Archaeal histones

However, the packing of DNA into nucleosome-like structures is not unique to eukarya similar structures appear in archaea (reviewed in Reeve et al., 1997). Additionally, histones and minichromosome maintenance proteins (MCM) are widespread among eukarya and archaea and absent in prokarya, and the eukaryotic chromo domain has a structure that is highly reminiscent of archaeal histones that are involved in formation of archaeal chromatin (Ball et al., 1997). Consequently, it is possible that chromatin remodeling in eukaryotes is an elaboration of a similar cellular mechanism in archaea. 

To obtain purified preparations of single histone homodimers, a system to synthesize and purify recombinant archaeal histones from Escherichia coli was... 

Quantitation. Archaeal histones are enriched in arginine and lysine residues, and the methods of protein quantitation based on Coomassie Brilliant Blue G250 dye binding are inaccurate because the dye preferentially binds to these basic residues. Most archaeal histones also lack tyrosine or tryptophan residues, precluding A280 measurement. Therefore, reliable quantitation of archaeal histone solutions requires a determination of the total amino acid composition after acid hydrolysis. ... 

Several E. coli heterologous gene expression systems based on the tac, T7, and Hlac promoters have been used successfrilly to obtain recombinant archaeal histones, by following the commercial protocols specified for each expression system. The purification protocol detailed above for native archaeal histones also works well for recombinant archaeal histones, although the protease inhil >itor phenylmethylsulfonyl fluoride (PMSF) must be added to a final concentration of 0.1 mAf during the DNase I digestion. Almost 100% purity can be achieved from E. coli crude extracts. 

Modifications to Purification Protocol. Most archaeal histones are positively charged at pH 8.0 in the Tris buffer recommended above however, some histones have more neutral isoelectric points, when calculated using the Molecular Weight/Isoelectric Point program found at www.expasy.ch/tools/pi tools.html. The buffer used should be at least one pH unit lower than the calculated isoelectric point for efficient binding to the heparin column. 

Four assays are described for archaeal histone binding to DNA. Two are electrophoretic mobility shift assays one using agarose gels to detect compaction of... 

Different archaeal histones have different saturation points in this assay, both in terms of the histone DNA ratio that results in maximal enhancement of mobility, and in terms of the maximum extent of the gel shift (Fig. lA). 

Gel Mobility Retardation in Polyacrylamide. This procedure assays the association of archaeal histones with linear DNA molecules 60-500 bp in length and is used to monitor archaeal nucleosome assembly under different experimental conditions and to determine the dissociation constants Kd) of different histone-DNA complexes. ... 

Archaeal histones can be isolated from purified archaeal nucleosomes following exciaon from the agarose gel and agarase diction of the gel slice. The gel... 

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