Micrococcal nuclease digestion

Similar results were obtained from reconstitution experiments with DNA and a non-cross-linked octamer (Thomas and Butler, 1978). Nucleosome-like particles were observed in the EM and a pattern of histone cross-linking comparable to that of native chromatin was obtained. However, only 140-base-pair repeats were obtained upon micrococcal nuclease digestion instead of 200-base-pair repeats obtained for native rat liver chromatin (Noll and Komberg, 1977). This indicates that, in the absence of HI, only core particles can be reconstituted. Nevertheless, these studies with both cross-linked and reassembled un-cross-linked histones demonstrate that the octamer is a complete biological functional unit retaining the information for folding the DNA around the histone core. 

Antibiotic Mg complex induced alteration in the ultrastructural changes in the native and HI depleted chromatin were monitored by thermal melting analysis, polyacrylamide gel mobility assay, dynamic light scattering experiments and transmission electron microscopic studies. Micrococcal nuclease digestion is the biochemical probe to assess the accessibility of the antibiotic Mg + complexes to nucleosomal DNA. 

Marx, K.A. and Reynolds, T.C. (1983) Ion competition and micrococcal nuclease digestion studies of spermidine-condensed calf thymus DNA. Evidence for toms organization by circumferential DNA wrapping. Biochim. Biophys. Acta, 741, 279-287. 

Partial digestion by DNAase-I is widely used in the study of chromatin structure. Micrococcal nuclease (also called Staphyloccocal nuclease) makes double-stranded cuts initially in the linker DNA between nucleosomes, useful for mapping the positions of nucleosomes. Micrococcal nuclease digests must be interpreted carefully because this nuclease shows some sequence specificity when partial digestion of purified DNA is carried out as a control. In addition to these enzyme probes, there are chemical reagents that cut DNA. These reagents are much less sequence-dependent than even DNAase-I. Accessible DNA can also be labeled by methylating enzymes. 

In general, the remodeling of sperm chromatin that accompanies stage I decondensation involves the replacement of sperm-specific basic proteins with histones from the egg and results in the formation of nucleosomes. It now seems clear, at least in amphibian egg and Drosophila embryo extracts, that this remodeling is mediated by specific factors that participate in both assembly and disassembly processes. These changes in chromatin composition and structure can be analyzed by one- or two-dimensional polyacrylamide gel electrophoresis and micrococcal nuclease digestion. 

Analysis of Chromatin Structure by Micrococcal Nuclease Digestion... 

Fig. 3 Appearance of fragments protected from micrococcal nuclease digestion following incubation of Xenopus sperm chromatin in homologous egg extract. Sperm nuclei were used without extract incubation (lane 2) or following incubation of 50 ng DNAZ/il egg LSS for 3 min (lane 3), 10 min (lane 4), 30 rain (lane 5), or 120 min (lane 6). Samples were diluted and the sperm nuclei were isolated by centrifugation. The samples were digested with micrococcal nuclease for 1 min and the DNA purified and separated by a 1.4% Tris-borate-EDTA agarose gel. Lane I shows molecular weight markers in lOO-base-pair increments.

Micrococcal Nuclease Digestion of the Poly(ADP-Ribose) Polymerase — sDNA Complex... 

Fig. 4A-F. Visualization by dark field electron microscopy of the progressive micrococcal nuclease digestion of DNA-bound poly(ADP-ribose) polymerase [15]. DNA-bound poly(ADP-ribose) polymerase was incubated without A or with B-F micrococcal nuclease under the conditions described in Fig. 3. B 2 min C 5 min D 10 min E 20 min, A and F 30 min. The bar represents 100 nm for all pictures...

Preliminary results indicate that the DNA purified after micrococcal nuclease digestion of the enzyme—DNA complex activates more efficiently the DNA-free poly(ADP-ribose) polymerase than the total purified sDNA and much more than the purified nucleosomal core particles DNA (data not shown). Ohgushi et al. [8] and Benjamin and Gill [7] correlate the activation of poly(ADP-ribose) polymerase only to its binding to nicks or ends and not to a specific DNA sequence. Although our observations of poly(ADP-ribose) polymerase-sDNA complexes by electron microscopy and by polyacrylamide gel analysis do not exclude the possibUity that the enzyme is activated preferentially by internal nicks on sDNA fragments, they raise the question whether... 

The bands observed at 69 and 64 kD may be due to ADP-ribosylation of lamin A and lamin B, major constituents of the nuclear matrix, as was previously suggested by Song and Adolph [26]. They reported that nuclear scaffolds isolated by mild micrococcal nuclease digestion from in vitro labeled HeLa cell nuclei contained labeled proteins at 116 kD and in the 65 to 70 kD range. 

In order to demonstrate that the inhibitory property of vaccinia virus in vitro transcripts is RNA, we subjected these preparations to alkaline hydrolysis, and micrococcal nuclease digestion. The resulting hydrolysate and digest no longer had an inhibitory effect on HeLa cell mRNA function indicating that the integrity of the transcripts is necessary for this inhibition (Coppola and Bablanian, 1983). 

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