Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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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)

Four different footprinting techniques have been used to probe the DNA sequence selectivity of Thia-Net, a bis-cationic analogue of the minor groove binder netropsin in which the N-methylpyrrole moieties are replaced by thiazole groups. In Thia-Net the ring nitrogen atoms are directed into the minor groove where they could accept hydrogen bonds from the exocyclic 2-amino group of guanine. Three nucleases (DNAase I, DNAase II, and micrococcal nuclease) were employed to detect binding sites on the 160bp tyr T fragment obtained from plasmid pKM delta-98, and further experiments were performed with 117mer and 253mer fragments cut out of the plasmid pBS. MPE.Fe(II) was used to footprint binding sites on an EcoRI/HindIII fragment from pBR322. Thia-Net binds to sites in the minor groove containing 4 or 5 base pairs which are predominantly composed of alternating A and T residues, but with significant acceptance of intrusive GC base pairs. Unlike the parent antibiotic netropsin, Thia-Net discriminates against homooligomeric runs of A and T. The evident preference of Thia-Net for AT-rich sites, despite its containing thiazole nitrogens capable of accepting GC sites by hydrogen bonding, supports the view that the biscationic nature of the ligand imposes a bias due to the electrostatic potential differences in the receptor which favour the ligand reading alternating AT sequences.
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PMID:DNA-sequence specific recognition by a thiazole analogue of netropsin: a comparative footprinting study. 165 46

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.
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PMID:Sequence-specific positioning of nucleosomes over the steroid-inducible MMTV promoter. 282 86

We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.
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PMID:Methidiumpropyl-EDTA-iron(II) cleavage of ribosomal DNA chromatin from Dictyostelium discoideum. 300 86

The chromatin structure of the larval cuticle gene cluster at 44D was characterized in embryos from wild-type (Oregon R) and a variant line (2/3) of Drosophila melanogaster. A major DNase I hypersensitive (DH) site was found between genes II and III in the chromatin, in a position 5' to the transcriptional start of the genes in the cluster. The introduction of a 7.3 kilobase transposable element into the cluster in the 2/3 variant enhanced the sensitivity of the major site in 2/3 chromatin but had no other effect upon the pattern of DH sites associated with the wild-type sequences. The wild-type sequences were packaged into an ordered nucleosome-like array in embryos, as revealed by digestion with the chemical cleavage reagent (methidiumpropyl-EDTA) iron (II) [MPE . Fe(II)]. Nucleolytic cleavage within the transposable element chromatin shows it to be organized in an ordered array punctuated by several DH sites. While the patterns of DNase I hypersensitivity are similar in the vicinity of the direct terminal repeats, the patterns revealed by micrococcal nuclease and MPE . Fe(II) are not, indicating a different chromatin organization of these two identical sequences.
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PMID:Chromatin structure at the 44D larval cuticle gene locus in Drosophila: the effect of a transposable element insertion. 609 16

Methidiumpropyl-EDTA . iron(II) [MPE . Fe (II)] cleaves double-helical DNA with considerably lower sequence specificity than micrococcal nuclease. Moreover, digestions with MPE . Fe(II) can be performed in the presence of certain metal chelators, which will minimize the action of many endogenous nucleases. Because of these properties MPE . Fe(II) would appear to be a superior tool for probing chromatin structure. We have compared the patterns generated from the 1.688 g/cm3 complex satellite, 5S ribosomal RNA, and histone gene sequences of Drosophila melanogaster chromatin and protein-free DNA by MPE . Fe(II) and micrococcal nuclease cleavage. MPE . Fe(II) at low concentrations recognizes the nucleosome array, efficiently introducing a regular series of single-stranded (and some double-stranded) cleavages in chromatin DNA. Subsequent S1 nuclease digestion of the purified DNA produces a typical extended oligonucleosome pattern, with a repeating unit of ca. 190 base pairs. Under suitable conditions, relatively little other nicking is observed. Unlike micrococcal nuclease, which has a noticeable sequence preference in introducing cleavages, MPE . Fe(II) cleaves protein-free tandemly repetitive satellite and 5S DNA sequences in a near-random fashion. The spacing of cleavage sites in chromatin, however, bears a direct relationship to the length of the respective sequence repeats. In the case of the histone gene sequences a faint, but detectable, MPE . Fe(II) cleavage pattern is observed on DNA, in some regions similar to and in some regions different from the strong chromatin-specified pattern. The results indicate that MPE . Fe(II) will be very useful in the analysis of chromatin structure.
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PMID:Cleavage of chromatin with methidiumpropyl-EDTA . iron(II). 640 8

