cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity
Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization...
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| Veröffentlicht in: | Nucleic acids research 1998-03, Vol.26 (5), p.1160-1166 |
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| container_title | Nucleic acids research |
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| creator | Keck, Thomas Glaser, Tova Rothbarth, Karsten Stammer, Hermann Werner, Dieter Spiess, Eberhard Nehls, Peter Greferath, Ruth |
| description | Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine λgt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by 125Iodine labeling and SDS-PAGE. |
| doi_str_mv | 10.1093/nar/26.5.1160 |
| format | Article |
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Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine λgt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by 125Iodine labeling and SDS-PAGE.</description><identifier>ISSN: 0305-1048</identifier><identifier>ISSN: 1362-4962</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/26.5.1160</identifier><identifier>PMID: 9469821</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Amino Acid Sequence ; Animals ; Antibodies ; Cloning, Molecular ; DNA, Complementary - genetics ; DNA-Binding Proteins - genetics ; DNA-Binding Proteins - immunology ; DNA-Binding Proteins - metabolism ; Gene Expression ; Humans ; Mice ; Molecular Sequence Data ; Peptides - genetics ; Peptides - immunology ; Peptides - metabolism ; Recombinant Proteins - genetics ; Recombinant Proteins - immunology ; Recombinant Proteins - metabolism ; Sequence Homology, Amino Acid</subject><ispartof>Nucleic acids research, 1998-03, Vol.26 (5), p.1160-1166</ispartof><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c3650-7057a8e71c9ff46f52249611a0ccb86cbf5440efee73c014a56fa983092c84ea3</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/PMC147382/pdf/$$EPDF$$P50$$Gpubmedcentral$$H</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC147382/$$EHTML$$P50$$Gpubmedcentral$$H</linktohtml><link.rule.ids>230,314,727,780,784,885,27924,27925,53791,53793</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/9469821$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Keck, Thomas</creatorcontrib><creatorcontrib>Glaser, Tova</creatorcontrib><creatorcontrib>Rothbarth, Karsten</creatorcontrib><creatorcontrib>Stammer, Hermann</creatorcontrib><creatorcontrib>Werner, Dieter</creatorcontrib><creatorcontrib>Spiess, Eberhard</creatorcontrib><creatorcontrib>Nehls, Peter</creatorcontrib><creatorcontrib>Greferath, Ruth</creatorcontrib><title>cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Research</addtitle><description>Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine λgt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by 125Iodine labeling and SDS-PAGE.</description><subject>Amino Acid Sequence</subject><subject>Animals</subject><subject>Antibodies</subject><subject>Cloning, Molecular</subject><subject>DNA, Complementary - genetics</subject><subject>DNA-Binding Proteins - genetics</subject><subject>DNA-Binding Proteins - immunology</subject><subject>DNA-Binding Proteins - metabolism</subject><subject>Gene Expression</subject><subject>Humans</subject><subject>Mice</subject><subject>Molecular Sequence Data</subject><subject>Peptides - genetics</subject><subject>Peptides - immunology</subject><subject>Peptides - metabolism</subject><subject>Recombinant Proteins - genetics</subject><subject>Recombinant Proteins - immunology</subject><subject>Recombinant Proteins - metabolism</subject><subject>Sequence Homology, Amino Acid</subject><issn>0305-1048</issn><issn>1362-4962</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1998</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkUtvEzEUhS1EVUJhyRJpVqyY1O-ZWbCoyiOVqrLhUbGxbpzrxjCxp7YDDb-eGTUKdMXK0j3fOfLRIeQFo3NGO3EaIJ1yPVdzxjR9RGZMaF7LTvPHZEYFVTWjsn1Cnub8nVImmZLH5LiTums5mxFn316dVbaPwYeb11VCGzdLHyCUCu-GhDn7GCoIq8quIYEtmPxvKNMxumqI_W7A4leYq1--rEePxWFSoa-mYHDOB192z8iRgz7j8_17Qj6_f_fpfFFffvxwcX52WVuhFa0bqhposWG2c05qpzgfmzAG1Nplq-3SKSkpOsRG2LEMKO2gawXtuG0lgjghb-5zh-1ygyuLoSTozZD8BtLORPDmoRL82tzEn4bJRrR89L_a-1O83WIuZuOzxb6HgHGbTdM1lClF_wsyLRRl3QTW96BNMeeE7vAZRs00oBkHNFwbZaYBR_7lvw0O9H6xv3k-F7w7yJB-GN2IRpnF9TdDv1wvvkqujBJ_AHTKqFA</recordid><startdate>199803</startdate><enddate>199803</enddate><creator>Keck, Thomas</creator><creator>Glaser, Tova</creator><creator>Rothbarth, Karsten</creator><creator>Stammer, Hermann</creator><creator>Werner, Dieter</creator><creator>Spiess, Eberhard</creator><creator>Nehls, Peter</creator><creator>Greferath, Ruth</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>7TM</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>199803</creationdate><title>cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity</title><author>Keck, Thomas ; Glaser, Tova ; Rothbarth, Karsten ; Stammer, Hermann ; Werner, Dieter ; Spiess, Eberhard ; Nehls, Peter ; Greferath, Ruth</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c3650-7057a8e71c9ff46f52249611a0ccb86cbf5440efee73c014a56fa983092c84ea3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1998</creationdate><topic>Amino Acid Sequence</topic><topic>Animals</topic><topic>Antibodies</topic><topic>Cloning, Molecular</topic><topic>DNA, Complementary - genetics</topic><topic>DNA-Binding Proteins - genetics</topic><topic>DNA-Binding Proteins - immunology</topic><topic>DNA-Binding Proteins - metabolism</topic><topic>Gene Expression</topic><topic>Humans</topic><topic>Mice</topic><topic>Molecular Sequence Data</topic><topic>Peptides - genetics</topic><topic>Peptides - immunology</topic><topic>Peptides - metabolism</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - immunology</topic><topic>Recombinant Proteins - metabolism</topic><topic>Sequence Homology, Amino Acid</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Keck, Thomas</creatorcontrib><creatorcontrib>Glaser, Tova</creatorcontrib><creatorcontrib>Rothbarth, Karsten</creatorcontrib><creatorcontrib>Stammer, Hermann</creatorcontrib><creatorcontrib>Werner, Dieter</creatorcontrib><creatorcontrib>Spiess, Eberhard</creatorcontrib><creatorcontrib>Nehls, Peter</creatorcontrib><creatorcontrib>Greferath, Ruth</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>Nucleic Acids 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>Keck, Thomas</au><au>Glaser, Tova</au><au>Rothbarth, Karsten</au><au>Stammer, Hermann</au><au>Werner, Dieter</au><au>Spiess, Eberhard</au><au>Nehls, Peter</au><au>Greferath, Ruth</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Research</addtitle><date>1998-03</date><risdate>1998</risdate><volume>26</volume><issue>5</issue><spage>1160</spage><epage>1166</epage><pages>1160-1166</pages><issn>0305-1048</issn><issn>1362-4962</issn><eissn>1362-4962</eissn><abstract>Polypeptides remaining tightly associated with isolated genomic DNA are of interest with respect to their potential involvement in the topological organization and/or function of genomic DNA. Such residual DNA-polypeptide complexes were used for raising monoclonal antibodies by in vitro immunization. Screening of a murine λgt11 cDNA library with these antibodies released a positive cDNA (MC1D) encoding a 16 kDa polypeptide. The cloned homologous human cDNA (HC1D) was identified in the dbest data base by partial sequence comparison, and it was sequenced full length. The cDNA-derived amino acid sequences comprise nuclear location signals but none of the known DNA-binding motifs. However, the recombinantly expressed proteins show in vitro DNA binding affinities. A polyclonal antiserum to the recombinant MC1D protein immunostains sub-nuclear structures, and it detects a residual 16 kDa polypeptide on western blots of DNA digests. These results support the conclusion that the cloned cDNAs reflect mRNAs encoding one of the chemically-resistant polypeptides which can be detected in isolated genomic DNA by sensitive techniques, e.g. by 125Iodine labeling and SDS-PAGE.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>9469821</pmid><doi>10.1093/nar/26.5.1160</doi><tpages>7</tpages><oa>free_for_read</oa></addata></record> |
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| source | MEDLINE; Access via Oxford University Press (Open Access Collection); PubMed Central; Free Full-Text Journals in Chemistry |
| subjects | Amino Acid Sequence Animals Antibodies Cloning, Molecular DNA, Complementary - genetics DNA-Binding Proteins - genetics DNA-Binding Proteins - immunology DNA-Binding Proteins - metabolism Gene Expression Humans Mice Molecular Sequence Data Peptides - genetics Peptides - immunology Peptides - metabolism Recombinant Proteins - genetics Recombinant Proteins - immunology Recombinant Proteins - metabolism Sequence Homology, Amino Acid |
| title | cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity |
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