Micrococcal nuclease

About: Micrococcal nuclease is a research topic. Over the lifetime, 1862 publications have been published within this topic receiving 77597 citations.

An efficient mRNA-dependent translation system from reticulocyte lysates.

Abstract: A simple method is described for converting a standard rabbit reticulocyte cell-free extract (lysate) into an mRNA-dependent protein synthesis system. The lysate is preincubated with CaCl2 and micrococcal nuclease, and then excess ethyleneglycol-bis(2-aminoethylether)-N,N′-tetraacetic acid is added to chelate the Ca2+ and inactivate the nuclease. Lysates treated in this way have negligible endogenous amino acid incorporation activity, but 75% of the activity of the original lysate can be recovered by the addition of globin mRNA. The efficiency utilisation of added mRNA and the sensitivity of the system are both very high. No residual nuclease activity could be detected, and the tRNA is functionally unimpaired. Several different species of mRNA have been shown to be translated efficiently into full-sized products of the expected molecular weight up to about 200 000, and there is no detectable accumulation of incomplete protein products. The efficient translation of RNA from two plant viruses (tobacco mosaic virus and cowpea mosaic virus) required heterologous tRNA.

OB(oligonucleotide/oligosaccharide binding)-fold : common structural and functional solution for non-homologous sequences

Abstract: A novel folding motif has been observed in four different proteins which bind oligonucleotides or oligosaccharides: staphylococcal nuclease, anticodon binding domain of asp-tRNA synthetase and B-subunits of heat-labile enterotoxin and verotoxin-1. The common fold of the four proteins, which we call the OB-fold, has a five-stranded beta-sheet coiled to form a closed beta-barrel. This barrel is capped by an alpha-helix located between the third and fourth strands. The barrel-helix frameworks can be superimposed with r.m.s. deviations of 1.4-2.2 A, but no similarities can be observed in the corresponding alignment of the four sequences. The nucleotide or sugar binding sites, known for three of the four proteins, are located in nearly the same position in each protein: on the side surface of the beta-barrel, where three loops come together. Here we describe the determinants of the OB-fold, based on an analysis of all four structures. These proposed determinants explain how very different sequences adopt the OB-fold. They also suggest a reinterpretation of the controversial structure of gene 5 ssDNA binding protein, which exhibits some topological and functional similarities with the OB-fold proteins.

Purification and further properties of single-strand-specific nuclease from Aspergillus oryzae.

Abstract: A simple scheme has been devised for the purification of Aspergillus nuclease S1 from commercially available crude amylase powder. Five purification steps yield 90% pure nuclease with an overall yield of 27%. The enzyme is shown to be absolutely specific for single-stranded nucleic acids. It degrades both DNA and RNA. The nuclease exhibits a sharp pH optimum at pH 4.2 and is active in low concentrations of dodecylsulfate. Aspergillus nuclease S1 is a metalloprotein of molecular weight 32000. The utility of this nuclease for measuring annealing of labeled DNA is discussed.

Action of micrococcal nuclease on chromatin and the location of histone H1

Abstract: Digestion of rat liver chromatin with micrococcal nuclease at 2°C yields fragments containing multiples of 198±6 base-pairs, which represents the DNA content of a unit of the structure. Digestion at 37°C results in degradation of these fragments from the ends and also in the release of a histone, H1. This suggests an association of H1 with the region of DNA that links adjacent units of the structure. Two further lines of evidence lead to the same idea. If H1 is removed before digestion, then cleavage between units is eightfold more rapid. And while the removal of H1 has little effect on the sedimentation coefficient of the smallest, or monomer, fragment, it causes a marked reduction in the sedimentation coefficients of dimers and higher multimers.

Sequence-specific positioning of nucleosomes over the steroid-inducible MMTV promoter.

Abstract: We have investigated chromatin organization over the MMTV LTR, a promoter regulated by steroid hormones. The studies were performed on cell lines containing BPV-based episomal constructs. Nucleosome positioning was determined by localization of sites sensitive to the enzyme micrococcal nuclease, or to the chemical MPE-Fe(II). Experiments with both reagents indicate that nucleosomes are specifically positioned in MMTV LTR chromatin. In the absence of hormone a regular cutting pattern is obtained, with cleavage sites at +136, -60, -250, -444, -651, -826 and -1019 relative to the Cap site. In the presence of hormone the cutting pattern is unchanged, except for a region between -60 and -250 that becomes hypersensitive to MPE-Fe(II). This region contains the DNA sequences to which steroid receptor complexes bind during transcriptional activation. Our results indicate that this region is associated in chromatin from uninduced cells with a macromolecular complex (probably a nucleosome core), and this complex is displaced (or modified) upon binding of activated receptor.