268</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3999-65eb1e74201f6dad10694b0cf50f2787455ee2fabf7d8926816fdfd9a1a513bd3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC307016/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC307016/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/7610054$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Thompson, James D.</creatorcontrib><creatorcontrib>Ayers, David F.</creatorcontrib><creatorcontrib>Malmstrom, Terra A.</creatorcontrib><creatorcontrib>McKenzie, Timothy L.</creatorcontrib><creatorcontrib>Ganousis, Louis</creatorcontrib><creatorcontrib>Chowrira, Bharat M.</creatorcontrib><creatorcontrib>Couture, Larry</creatorcontrib><creatorcontrib>Stinchcomb, Dan T.</creatorcontrib><title>Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human met1 tRNA-derived pol III promoter >100-fold by modifying the 3′ terminus of the transcripts to hybridize to the 5′ terminus. This terminal duplex Includes the 8 nt leader sequence present in the primary wild-type met) tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed ‘TRZ’, was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.</description><subject>AIDS/HIV</subject><subject>Base Sequence</subject><subject>Blotting, Northern</subject><subject>Cell Line</subject><subject>Gene Expression</subject><subject>Human immunodeficiency virus</subject><subject>Humans</subject><subject>Molecular Sequence Data</subject><subject>Nucleic Acid Conformation</subject><subject>Plasmids</subject><subject>Promoter Regions, Genetic</subject><subject>Retroviridae - genetics</subject><subject>RNA Polymerase III - genetics</subject><subject>RNA Probes</subject><subject>RNA, Catalytic - genetics</subject><subject>RNA, Catalytic - metabolism</subject><subject>RNA, Messenger - chemistry</subject><subject>RNA, Messenger - metabolism</subject><subject>RNA, Transfer, Met - genetics</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1995</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtv1DAUhS0EKkNhyw7JK3aZ-voZL1hUI6CRKvpQkVA3lpPYJZDEwU5GHX49iWY0KquubN_znaNrHYTeA1kD0eyst_GMsjXQNaVCv0ArYJJmXEv6Eq0IIyIDwvPX6E1KvwgBDoKfoBMlgRDBV-ih6IYYtq7Gtqqmbmrt2IQe234ZjM22GXc4eBybMvzddS5h9zhEl9Js8DF02OLx9tt5VtplMt_wENqZi_MbF0WB5_AujC6-Ra-8bZN7dzhP0fcvn-82F9nl1ddic36ZVUxrnUnhSnCKUwJe1rYGIjUvSeUF8VTligvhHPW29KrONZU5SF_7WluwAlhZs1P0aZ87TGXn6sr1Y7StGWLT2bgzwTbmf6VvfpqHsDWMKAJy9n88-GP4M7k0mq5JlWtb27swJaMUy7mg_FmQEsKZoPAsCDJnQoslcb0HqxhSis4ftwZilq7N3LWhzAA1S9ez4cPTvx7xQ7mznu31Jo3u8Sjb-NtIxZQwFz_uzfXm9uae3WwMsH8ixrY9</recordid><startdate>19950625</startdate><enddate>19950625</enddate><creator>Thompson, James D.</creator><creator>Ayers, David F.</creator><creator>Malmstrom, Terra A.</creator><creator>McKenzie, Timothy L.</creator><creator>Ganousis, Louis</creator><creator>Chowrira, Bharat M.</creator><creator>Couture, Larry</creator><creator>Stinchcomb, Dan T.</creator><general>Oxford University Press</general><scope>BSCLL</scope><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>7QO</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>FR3</scope><scope>H94</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>19950625</creationdate><title>Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter</title><author>Thompson, James D. ; Ayers, David F. ; Malmstrom, Terra A. ; McKenzie, Timothy L. ; Ganousis, Louis ; Chowrira, Bharat M. ; Couture, Larry ; Stinchcomb, Dan T.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3999-65eb1e74201f6dad10694b0cf50f2787455ee2fabf7d8926816fdfd9a1a513bd3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1995</creationdate><topic>AIDS/HIV</topic><topic>Base Sequence</topic><topic>Blotting, Northern</topic><topic>Cell Line</topic><topic>Gene Expression</topic><topic>Human immunodeficiency virus</topic><topic>Humans</topic><topic>Molecular Sequence Data</topic><topic>Nucleic Acid Conformation</topic><topic>Plasmids</topic><topic>Promoter Regions, Genetic</topic><topic>Retroviridae - genetics</topic><topic>RNA Polymerase III - genetics</topic><topic>RNA Probes</topic><topic>RNA, Catalytic - genetics</topic><topic>RNA, Catalytic - metabolism</topic><topic>RNA, Messenger - chemistry</topic><topic>RNA, Messenger - metabolism</topic><topic>RNA, Transfer, Met - genetics</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Thompson, James D.</creatorcontrib><creatorcontrib>Ayers, David F.</creatorcontrib><creatorcontrib>Malmstrom, Terra A.</creatorcontrib><creatorcontrib>McKenzie, Timothy L.</creatorcontrib><creatorcontrib>Ganousis, Louis</creatorcontrib><creatorcontrib>Chowrira, Bharat M.</creatorcontrib><creatorcontrib>Couture, Larry</creatorcontrib><creatorcontrib>Stinchcomb, Dan T.</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</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>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Thompson, James D.</au><au>Ayers, David F.</au><au>Malmstrom, Terra A.</au><au>McKenzie, Timothy L.</au><au>Ganousis, Louis</au><au>Chowrira, Bharat M.</au><au>Couture, Larry</au><au>Stinchcomb, Dan T.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>1995-06-25</date><risdate>1995</risdate><volume>23</volume><issue>12</issue><spage>2259</spage><epage>2268</epage><pages>2259-2268</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><abstract>RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human met1 tRNA-derived pol III promoter >100-fold by modifying the 3′ terminus of the transcripts to hybridize to the 5′ terminus. This terminal duplex Includes the 8 nt leader sequence present in the primary wild-type met) tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed ‘TRZ’, was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>7610054</pmid><doi>10.1093/nar/23.12.2259</doi><tpages>10</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; PubMed Central; Oxford University Press Journals Digital Archive Legacy |
| subjects | AIDS/HIV Base Sequence Blotting, Northern Cell Line Gene Expression Human immunodeficiency virus Humans Molecular Sequence Data Nucleic Acid Conformation Plasmids Promoter Regions, Genetic Retroviridae - genetics RNA Polymerase III - genetics RNA Probes RNA, Catalytic - genetics RNA, Catalytic - metabolism RNA, Messenger - chemistry RNA, Messenger - metabolism RNA, Transfer, Met - genetics |
| title | Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter |
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