Oxytricha macronuclear DNA exists as approximately 24 X 10(6) gene-sized molecules terminating with a C4A4 repeat. DNA-protein interactions at the ends of bulk macronuclear molecules were probed with micrococcal nuclease and methidiumpropyl-EDTA X Fe(II) (MPE X Fe[II]). The ends were indirectly labeled by hybridizing with (C4A4)2. Alternatively, a novel method using MPE X FE(II) as a probe and directly labeling the 3' ends with terminal transferase was implemented. A terminal complex involving approximately 100 bp with nucleosomes phased inward from the complex was found to be characteristic of most or all of the ends. Analysis of two specific genes confirmed the pattern and showed that the special structure was on both ends of each molecule. We conclude that a DNA-protein complex involving 100 bp and terminating with the C4A4 repeat can be sufficient to provide the fundamental functions of telomeres, allowing linear DNA replication and conferring stability of linear DNA.
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PMID:Chromatin structure of the molecular ends of Oxytricha macronuclear DNA: phased nucleosomes and a telomeric complex. 643 44

We prepared synthetic 50-mer DNA duplexes, each containing four mismatched base-pairs in similar positions. We examined their cleavage by DNases I and II, micrococcal nuclease (MNase), methidiumpropyl-EDTA-Fe(II) [MPE-Fe(II)] and hydroxyl radicals. We find that single mismatches only produce subtle changes in the DNase I-cleavage pattern, the most common of which is attenuated cleavage at locations 2-3 bases on the 3'-side of the mismatch. Subtle changes are also observed in most of the DNase II-cleavage patterns, although GT and GG inhibit the cleavage over longer regions and generate patterns that resemble footprints. MNase cleaves the heteroduplexes at the mismatches themselves (except for CC), and in some cases cleaves CpG and CpC steps. None of the mismatches causes any change in the cleavage patterns produced by hydroxyl radicals or MPE-Fe(II). We also examined the cleavage patterns of fragments containing tandem GA mismatches in the sequences RGAY/RGAY and YGAR/YGAR (R, purine; Y, pyrimidine). RGAY causes only subtle changes in the cleavage patterns, which are similar to those seen with single mismatches, except that there are no changes in MNase cleavage. However, YGAR inhibits DNases I and II cleavage over 4-6 bases, and attenuates MPE-Fe(II) and hydroxyl radical cleavage at 2 bases. These changes suggest that this mismatch has a more pronounced effect on the local DNA structure. These changes are discussed in terms of the structural and dynamic effects of each mismatch.
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PMID:Cleavage of fragments containing DNA mismatches by enzymic and chemical probes. 1255 99

The analysis of chromatin structure is essential for the understanding of transcriptional regulation in eukaryotes. Here we describe methidiumpropyl-EDTA sequencing (MPE-seq), a method for the genome-wide characterization of chromatin that involves the digestion of nuclei withMPE-Fe(II) followed by massively parallel sequencing. Like micrococcal nuclease (MNase), MPE-Fe(II) preferentially cleaves the linker DNA between nucleosomes. However, there are differences in the cleavage of nuclear chromatin by MPE-Fe(II) relative to MNase. Most notably, immediately upstream of the transcription start site of active promoters, we frequently observed nucleosome-sized (141-190 bp) and subnucleosome-sized (such as 101-140 bp) peaks of digested chromatin fragments with MPE-seq but not with MNase-seq. These peaks also correlate with the presence of core histones and could thus be due, at least in part, to noncanonical chromatin structures such as labile nucleosome-like particles that have been observed in other contexts. The subnucleosome-sized MPE-seq peaks exhibit a particularly distinct association with active promoters. In addition, unlike MNase, MPE-Fe(II) cleaves nuclear DNA with little sequence bias. In this regard, we found that DNA sequences at RNA splice sites are hypersensitive to digestion by MNase but not by MPE-Fe(II). This phenomenon may have affected the analysis of nucleosome occupancy over exons. These findings collectively indicate that MPE-seq provides a unique and straightforward means for the genome-wide analysis of chromatin structure with minimal DNA sequence bias. In particular, the combined use of MPE-seq and MNase-seq enables the identification of noncanonical chromatin structures that are likely to be important for the regulation of gene expression.
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PMID:MPE-seq, a new method for the genome-wide analysis of chromatin structure. 3159 Dec 51

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