Properties of nucleosomes from divergent eukaryotes
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It is widely assumed that eukaryotes package their genomes in equivalent chromatin structures. To test whether divergent eukaryotes form equivalent nucleosomes we selected eight organisms with completely sequenced genomes representing distant positions in eukaryote evolution and distinctive genome properties. These were Homo sapiens, Xenopus laevis, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Plasmodium falciparum, Leishmania major, Giardia lamblia and Encephalitozoon cuniculi. Histone genes were synthesised for high level recombinant expression in Escherichia coli and methods optimised for high yield and purity. The ability to form equivalent histone (H3-H4)2 tetramer and (H2A-H2B-H3-H4)2 octamer complexes was monitored by gel filtration chromatography and crosslinking. P. falciparum histones behaved similarly to X. laevis, H. sapiens and S. cerevisiae equivalents but the more divergent histones precipitated as tetramers. G. lamblia and E. cuniculi histones, but not those from L. major, formed octamers with good efficiency. Differences in four helix bundle packing interface residues differ and may allow the divergent organism tetramers to form insoluble extended polymers. All organisms could be shown to form nucleosomes on two different 147 bp metazoan nucleosome positioning DNAs with similar albeit distinct hydrodynamic properties and DNA footprints. Assembly of E. cuniculi tetrasomes on metazoan nucleosome positioning sequences revealed a distinctive DNA sequence specificity. To compare sequence preferences of divergent organism nucleosome assembly, a library of human mononucleosomal DNAs was used for systematic evolution of ligands by exponential enrichment (SELEX) with H. sapiens, P. falciparum and E. cuniculi histone octamers. The selected DNA fragments were submitted for deep sequencing and will be analysed to test whether genome properties are linked with histone sequence variation in the organisms.