A procedure for the isolation of specific point mutants of influenza virus
Identifieur interne : 001582 ( Istex/Corpus ); précédent : 001581; suivant : 001583A procedure for the isolation of specific point mutants of influenza virus
Auteurs : Bradley M. Mitchell ; Dan C. DebordeSource :
- Journal of Virological Methods [ 0166-0934 ] ; 1993.
English descriptors
- Teeft :
- Allantoic fluid, Amplification, Congenic influenza virus, Consensus sequence, Deborde, Dextran sulfate, Embryonated, Embryonated chicken eggs, Embryonated eggs, Embryonated eggs inoculated, Enami, Equal volume, Gene sequence, Growth advantage, Hybridization, Individual eggs, Infectious virions, Infectious virus, Influenza, Influenza virus, Influenza viruses, Inoculum, Maassab, Major sequence, Mdck, Mdck cells, Mutant, Mutant virus, Mutation, Nitrocellulose paper, Oligodeoxynucleotide, Oligodeoxynucleotide probe, Palese, Particular probe, Phenotypic significance, Plaque, Point mutation, Polynucleotide kinase, Positive hybridization signal, Positive signal, Prehybridization buffer, Primer extension, Probe, Probe panel, Rare point, Rare sequence, Rare transfectant, Relative abundance, Room temperature, Screen vrna, Screening step, Selective conditions, Selective pressure, Sequencing, Several rounds, Single base, Sodium acetate, Specific point mutants, Temperature sensitivity, Tissue culture cells, Viral, Virion, Virus, Virus sample, Vrna.
Abstract
Abstract: Procedures were developed for the screening and isolation of RNA viruses that vary from the consensus population by a single point mutation and are present in low abundance. We tested these procedures using a mixture of the vaccine donor line cold-adapted (ca) B/Ann Arbor/1/66 and its wild type (wt) progenitor at a ratio of 1,000:1. A 13-mer oligodeoxynucleotide was prepared as a [32P]-radiolabeled probe which specifically hybridized to wt viral RNA (vRNA) at a region that varied between the ca and wt sequence by a single base at position 1,320 of the PA gene. This probe was able to detect as little as 88 pg of total wt vRNA blotted to nitrocellulose, while giving no positive signal with as much as 1 μg of total ca vRNA. We were able to isolate virus containing the wt PA gene sequence from the mixed pool of ca and wt viruses by two successive rounds of amplification in embryonated eggs inoculated with a controlled number of infectious virions. During each round of amplification the abundance of the virus containing the wt PA gene sequence was increased about ten-fold. Once a relative abundance of 1:10 was reached, the virus was cloned by plaquing in Madin-Darby canine kidney cells. These procedures, which allow the isolation of specific point mutants, utilize no selective pressure conditions and are suitable for analyzing the phenotypic importance of known mutations in biologically important viruses.
