The DNase II, Mg2+ procedure (Gottesfeld, J. M., Phil. Trans. R. Soc. Lond. B. 283:343-357, 1978) was used to cleave and separate fractions from maize seedling chromatin. Under gentle conditions of digestion with this nuclease, a fraction with a less compact structure can be isolated from chromatin; the fraction is also enriched in RNA polymerase activity and nascent mRNA (Mathis, D., et al., Prog. Nucleic Acid Res. Mol. Biol. 24:1-55, 1980). This fraction meets all criteria for transcriptionally-active chromatin, and probably corresponds to the euchromatin portions of the genome. Although much is known about fractionated animal chromatin, little has been reported on corresponding fractions from plants.
When 72-hr. maize seedling chromatin (FRMO17 X FRN28 hybrid strain) was digested with DNase II, a longer incubation period (90 min. vs. 15 min.) and a lower DNA concentration (from 370 µg/ml to 75 µg/ml) than required for animal chromatin was necessary to achieve proper digestion kinetics. The amount of Mg2+-soluble "euchromatin" released, at 14%, is comparable to animal chromatin fractions.
The distribution of chromatin proteins in fractionated animal chromatin has been well characterized, but data on plants are lacking. In the present study, total chromatin proteins in each fraction were dissociated with SDS, then reduced with 2-mercaptoethanol prior to electrophoresis on 12% polyacrylamide SDS slab gels. Molecular weights (MW) were determined from histones and other standard proteins. Positions of maize histones were confirmed by prior extraction with 0.4 N H2SO4. The results are shown in Table 1. Histories H1, H2A, H2B, H3, and H4 were present in both euchromatin and heterochromatin fractions, but each had common and unique H1 subfractions. Also found were low MW nonhistone proteins (NHCP) in the MW range of 15,000-20,000. To determine the existence of HMG proteins in this range, chromatin was extracted by standard protocol (Spiker S. et al., PNAS 80:815-819, 1983). Two such proteins were found at 19,000 and 15,000 MW, respectively, in each fraction; on the basis of MW, extraction in 0.35 M NaCl, and solubility in 2% trichloroacetic acid (w/v), it is possible that these nonhistones correspond to HMG proteins.
Each fraction contained distinct electrophoretic NHCP patterns. Table 1 indicates the various MW range positions of NHCP in euchromatin and heterochromatin fractions; individual proteins are not listed. Relatively few proteins were common to both fractions. Heterochromatin contained over 15 distinct NHCP bands and various fainter ones, while the other fraction contained fewer. However, the NHCP are the predominant proteins in the Mg2+-soluble fraction.
It appears that NHCP and histones are distributed among maize chromatin fractions in a manner similar to those of animal chromatin, as reflected by the variety of unique NHCP as well as unique H1 subfractions in each chromatin fraction. However, HMG proteins are enriched in animal Mg2+-soluble chromatin, but this was not observed with maize chromatin.
Table 1. Summary of proteins found in fractionated maize chromatin.
S. E. Palmer and V. Ulrich
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