Efficient solubilization and partial purification of sea urchin histone genes as chromatin.
Soluble chromatin fragments are rapidly and efficiently produced when nuclei are digested with restriction endonucleases in buffers containing very low concentrations of magnesium. Under these conditions, the sequence specificity of the restriction endonucleases is maintained, resulting in release of specific genes as fragments with discrete molecular weights that can be fractionated by size on glycerol gradients. Gradient fractions can be chosen to be significantly enriched in specific genes and their associated proteins. For instance, we can achieve a 16-fold enrichment of the chromatin containing the early histone genes of sea urchin. The enrichments produced by these methods are useful as a first step in techniques to purify specific genes as chromatin. Glycerol gradient analyses can also be used to test whether putative gene-specific proteins are actually bound to the same sequences in vivo.
D000818:Animals; D002467:Cell Nucleus / Q000648:ultrastructure; D002645:Chickens; D002843:Chromatin / Q000302:isolation & purification* / Q000648:ultrastructure; D004247:DNA / Q000302:isolation & purification*; D004262:DNA Restriction Enzymes; D004625:Embryo, Nonmammalian; D004912:Erythrocytes / Q000166:cytology; D005260:Female; D005796:Genes; D006657:Histones / Q000235:genetics*; D008274:Magnesium / Q000494:pharmacology; D010957:Plasmids; D012617:Sea Urchins; D012995:Solubility
D002843:Chromatin; D006657:Histones; D004247:DNA; D004262:DNA Restriction Enzymes; D008274:Magnesium