Url:
DOI: 10.1016/0166-0934(93)90031-L
Links to Exploration step
ISTEX:23AF5E6ED1B730CEAE73718BA765DA331125E0B6Le document en format XML
<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>A procedure for the isolation of specific point mutants of influenza virus</title><author><name sortKey="Mitchell, Bradley M" sort="Mitchell, Bradley M" uniqKey="Mitchell B" first="Bradley M." last="Mitchell">Bradley M. Mitchell</name><affiliation><mods:affiliation>Division of Biological Sciences, University of Montana, Missoula, MTUSA</mods:affiliation></affiliation></author><author><name sortKey="Deborde, Dan C" sort="Deborde, Dan C" uniqKey="Deborde D" first="Dan C." last="Deborde">Dan C. Deborde</name><affiliation><mods:affiliation>Division of Biological Sciences, University of Montana, Missoula, MTUSA</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:23AF5E6ED1B730CEAE73718BA765DA331125E0B6</idno><date when="1993" year="1993">1993</date><idno type="doi">10.1016/0166-0934(93)90031-L</idno><idno type="url">https://api.istex.fr/ark:/67375/6H6-KLP9P48M-J/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001582</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001582</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a">A procedure for the isolation of specific point mutants of influenza virus</title><author><name sortKey="Mitchell, Bradley M" sort="Mitchell, Bradley M" uniqKey="Mitchell B" first="Bradley M." last="Mitchell">Bradley M. Mitchell</name><affiliation><mods:affiliation>Division of Biological Sciences, University of Montana, Missoula, MTUSA</mods:affiliation></affiliation></author><author><name sortKey="Deborde, Dan C" sort="Deborde, Dan C" uniqKey="Deborde D" first="Dan C." last="Deborde">Dan C. Deborde</name><affiliation><mods:affiliation>Division of Biological Sciences, University of Montana, Missoula, MTUSA</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j">Journal of Virological Methods</title><title level="j" type="abbrev">VIRMET</title><idno type="ISSN">0166-0934</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1993">1993</date><biblScope unit="volume">42</biblScope><biblScope unit="issue">2–3</biblScope><biblScope unit="page" from="181">181</biblScope><biblScope unit="page" to="191">191</biblScope></imprint><idno type="ISSN">0166-0934</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0166-0934</idno></seriesStmt></fileDesc><profileDesc><textClass><keywords scheme="Teeft" xml:lang="en"><term>Allantoic fluid</term><term>Amplification</term><term>Congenic influenza virus</term><term>Consensus sequence</term><term>Deborde</term><term>Dextran sulfate</term><term>Embryonated</term><term>Embryonated chicken eggs</term><term>Embryonated eggs</term><term>Embryonated eggs inoculated</term><term>Enami</term><term>Equal volume</term><term>Gene sequence</term><term>Growth advantage</term><term>Hybridization</term><term>Individual eggs</term><term>Infectious virions</term><term>Infectious virus</term><term>Influenza</term><term>Influenza virus</term><term>Influenza viruses</term><term>Inoculum</term><term>Maassab</term><term>Major sequence</term><term>Mdck</term><term>Mdck cells</term><term>Mutant</term><term>Mutant virus</term><term>Mutation</term><term>Nitrocellulose paper</term><term>Oligodeoxynucleotide</term><term>Oligodeoxynucleotide probe</term><term>Palese</term><term>Particular probe</term><term>Phenotypic significance</term><term>Plaque</term><term>Point mutation</term><term>Polynucleotide kinase</term><term>Positive hybridization signal</term><term>Positive signal</term><term>Prehybridization buffer</term><term>Primer extension</term><term>Probe</term><term>Probe panel</term><term>Rare point</term><term>Rare sequence</term><term>Rare transfectant</term><term>Relative abundance</term><term>Room temperature</term><term>Screen vrna</term><term>Screening step</term><term>Selective conditions</term><term>Selective pressure</term><term>Sequencing</term><term>Several rounds</term><term>Single base</term><term>Sodium acetate</term><term>Specific point mutants</term><term>Temperature sensitivity</term><term>Tissue culture cells</term><term>Viral</term><term>Virion</term><term>Virus</term><term>Virus sample</term><term>Vrna</term></keywords></textClass><langUsage><language ident="en">en</language></langUsage></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">Abstract: Procedures were developed for the screening and isolation of RNA viruses that vary from the consensus population by a single point mutation and are present in low abundance. We tested these procedures using a mixture of the vaccine donor line cold-adapted (ca) B/Ann Arbor/1/66 and its wild type (wt) progenitor at a ratio of 1,000:1. A 13-mer oligodeoxynucleotide was prepared as a [32P]-radiolabeled probe which specifically hybridized to wt viral RNA (vRNA) at a region that varied between the ca and wt sequence by a single base at position 1,320 of the PA gene. This probe was able to detect as little as 88 pg of total wt vRNA blotted to nitrocellulose, while giving no positive signal with as much as 1 μg of total ca vRNA. We were able to isolate virus containing the wt PA gene sequence from the mixed pool of ca and wt viruses by two successive rounds of amplification in embryonated eggs inoculated with a controlled number of infectious virions. During each round of amplification the abundance of the virus containing the wt PA gene sequence was increased about ten-fold. Once a relative abundance of 1:10 was reached, the virus was cloned by plaquing in Madin-Darby canine kidney cells. These procedures, which allow the isolation of specific point mutants, utilize no selective pressure conditions and are suitable for analyzing the phenotypic importance of known mutations in biologically important viruses.</div></front></TEI><istex><corpusName>elsevier</corpusName><keywords><teeft><json:string>vrna</json:string><json:string>mutation</json:string><json:string>mutant</json:string><json:string>deborde</json:string><json:string>embryonated</json:string><json:string>hybridization</json:string><json:string>maassab</json:string><json:string>virion</json:string><json:string>viral</json:string><json:string>oligodeoxynucleotide</json:string><json:string>sequencing</json:string><json:string>enami</json:string><json:string>mdck</json:string><json:string>palese</json:string><json:string>inoculum</json:string><json:string>infectious virions</json:string><json:string>influenza</json:string><json:string>relative abundance</json:string><json:string>influenza virus</json:string><json:string>influenza viruses</json:string><json:string>positive signal</json:string><json:string>virus</json:string><json:string>individual eggs</json:string><json:string>mutant virus</json:string><json:string>nitrocellulose paper</json:string><json:string>embryonated eggs</json:string><json:string>oligodeoxynucleotide probe</json:string><json:string>selective conditions</json:string><json:string>mdck cells</json:string><json:string>allantoic fluid</json:string><json:string>embryonated eggs inoculated</json:string><json:string>several rounds</json:string><json:string>growth advantage</json:string><json:string>gene sequence</json:string><json:string>virus sample</json:string><json:string>single base</json:string><json:string>amplification</json:string><json:string>plaque</json:string><json:string>probe</json:string><json:string>rare transfectant</json:string><json:string>embryonated chicken eggs</json:string><json:string>specific point mutants</json:string><json:string>temperature sensitivity</json:string><json:string>sodium acetate</json:string><json:string>equal volume</json:string><json:string>room temperature</json:string><json:string>congenic influenza virus</json:string><json:string>polynucleotide kinase</json:string><json:string>prehybridization buffer</json:string><json:string>dextran sulfate</json:string><json:string>screening step</json:string><json:string>primer extension</json:string><json:string>rare point</json:string><json:string>phenotypic significance</json:string><json:string>selective pressure</json:string><json:string>probe panel</json:string><json:string>point mutation</json:string><json:string>tissue culture cells</json:string><json:string>screen vrna</json:string><json:string>consensus sequence</json:string><json:string>infectious virus</json:string><json:string>particular probe</json:string><json:string>rare sequence</json:string><json:string>major sequence</json:string><json:string>positive hybridization signal</json:string></teeft></keywords><author><json:item><name>Bradley M. Mitchell</name><affiliations><json:string>Division of Biological Sciences, University of Montana, Missoula, MTUSA</json:string></affiliations></json:item><json:item><name>Dan C. DeBorde</name><affiliations><json:string>Division of Biological Sciences, University of Montana, Missoula, MTUSA</json:string></affiliations></json:item></author><subject><json:item><lang><json:string>eng</json:string></lang><value>Influenza type B virus</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Oligodeoxynucleotide probe</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Hybridization</value></json:item><json:item><lang><json:string>eng</json:string></lang><value>Point mutant</value></json:item></subject><arkIstex>ark:/67375/6H6-KLP9P48M-J</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>Full-length article</json:string></originalGenre><abstract>Abstract: Procedures were developed for the screening and isolation of RNA viruses that vary from the consensus population by a single point mutation and are present in low abundance. We tested these procedures using a mixture of the vaccine donor line cold-adapted (ca) B/Ann Arbor/1/66 and its wild type (wt) progenitor at a ratio of 1,000:1. A 13-mer oligodeoxynucleotide was prepared as a [32P]-radiolabeled probe which specifically hybridized to wt viral RNA (vRNA) at a region that varied between the ca and wt sequence by a single base at position 1,320 of the PA gene. This probe was able to detect as little as 88 pg of total wt vRNA blotted to nitrocellulose, while giving no positive signal with as much as 1 μg of total ca vRNA. We were able to isolate virus containing the wt PA gene sequence from the mixed pool of ca and wt viruses by two successive rounds of amplification in embryonated eggs inoculated with a controlled number of infectious virions. During each round of amplification the abundance of the virus containing the wt PA gene sequence was increased about ten-fold. Once a relative abundance of 1:10 was reached, the virus was cloned by plaquing in Madin-Darby canine kidney cells. These procedures, which allow the isolation of specific point mutants, utilize no selective pressure conditions and are suitable for analyzing the phenotypic importance of known mutations in biologically important viruses.</abstract><qualityIndicators><score>8.675</score><pdfWordCount>3855</pdfWordCount><pdfCharCount>22919</pdfCharCount><pdfVersion>1.4</pdfVersion><pdfPageCount>11</pdfPageCount><pdfPageSize>432 x 612 pts</pdfPageSize><refBibsNative>true</refBibsNative><abstractWordCount>235</abstractWordCount><abstractCharCount>1433</abstractCharCount><keywordCount>4</keywordCount></qualityIndicators><title>A procedure for the isolation of specific point mutants of influenza virus</title><pmid><json:string>8514840</json:string></pmid><pii><json:string>0166-0934(93)90031-L</json:string></pii><genre><json:string>research-article</json:string></genre><serie><title>Proc. 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USA</title><language><json:string>unknown</json:string></language><volume>87</volume><pages><first>3802</first><last>3805</last></pages></serie><host><title>Journal of Virological Methods</title><language><json:string>unknown</json:string></language><publicationDate>1993</publicationDate><issn><json:string>0166-0934</json:string></issn><pii><json:string>S0166-0934(00)X0212-1</json:string></pii><volume>42</volume><issue>2–3</issue><pages><first>181</first><last>191</last></pages><genre><json:string>journal</json:string></genre></host><namedEntities><unitex><date><json:string>1993</json:string><json:string>3000</json:string></date><geogName></geogName><orgName><json:string>N International, Redmond</json:string><json:string>University of Montana</json:string><json:string>University of Montana Molecular Biology Facility</json:string><json:string>American Type Culture Collection</json:string><json:string>National Institute of Allergy</json:string><json:string>Infectious Diseases Grant</json:string></orgName><orgName_funder><json:string>National Institute of Allergy</json:string><json:string>Infectious Diseases Grant</json:string></orgName_funder><orgName_provider></orgName_provider><persName><json:string>Michael Minnick</json:string><json:string>H.F. Maassab</json:string><json:string>B. Panel</json:string><json:string>Dan C. DeBorde</json:string><json:string>Ann Arbor</json:string><json:string>George Card</json:string><json:string>Speedvac</json:string></persName><placeName></placeName><ref_url></ref_url><ref_bibl><json:string>Enami and Palese, 1991</json:string><json:string>Appleyard and Maber, 1974</json:string><json:string>Luytjes et al., 1989</json:string><json:string>Maassab and DeBorde (1985)</json:string><json:string>Zeff and Geliebter (1987)</json:string><json:string>Zeff and Geliebter, 1987</json:string><json:string>Wahl et al., 1979</json:string><json:string>Donabedian et al., 1987</json:string><json:string>Geliebter, 1987</json:string><json:string>Thomas, 1983</json:string><json:string>DeBorde et al. (1986)</json:string><json:string>Muster et al., 1991</json:string><json:string>Odagiri et al., 1982</json:string><json:string>DeBorde et al., 1988</json:string><json:string>Enami et al., 1990</json:string><json:string>Steinhauer and Holland, 1987</json:string><json:string>Mills and Chanock, 1971</json:string><json:string>Parvin et al., 1986</json:string><json:string>Wardlaw, 1985</json:string><json:string>DeBorde et al., 1986</json:string></ref_bibl><bibl></bibl></unitex></namedEntities><ark><json:string>ark:/67375/6H6-KLP9P48M-J</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - virology</json:string><json:string>2 - biotechnology & applied microbiology</json:string><json:string>2 - biochemical research methods</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - virology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Immunology and Microbiology</json:string><json:string>3 - Virology</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>1993</publicationDate><copyrightDate>1993</copyrightDate><doi><json:string>10.1016/0166-0934(93)90031-L</json:string></doi><id>23AF5E6ED1B730CEAE73718BA765DA331125E0B6</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-KLP9P48M-J/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-KLP9P48M-J/bundle.zip</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/6H6-KLP9P48M-J/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a">A procedure for the isolation of specific point mutants of influenza virus</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher scheme="https://scientific-publisher.data.istex.fr">ELSEVIER</publisher><availability><licence><p>elsevier</p></licence></availability><p scheme="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M"></p><date>1993</date></publicationStmt><notesStmt><note type="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="journal" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="inbook"><analytic><title level="a">A procedure for the isolation of specific point mutants of influenza virus</title><author xml:id="author-0000"><persName><forename type="first">Bradley M.</forename><surname>Mitchell</surname></persName><affiliation>Division of Biological Sciences, University of Montana, Missoula, MTUSA</affiliation></author><author xml:id="author-0001"><persName><forename type="first">Dan C.</forename><surname>DeBorde</surname></persName><note type="correspondence"><p>Correspondence to: Dan C. DeBorde, Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA.</p></note><affiliation>Division of Biological Sciences, University of Montana, Missoula, MTUSA</affiliation></author><idno type="istex">23AF5E6ED1B730CEAE73718BA765DA331125E0B6</idno><idno type="ark">ark:/67375/6H6-KLP9P48M-J</idno><idno type="DOI">10.1016/0166-0934(93)90031-L</idno><idno type="PII">0166-0934(93)90031-L</idno></analytic><monogr><title level="j">Journal of Virological Methods</title><title level="j" type="abbrev">VIRMET</title><idno type="pISSN">0166-0934</idno><idno type="PII">S0166-0934(00)X0212-1</idno><imprint><publisher>ELSEVIER</publisher><date type="published" when="1993"></date><biblScope unit="volume">42</biblScope><biblScope unit="issue">2–3</biblScope><biblScope unit="page" from="181">181</biblScope><biblScope unit="page" to="191">191</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><profileDesc><creation><date>1993</date></creation><langUsage><language ident="en">en</language></langUsage><abstract xml:lang="en"><p>Abstract: Procedures were developed for the screening and isolation of RNA viruses that vary from the consensus population by a single point mutation and are present in low abundance. We tested these procedures using a mixture of the vaccine donor line cold-adapted (ca) B/Ann Arbor/1/66 and its wild type (wt) progenitor at a ratio of 1,000:1. A 13-mer oligodeoxynucleotide was prepared as a [32P]-radiolabeled probe which specifically hybridized to wt viral RNA (vRNA) at a region that varied between the ca and wt sequence by a single base at position 1,320 of the PA gene. This probe was able to detect as little as 88 pg of total wt vRNA blotted to nitrocellulose, while giving no positive signal with as much as 1 μg of total ca vRNA. We were able to isolate virus containing the wt PA gene sequence from the mixed pool of ca and wt viruses by two successive rounds of amplification in embryonated eggs inoculated with a controlled number of infectious virions. During each round of amplification the abundance of the virus containing the wt PA gene sequence was increased about ten-fold. Once a relative abundance of 1:10 was reached, the virus was cloned by plaquing in Madin-Darby canine kidney cells. These procedures, which allow the isolation of specific point mutants, utilize no selective pressure conditions and are suitable for analyzing the phenotypic importance of known mutations in biologically important viruses.</p></abstract><textClass><keywords scheme="keyword"><list><head>Keywords</head><item><term>Influenza type B virus</term></item><item><term>Oligodeoxynucleotide probe</term></item><item><term>Hybridization</term></item><item><term>Point mutant</term></item></list></keywords></textClass></profileDesc><revisionDesc><change when="1993">Published</change></revisionDesc></teiHeader></istex:fulltextTEI><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-KLP9P48M-J/fulltext.txt</uri></json:item></fulltext><metadata><istex:metadataXml wicri:clean="Elsevier, elements deleted: 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:docType><istex:document><converted-article version="4.5.2" docsubtype="fla"><item-info><jid>VIRMET</jid><aid>9390031L</aid><ce:pii>0166-0934(93)90031-L</ce:pii><ce:doi>10.1016/0166-0934(93)90031-L</ce:doi><ce:copyright type="unknown" year="1993"></ce:copyright></item-info><head><ce:title>A procedure for the isolation of specific point mutants of influenza virus</ce:title><ce:author-group><ce:author><ce:given-name>Bradley M.</ce:given-name><ce:surname>Mitchell</ce:surname></ce:author><ce:author><ce:given-name>Dan C.</ce:given-name><ce:surname>DeBorde</ce:surname><ce:cross-ref refid="COR1"><ce:sup>∗</ce:sup></ce:cross-ref></ce:author><ce:affiliation><ce:textfn>Division of Biological Sciences, University of Montana, Missoula, MTUSA</ce:textfn></ce:affiliation><ce:correspondence id="COR1"><ce:label>∗</ce:label><ce:text>Correspondence to: Dan C. DeBorde, Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA.</ce:text></ce:correspondence></ce:author-group><ce:date-accepted day="5" month="11" year="1992"></ce:date-accepted><ce:abstract><ce:section-title>Abstract</ce:section-title><ce:abstract-sec><ce:simple-para>Procedures were developed for the screening and isolation of RNA viruses that vary from the consensus population by a single point mutation and are present in low abundance. We tested these procedures using a mixture of the vaccine donor line cold-adapted (ca) B/Ann Arbor/1/66 and its wild type (wt) progenitor at a ratio of 1,000:1. A 13-mer oligodeoxynucleotide was prepared as a [<ce:sup loc="pre">32</ce:sup>P]-radiolabeled probe which specifically hybridized to wt viral RNA (vRNA) at a region that varied between the ca and wt sequence by a single base at position 1,320 of the PA gene. This probe was able to detect as little as 88 pg of total wt vRNA blotted to nitrocellulose, while giving no positive signal with as much as 1 μg of total ca vRNA. We were able to isolate virus containing the wt PA gene sequence from the mixed pool of ca and wt viruses by two successive rounds of amplification in embryonated eggs inoculated with a controlled number of infectious virions. During each round of amplification the abundance of the virus containing the wt PA gene sequence was increased about ten-fold. Once a relative abundance of 1:10 was reached, the virus was cloned by plaquing in Madin-Darby canine kidney cells. These procedures, which allow the isolation of specific point mutants, utilize no selective pressure conditions and are suitable for analyzing the phenotypic importance of known mutations in biologically important viruses.</ce:simple-para></ce:abstract-sec></ce:abstract><ce:keywords><ce:section-title>Keywords</ce:section-title><ce:keyword><ce:text>Influenza type B virus</ce:text></ce:keyword><ce:keyword><ce:text>Oligodeoxynucleotide probe</ce:text></ce:keyword><ce:keyword><ce:text>Hybridization</ce:text></ce:keyword><ce:keyword><ce:text>Point mutant</ce:text></ce:keyword></ce:keywords></head></converted-article></istex:document></istex:metadataXml><mods version="3.6"><titleInfo><title>A procedure for the isolation of specific point mutants of influenza virus</title></titleInfo><titleInfo type="alternative" contentType="CDATA"><title>A procedure for the isolation of specific point mutants of influenza virus</title></titleInfo><name type="personal"><namePart type="given">Bradley M.</namePart><namePart type="family">Mitchell</namePart><affiliation>Division of Biological Sciences, University of Montana, Missoula, MTUSA</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="given">Dan C.</namePart><namePart type="family">DeBorde</namePart><affiliation>Division of Biological Sciences, University of Montana, Missoula, MTUSA</affiliation><description>Correspondence to: Dan C. DeBorde, Division of Biological Sciences, University of Montana, Missoula, MT 59812, USA.</description><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="Full-length article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1993</dateIssued><copyrightDate encoding="w3cdtf">1993</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract lang="en">Abstract: Procedures were developed for the screening and isolation of RNA viruses that vary from the consensus population by a single point mutation and are present in low abundance. We tested these procedures using a mixture of the vaccine donor line cold-adapted (ca) B/Ann Arbor/1/66 and its wild type (wt) progenitor at a ratio of 1,000:1. A 13-mer oligodeoxynucleotide was prepared as a [32P]-radiolabeled probe which specifically hybridized to wt viral RNA (vRNA) at a region that varied between the ca and wt sequence by a single base at position 1,320 of the PA gene. This probe was able to detect as little as 88 pg of total wt vRNA blotted to nitrocellulose, while giving no positive signal with as much as 1 μg of total ca vRNA. We were able to isolate virus containing the wt PA gene sequence from the mixed pool of ca and wt viruses by two successive rounds of amplification in embryonated eggs inoculated with a controlled number of infectious virions. During each round of amplification the abundance of the virus containing the wt PA gene sequence was increased about ten-fold. Once a relative abundance of 1:10 was reached, the virus was cloned by plaquing in Madin-Darby canine kidney cells. These procedures, which allow the isolation of specific point mutants, utilize no selective pressure conditions and are suitable for analyzing the phenotypic importance of known mutations in biologically important viruses.</abstract><subject><genre>Keywords</genre><topic>Influenza type B virus</topic><topic>Oligodeoxynucleotide probe</topic><topic>Hybridization</topic><topic>Point mutant</topic></subject><relatedItem type="host"><titleInfo><title>Journal of Virological Methods</title></titleInfo><titleInfo type="abbreviated"><title>VIRMET</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><originInfo><publisher>ELSEVIER</publisher><dateIssued encoding="w3cdtf">1993</dateIssued></originInfo><identifier type="ISSN">0166-0934</identifier><identifier type="PII">S0166-0934(00)X0212-1</identifier><part><date>1993</date><detail type="volume"><number>42</number><caption>vol.</caption></detail><detail type="issue"><number>2–3</number><caption>no.</caption></detail><extent unit="issue-pages"><start>137</start><end>366</end></extent><extent unit="pages"><start>181</start><end>191</end></extent></part></relatedItem><identifier type="istex">23AF5E6ED1B730CEAE73718BA765DA331125E0B6</identifier><identifier type="ark">ark:/67375/6H6-KLP9P48M-J</identifier><identifier type="DOI">10.1016/0166-0934(93)90031-L</identifier><identifier type="PII">0166-0934(93)90031-L</identifier><recordInfo><recordContentSource authority="ISTEX" authorityURI="https://loaded-corpus.data.istex.fr" valueURI="https://loaded-corpus.data.istex.fr/ark:/67375/XBH-HKKZVM7B-M">elsevier</recordContentSource></recordInfo></mods><json:item><extension>json</extension><original>false</original><mimetype>application/json</mimetype><uri>https://api.istex.fr/ark:/67375/6H6-KLP9P48M-J/record.json</uri></json:item></metadata></istex></record>Pour manipuler ce document sous Unix (Dilib)
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