Abstract
A nucleosome assembly protein (NAP-1) of Saccharomyces cerevisiae facilitates the association of histones with DNA to form nucleosomes in vitro at physiological ionic conditions. The cloned gene was expressed in Escherichia coli using a T7 expression system, and the protein (417 amino acid residues) was purified by Mono Q column chromatography. Various deletion fragments of NAP-1 protein were also produced, and their nucleosome assembly activity was examined by supercoiling assay. The internal fragment containing the residues 43-365 was necessary and sufficient for the activity, and a long stretch of negatively charged region near the carboxyl terminus was dispensable. This minimal size fragment could form the 12 S NAP-1-histone complex as the whole protein could, whereas deleted fragments on either side could bind with core histones only to form aggregates.
MeSH terms
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Animals
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Bacteriophage T7 / genetics
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Base Sequence
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Cell Cycle Proteins
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Chromatography, Ion Exchange
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Cloning, Molecular
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Electrophoresis, Polyacrylamide Gel
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Escherichia coli / genetics
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Fungal Proteins / genetics*
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Fungal Proteins / isolation & purification
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Fungal Proteins / metabolism
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Genes, Fungal*
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Histones / metabolism
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Immunoblotting
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Mice
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Molecular Sequence Data
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Nuclear Proteins
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Nucleosome Assembly Protein 1
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Plasmids
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Proteins / genetics*
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Proteins / isolation & purification
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Proteins / metabolism
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Restriction Mapping
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Saccharomyces cerevisiae / genetics*
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Saccharomyces cerevisiae / metabolism
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Saccharomyces cerevisiae Proteins
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Sequence Deletion
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Sequence Homology, Amino Acid
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beta-Galactosidase / genetics
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beta-Galactosidase / metabolism
Substances
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Cell Cycle Proteins
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Fungal Proteins
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Histones
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NAP1 protein, S cerevisiae
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Nap1l1 protein, mouse
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Nuclear Proteins
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Nucleosome Assembly Protein 1
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Proteins
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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beta-Galactosidase