Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1985 Apr;82(8):2296–2300. doi: 10.1073/pnas.82.8.2296

A method for mapping intranuclear protein-DNA interactions and its application to a nuclease hypersensitive site.

P D Jackson, G Felsenfeld
PMCID: PMC397544  PMID: 3857580

Abstract

We have devised a method for mapping sites on DNA within the nucleus that are protected against nuclease attack by interaction with bound protein or other factors. This "footprinting" method uses an end-labeled sequence-specific DNA probe, which is annealed to the DNA from nuclear digests under carefully controlled conditions. The annealed complexes are treated with single-strand-specific nuclease, the resulting duplex molecules are electrophoresed on gels, and the gels are autoradiographed. The high sensitivity and resolution of the method have made it possible to obtain a detailed map of DNase I cutting patterns in the 5' flanking sequence of the chicken adult beta (beta A)-globin gene within nuclei from various tissues. In nuclei from adult erythrocytes, this domain is hypersensitive to nucleases. However, we detect within the domain two well-defined regions that are protected against attack, indicating the presence of one or more bound factors. Nuclei from oviduct or 5-day-old embryonic erythrocytes, in which the domain is not hypersensitive, show limited and different patterns of protection.

Full text

PDF
2296

Images in this article

2298Image
on p.2298
2299Image
on p.2299

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  2. Bloom K. S., Anderson J. N. Hormonal regulation of the conformation of the ovalbumin gene in chick oviduct chromatin. J Biol Chem. 1982 Nov 10;257(21):13018–13027. [PubMed] [Google Scholar]
  3. Borgmeyer U., Nowock J., Sippel A. E. The TGGCA-binding protein: a eukaryotic nuclear protein recognizing a symmetrical sequence on double-stranded linear DNA. Nucleic Acids Res. 1984 May 25;12(10):4295–4311. doi: 10.1093/nar/12.10.4295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Camerini-Otero R. D., Sollner-Webb B., Simon R. H., Williamson P., Zasloff M., Felsenfeld G. Nucleosome structure, DNA folding, and gene activity. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 1):57–75. doi: 10.1101/sqb.1978.042.01.008. [DOI] [PubMed] [Google Scholar]
  5. Church G. M., Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984 Apr;81(7):1991–1995. doi: 10.1073/pnas.81.7.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. DeLucia A. L., Lewton B. A., Tjian R., Tegtmeyer P. Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication. J Virol. 1983 Apr;46(1):143–150. doi: 10.1128/jvi.46.1.143-150.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dolan M., Dodgson J. B., Engel J. D. Analysis of the adult chicken beta-globin gene. Nucleotide sequence of the locus, microheterogeneity at the 5'-end of beta-globin mRNA, and aberrant nuclear RNA species. J Biol Chem. 1983 Mar 25;258(6):3983–3990. [PubMed] [Google Scholar]
  8. Dretzen G., Bellard M., Sassone-Corsi P., Chambon P. A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem. 1981 Apr;112(2):295–298. doi: 10.1016/0003-2697(81)90296-7. [DOI] [PubMed] [Google Scholar]
  9. Dynan W. S., Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983 Nov;35(1):79–87. doi: 10.1016/0092-8674(83)90210-6. [DOI] [PubMed] [Google Scholar]
  10. Emerson B. M., Felsenfeld G. Specific factor conferring nuclease hypersensitivity at the 5' end of the chicken adult beta-globin gene. Proc Natl Acad Sci U S A. 1984 Jan;81(1):95–99. doi: 10.1073/pnas.81.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Laskowski M., Sr Purification and properties of the mung bean nuclease. Methods Enzymol. 1980;65(1):263–276. doi: 10.1016/s0076-6879(80)65036-8. [DOI] [PubMed] [Google Scholar]
  12. McGhee J. D., Wood W. I., Dolan M., Engel J. D., Felsenfeld G. A 200 base pair region at the 5' end of the chicken adult beta-globin gene is accessible to nuclease digestion. Cell. 1981 Nov;27(1 Pt 2):45–55. doi: 10.1016/0092-8674(81)90359-7. [DOI] [PubMed] [Google Scholar]
  13. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  14. Mulvihill E. R., Palmiter R. D. Relationship of nuclear estrogen receptor levels to induction of ovalbumin and conalbumin mRNA in chick oviduct. J Biol Chem. 1977 Mar 25;252(6):2060–2068. [PubMed] [Google Scholar]
  15. Parker C. S., Topol J. A Drosophila RNA polymerase II transcription factor binds to the regulatory site of an hsp 70 gene. Cell. 1984 May;37(1):273–283. doi: 10.1016/0092-8674(84)90323-4. [DOI] [PubMed] [Google Scholar]
  16. Payvar F., DeFranco D., Firestone G. L., Edgar B., Wrange O., Okret S., Gustafsson J. A., Yamamoto K. R. Sequence-specific binding of glucocorticoid receptor to MTV DNA at sites within and upstream of the transcribed region. Cell. 1983 Dec;35(2 Pt 1):381–392. doi: 10.1016/0092-8674(83)90171-x. [DOI] [PubMed] [Google Scholar]
  17. Rawlins D. R., Rosenfeld P. J., Wides R. J., Challberg M. D., Kelly T. J., Jr Structure and function of the adenovirus origin of replication. Cell. 1984 May;37(1):309–319. doi: 10.1016/0092-8674(84)90327-1. [DOI] [PubMed] [Google Scholar]
  18. Southern E. Gel electrophoresis of restriction fragments. Methods Enzymol. 1979;68:152–176. doi: 10.1016/0076-6879(79)68011-4. [DOI] [PubMed] [Google Scholar]
  19. Swanstrom R., Shank P. R. X-Ray Intensifying Screens Greatly Enhance the Detection by Autoradiography of the Radioactive Isotopes 32P and 125I. Anal Biochem. 1978 May;86(1):184–192. doi: 10.1016/0003-2697(78)90333-0. [DOI] [PubMed] [Google Scholar]
  20. Tegtmeyer P., Lewton B. A., DeLucia A. L., Wilson V. G., Ryder K. Topography of simian virus 40 A protein-DNA complexes: arrangement of protein bound to the origin of replication. J Virol. 1983 Apr;46(1):151–161. doi: 10.1128/jvi.46.1.151-161.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Wood W. I., Felsenfeld G. Chromatin structure of the chicken beta-globin gene region. Sensitivity to DNase I, micrococcal nuclease, and DNase II. J Biol Chem. 1982 Jul 10;257(13):7730–7736. [PubMed] [Google Scholar]
  22. Wood W. I., Nickol J., Felsenfeld G. Repeated sequence organization and RNA transcription map of the chicken adult beta-globin gene region. J Biol Chem. 1981 Feb 25;256(4):1502–1506. [PubMed] [Google Scholar]
  23. Wu C. Activating protein factor binds in vitro to upstream control sequences in heat shock gene chromatin. Nature. 1984 Sep 6;311(5981):81–84. doi: 10.1038/311081a0. [DOI] [PubMed] [Google Scholar]
  24. Wu C. Two protein-binding sites in chromatin implicated in the activation of heat-shock genes. Nature. 1984 May 17;309(5965):229–234. doi: 10.1038/309229a0. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES