Isolation of genes as chromatin by nucleoprotein hybridization.

Abstract

The developmentally regulated sea urchin early histone gene repeat (SUEHGR) from Strongylocentrotus purpuratus was isolated as chromatin by nucleoprotein hybridization. This technique is a novel method to isolate specific sequences as chromatin. Because the purification scheme is based on the gene sequence, I was able to isolate the same gene in different functional states. Gene size chromatin fragments were solubilized by restriction endonuclease digestion of cell nuclei. Using T7 gene 6 exonuclease, the 3$\sp\prime$ termini of the fragments were exposed and then hybridized in solution to a biotinylated oligonucleotide complementary to one end of the SUEHGR fragment. The hybrids were bound to an Avidin D matrix and eluted by DTT cleavage of the biotin linker. The inactive genes could be enriched more than 700 fold in SUEHGR sequences in overall yields between 2-15% ($>$80% purity) and could be studied in the electron microscope. The purity of the active gene repeat was $\sp\sim$15% and yields were 1-3%. The changes occurring during isolation were characterized by micrococcal nuclease digestion, and MPE cleavage combined with indirect end-labeling. The MPE experiments were quantitatively analyzed by the use of an algorithm to calculate cleavage probabilities from the DNA size distribution. The changes in MPE pattern occurring during isolation on the inactive SUEHGR are interpreted as loss of non-histone proteins, while the positions of nucleosomes remain unchanged. Positioned nucleosomes were not found on the active gene repeat, and the only change observed was cutting by site specific and nonspecific endogenous nucleases. Two dimensional gel analysis of the copurified histone and non-histone proteins was made possible by the optimization of an in vitro iodination method. The inactive SUEHGR was depleted in late histone variants as compared to the bulk of the same developmental stage revealing an unequal distribution of histone sequence variants over the genome. Preliminary data indicate a different histone content for the active SUEHGR preparation. The active fraction was enriched in several histone H2A variants, depleted in H3 and H4, and several histones were hyperacetylated. The active and inactive SUEHGR also contained different amounts of a few low molecular weight proteins.