Folding of DNA into nucleosomes and higher order chromatin structures restricts its accessibility to proteins and drugs. Hence, the location of histone octamers on the DNA sequence (nucleosome positions) as well as structural and dynamic properties of nucleosomes may play important roles in gene regulation, replication and DNA repair. Conventional approaches to characterize chromatin structure include (partial) purification of chromatin and characterization of DNA accessibility to nucleases (micrococcal nuclease, DNaseI) and chemical cleavage reagents (hydroxyl radicals, methidium propyl-EDTA-iron, copper phenanthroline). The cleavage sites are monitored using low- and high-resolution footprinting protocols. These techniques, however, expose the problem that chromatin extraction procedures could alter chromatin composition and structure, including nucleosome positioning. To investigate chromatin structures in vivo, alternative approaches are applied, such as expression of prokaryotic methyltransferases in Saccharomyces cerevisiae, the genome of which contains no endogenous detectable methylation (1,2). The sites of methylation can be measured after DNA isolation using methylation-sensitive restriction enzymes. This approach, however, requires expression of a foreign gene, and the resolution is restricted because of the sequence specificity of the methyltransferases.
KeywordsVortex Phenol EDTA Electrophoresis Boiling
- 3.Friedberg, E. C., Walker, G. C., and Siede, W. (1995) DNA repair and mutagenesis. ASM Press, Washington, DC.Google Scholar
- 15.Maniatis, T., Fritsch, E., and Sambrook, J. (1982) Molecular cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY.Google Scholar
- 16.Sherman, F., Fink, G. R., and Hicks, J. B. (1986) Laboratory Course Manual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar