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)

Ligation-mediated polymerase chain reaction (LMPCR) provides adequate sensitivity for nucleotide-level analysis of single-copy genes. Here, we report that chromatin structure can be studied by enzyme treatment of permeabilized cells followed by LMPCR. DNase I treatment of lysolecithin-permeabilized cells was found to give very clear footprints and to show differences between active and inactive X chromosomes (Xa and Xi, respectively) at the human X-linked phosphoglycerate kinase (PGK-1) locus. Beginning 380 bp upstream and continuing 70 bp downstream of the major transcription start site of PGK-1, we analyzed both strands of this promoter and CpG island and discovered the following: (1) The transcriptionally active Xa in permeabilized cells has several upstream regions that are almost completely protected on both strands from DNase I nicking. (2) Nuclei isolated in polyamine-containing buffers lack these footprints, suggesting that data from isolated nuclei can be flawed; other buffers are less disruptive. (3) The Xa has no detectable footprints at the transcription start and HIP1 consensus sequence. (4) The heterochromatic and transcriptionally inactive Xi has no footprints but has two regions showing increased DNase I sensitivity at 10-bp intervals, suggesting that the DNA is wrapped on the surface of a particle; one nucleosome-sized particle seems to be positioned over the transcription start site and another is centered approximately 260 bp upstream. (5) Potassium permanganate and micrococcal nuclease (MNase) studies indicate no melted or otherwise unusual DNA structures in the region analyzed, and MNase, unlike restriction endonuclease MspI, does cut within the positioned particles on the Xi. Results are discussed in the context of X chromosome inactivation and the maintenance of protein and DNA methylation differences between euchromatin and facultative heterochromatin at CpG islands.
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PMID:Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR. 204 57

The reversible unfolding of two dissimilar proteins, phosphoglycerate kinase from Bacillus stearothermophilus (PGK) and Staphylococcus aureus nuclease (SAN), was induced with two denaturants, urea and guanidinium chloride (GuHCl). For each protein, structural transitions were monitored by intrinsic fluorescence intensity changes arising from a unique tryptophan residue. In the case of SAN the single, native tryptophan residue was used, whereas for PGK two versions, one with a tryptophan at position 315 and one at 379, were constructed genetically. The resultant folding curves were analyzed by considering the change in the solvation free energy of internal amino acid residues as the denaturant concentration was varied. We derive the following simple relationship: -RT ln K = delta Gw + n delta Gs,m[D]/Kden. + [D]) where K is the equilibrium constant describing the distribution of folded and unfolded forms at a given denaturant concentration [D], delta Gw is the free energy change for the transition in the absence of denaturant, and n is the number of internal side chains becoming exposed. delta Gs,m and Kden. are constants derived empirically from the solvation energies of model compounds and represent the behavior of an average internal side chain between 0 and 6 M GuHCl and 0 and 8 M urea. For proteins of known structure these values can easily be derived, and for others, average values in guanidinium chloride (delta Gs,m = 0.775 kcal/mol and Kden. = 5.4 M) or urea (delta Gs,m = 1.198 kcal/mol and Kden. = 25.25 M) can be used in the analysis. Results show that the parameters n and delta Gw are independent of the denaturant used for all 12 transitions studied. This supports the hypothesis that the unfolding activity of urea and GuHCl can be accounted for by their effect on the solvation energy of amino acid side chains which are buried in the folded but exposed in the unfolded protein. This simple analytical treatment allows the "cooperativity" of protein folding to be interpreted in terms of the number of side chains becoming exposed to the solvent in a given step and allows accurate estimation of the free energy irrespective of the denaturant concentration needed to induce the transition.
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PMID:The energetics and cooperativity of protein folding: a simple experimental analysis based upon the solvation of internal residues. 847 98