Higher order DNA structure in macronuclear chromatin of the hypotrichous ciliate Oxytricha nova (original) (raw)

Abstract

On lysis of macronuclei from the ciliated protozoan Oxytricha at 0.5-2 M NaCl, the DNA, which is normally found as discrete molecules ranging from 0.5 to 20 kilobases, appears in high molecular weight aggregates. Various treatments of the macronuclear lysate (i.e., nucleases, proteases, variation of salt, pH, and temperature) indicate that preservation of the aggregate structure depends on both nucleic acid-nucleic acid and nucleic acid-protein interactions. Purification of the DNA-protein complex after lysing the nuclei in 2 M NaCl shows that one major nuclear protein copurifies with the DNA. As shown by DNA-protein binding experiments, this protein has a high affinity for DNA; however, no evidence for sequence specificity of the protein binding was obtained. Chromatin reconstitution experiments suggest that the protein in itself is not sufficient for DNA aggregation in nuclei, but other factors, possibly the native chromatin structure, are required. Electron microscopy of the purified DNA-protein complex showed structures similar to those observed previously with in vitro-aggregated purified macronuclear DNA (14). A model is presented in which the terminal inverted repeat sequences found on all macronuclear DNA molecules interact with each other forming multistranded DNA complexes. The formation of these structures may be accelerated and stabilized by a protein in vivo.

2495

Images in this article

Selected References

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

  1. Alwine J. C., Kemp D. J., Stark G. R. Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5350–5354. doi: 10.1073/pnas.74.12.5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bowen B., Steinberg J., Laemmli U. K., Weintraub H. The detection of DNA-binding proteins by protein blotting. Nucleic Acids Res. 1980 Jan 11;8(1):1–20. doi: 10.1093/nar/8.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cassuto E., West S. C., Podell J., Howard-Flanders P. Genetic recombination: recA protein promotes homologous pairing between duplex DNA molecules without strand unwinding. Nucleic Acids Res. 1981 Aug 25;9(16):4201–4210. doi: 10.1093/nar/9.16.4201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DasGupta C., Wu A. M., Kahn R., Cunningham R. P., Radding C. M. Concerted strand exchange and formation of Holliday structures by E. coli RecA protein. Cell. 1981 Aug;25(2):507–516. doi: 10.1016/0092-8674(81)90069-6. [DOI] [PubMed] [Google Scholar]
  5. Elsevier S. M., Lipps H. J., Steinbrück G. Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus. Chromosoma. 1978 Dec 6;69(3):291–306. doi: 10.1007/BF00332133. [DOI] [PubMed] [Google Scholar]
  6. Johnson D., Morgan A. R. Unique structures formed by pyrimidine-purine DNAs which may be four-stranded. Proc Natl Acad Sci U S A. 1978 Apr;75(4):1637–1641. doi: 10.1073/pnas.75.4.1637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Klobutcher L. A., Swanton M. T., Donini P., Prescott D. M. All gene-sized DNA molecules in four species of hypotrichs have the same terminal sequence and an unusual 3' terminus. Proc Natl Acad Sci U S A. 1981 May;78(5):3015–3019. doi: 10.1073/pnas.78.5.3015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. 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]
  10. Lawn R. M., Heumann J. M., Herrick G., Prescott D. M. The gene-size DNA molecules in Oxytricha. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 1):483–492. doi: 10.1101/sqb.1978.042.01.051. [DOI] [PubMed] [Google Scholar]
  11. Lipps H. J. In vitro aggregation of the gene-sized DNA molecules of the ciliate Stylonychia mytilus. Proc Natl Acad Sci U S A. 1980 Jul;77(7):4104–4107. doi: 10.1073/pnas.77.7.4104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Lipps H. J., Morris N. R. Chromatin structure in the nuclei of the ciliate stylonychia mytilus. Biochem Biophys Res Commun. 1977 Jan 10;74(1):230–234. doi: 10.1016/0006-291x(77)91398-5. [DOI] [PubMed] [Google Scholar]
  13. Lipps H. J., Nock A., Riewe M., Steinbrück G. Chromatin structure in the macronucleus of the ciliate Stylonychia mytilus. Nucleic Acids Res. 1978 Dec;5(12):4699–4709. doi: 10.1093/nar/5.12.4699. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lipps H. J., Sapra G. R., Ammermann D. The histones of the ciliated protozoan Stylonychia mytilus. Chromosoma. 1974 Apr 9;45(3):273–280. doi: 10.1007/BF00283411. [DOI] [PubMed] [Google Scholar]
  15. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  16. Meyer G. F., Lipps H. J. Chromatin elimination in the hypotrichous ciliate Stylonychia mytilus. Chromosoma. 1980;77(3):285–297. doi: 10.1007/BF00286054. [DOI] [PubMed] [Google Scholar]
  17. Meyer G. F., Lipps H. J. The formation of polytene chromosomes during macronuclear development of the hypotrichous ciliate Stylonychia mytilus. Chromosoma. 1981;82(2):309–314. doi: 10.1007/BF00286113. [DOI] [PubMed] [Google Scholar]
  18. Oka Y., Shiota S., Nakai S., Nishida Y., Okubo S. Inverted terminal repeat sequence in the macronuclear DNA of Stylonychia pustulata. Gene. 1980 Sep;10(4):301–306. doi: 10.1016/0378-1119(80)90150-x. [DOI] [PubMed] [Google Scholar]
  19. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  20. Thomas J. O., Kornberg R. D. An octamer of histones in chromatin and free in solution. Proc Natl Acad Sci U S A. 1975 Jul;72(7):2626–2630. doi: 10.1073/pnas.72.7.2626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tu C. P., Cohen S. N. 3'-end labeling of DNA with [alpha-32P]cordycepin-5'-triphosphate. Gene. 1980 Jul;10(2):177–183. doi: 10.1016/0378-1119(80)90135-3. [DOI] [PubMed] [Google Scholar]
  22. Wilson J. H. Nick-free formation of reciprocal heteroduplexes: a simple solution to the topological problem. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3641–3645. doi: 10.1073/pnas.76.8.3641. [DOI] [PMC free article] [PubMed] [Google Scholar]