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. 1998 Mar 1;26(5):1160–1166. doi: 10.1093/nar/26.5.1160

cDNA cloning, recombinant expression and characterization of polypetides with exceptional DNA affinity.

P Nehls 1, T Keck 1, R Greferath 1, E Spiess 1, T Glaser 1, K Rothbarth 1, H Stammer 1, D Werner 1
PMCID: PMC147382  PMID: 9469821

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 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.

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Selected References

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  1. Avramova Z., Tsanev R. Stable DNA-protein complexes in eukaryotic chromatin. J Mol Biol. 1987 Jul 20;196(2):437–440. doi: 10.1016/0022-2836(87)90704-2. [DOI] [PubMed] [Google Scholar]
  2. Eils R., Bertin E., Saracoglu K., Rinke B., Schröck E., Parazza F., Usson Y., Robert-Nicoud M., Stelzer E. H., Chassery J. M. Application of confocal laser microscopy and three-dimensional Voronoi diagrams for volume and surface estimates of interphase chromosomes. J Microsc. 1995 Feb;177(Pt 2):150–161. doi: 10.1111/j.1365-2818.1995.tb03545.x. [DOI] [PubMed] [Google Scholar]
  3. Gross-Bellard M., Oudet P., Chambon P. Isolation of high-molecular-weight DNA from mammalian cells. Eur J Biochem. 1973 Jul 2;36(1):32–38. doi: 10.1111/j.1432-1033.1973.tb02881.x. [DOI] [PubMed] [Google Scholar]
  4. Hendrich B. D., Willard H. F. Epigenetic regulation of gene expression: the effect of altered chromatin structure from yeast to mammals. Hum Mol Genet. 1995;4(Spec No):1765–1777. doi: 10.1093/hmg/4.suppl_1.1765. [DOI] [PubMed] [Google Scholar]
  5. Juodka B., Pfütz M., Werner D. Chemical and enzymatic analysis of covalent bonds between peptides and chromosomal DNA. Nucleic Acids Res. 1991 Dec 11;19(23):6391–6398. doi: 10.1093/nar/19.23.6391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Krauth W., Werner D. Analysis of the most tightly bound proteins in eukaryotic DNA. Biochim Biophys Acta. 1979 Oct 25;564(3):390–401. doi: 10.1016/0005-2787(79)90030-3. [DOI] [PubMed] [Google Scholar]
  7. Kurz A., Lampel S., Nickolenko J. E., Bradl J., Benner A., Zirbel R. M., Cremer T., Lichter P. Active and inactive genes localize preferentially in the periphery of chromosome territories. J Cell Biol. 1996 Dec;135(5):1195–1205. doi: 10.1083/jcb.135.5.1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Lipman D. J., Pearson W. R. Rapid and sensitive protein similarity searches. Science. 1985 Mar 22;227(4693):1435–1441. doi: 10.1126/science.2983426. [DOI] [PubMed] [Google Scholar]
  10. Müller M., Spiess E., Werner D. Fragmentation of 'nuclear matrix' on a mica target. Eur J Cell Biol. 1983 Jul;31(1):158–166. [PubMed] [Google Scholar]
  11. Neuer B., Plagens U., Werner D. Phosphodiester bonds between polypeptides and chromosomal DNA. J Mol Biol. 1983 Feb 25;164(2):213–235. doi: 10.1016/0022-2836(83)90076-1. [DOI] [PubMed] [Google Scholar]
  12. Reading C. L. Theory and methods for immunization in culture and monoclonal antibody production. J Immunol Methods. 1982 Sep 30;53(3):261–291. doi: 10.1016/0022-1759(82)90175-2. [DOI] [PubMed] [Google Scholar]
  13. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Strohman R. C. The coming Kuhnian revolution in biology. Nat Biotechnol. 1997 Mar;15(3):194–200. doi: 10.1038/nbt0397-194. [DOI] [PubMed] [Google Scholar]

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