cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity
| Autor: | Karsten Rothbarth, Ruth Greferath, Tova Glaser, Eberhard Spiess, Thomas Keck, Peter Nehls, Hermann Stammer, Dieter Werner |
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| Rok vydání: | 1998 |
| Předmět: |
DNA
Complementary Library Molecular Sequence Data Gene Expression Biology Antibodies law.invention Mice chemistry.chemical_compound law Complementary DNA Genetics Animals Humans Amino Acid Sequence Cloning Molecular Antiserum Sequence Homology Amino Acid cDNA library Molecular biology Recombinant Proteins DNA-Binding Proteins genomic DNA chemistry Polyclonal antibodies Recombinant DNA biology.protein Peptides DNA Research Article |
| Zdroj: | Nucleic Acids Research. 26:1160-1166 |
| ISSN: | 1362-4962 |
| Popis: | 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 lambdagt11 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. by125Iodine labeling and SDS-PAGE. |
| Databáze: | OpenAIRE |
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