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Query: EC:3.1.31.1 (micrococcal nuclease)
 2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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 only on the gene sequence and is independent of other physical properties such as protein composition and transcriptional activity, we were 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'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. DTT cleavage of the biotin linker yielded a chromatin fraction greater than 700 fold enriched in SUEHGR. Overall yields were between 2% and 15%. The purity of the isolated material was independently measured to be greater than 80%. The homogeneous native structure of the inactive genes was preserved as shown by electron microscopy and micrococcal nuclease digestion of the purified SUEHGR. Minor heterogeneity was observed for the purified active genes by micrococcal nuclease digestion but the main features of the active chromatin were preserved during isolation. This isolation offers the first opportunity to study the structure of an RNA polymerase II gene at different stages of the cell cycle and development.
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PMID:Nucleoprotein hybridization: a method for isolating active and inactive genes as chromatin. 203 Sep 47

Prior to fertilization, sperm possess one of the longest nucleosome repeat lengths yet determined [approximately 250 base pairs (bp) for the sea urchin Strongylocentrotus purpuratus]. We show here that the two-cell embryo has an average repeat size of 189 +/- 2 bp as probed by micrococcal nuclease; this is the shortest average nucleosomal subunit reported for S. purpuratus. By the eight-cell stage, the average nucleosome repeat increases to 201 +/- 2 bp, and it subsequently increases further during development. These results indicate that a dramatic rearrangement of chromatin occurs upon fertilization and that this chromatin remodeling continues through early development. When two-cell embryos are labeled for 30 min with [3H]thymidine and digested briefly, they exhibit nuclease-hypersensitive fragments averaging 308 bp in size, which are consistent with the size of protected DNA units in replication intermediate complexes at blastula stage (as described by Levy and Jacob [Levy, A., & Jacob, K. M. (1978) Cell (Cambridge, Mass.) 14, 259]). Our results are consistent with two general propositions: (1) long repeat lengths are found in highly differentiated cells, and (2) short repeat lengths are characteristic of cells more active in cell division. Our data would also imply that a rapid increase in the DNA complement, e.g., in the transition from haploid to diploid state following fertilization, is accompanied by a shortening of the average size of DNA in a nucleosome after replication.
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PMID:Shortest nucleosomal repeat lengths during sea urchin development are found in two-cell embryos. 668 59

The Ars insulator is a boundary element identified in the upstream region of the arylsulfatase (HpArs) gene in the sea urchin, Hemicentrotus pulcherrimus, and possesses the ability to both block enhancer-promoter communications and protect transgenes from silent chromatin. To understand the molecular mechanism of the Ars insulator, we investigated the correlation between chromatin structure, DNA structure and insulator activity. Nuclease digestion of nuclei isolated from sea urchin embryos revealed the presence of a nuclease-hypersensitive site within the Ars insulator. Analysis of micrococcal nuclease-sensitive sites in the Ars insulator, reconstituted with nucleosomes, showed the exclusion of nucleosomes from the central AT-rich region. Furthermore, the central AT-rich region in naked DNA was sensitive to nucleotide base modification by diethylpyrocarbonate (DEPC). These observations suggest that non-B-DNA structures in the central AT-rich region may inhibit nucleosomal formation, which leads to nuclease hypersensitivity. Furthermore, comparison of nucleotide sequences between the HpArs gene and its ortholog in Strongylocentrotus purpuratus revealed that the central AT-rich region of the Ars insulator is conserved, and this conserved region showed significant enhancer blocking activity. These results suggest that the central AT-rich nucleosome-free region plays an important role in the function of the Ars insulator.
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PMID:Nucleosome exclusion from the interspecies-conserved central AT-rich region of the Ars insulator. 2193 Jun 54