During our work with maize chromatin, several existing methods for the isolation of plant nuclei have been employed. These methods have been optimized for other plant species and/or tissues. We have found most to be unsuitable for etiolated maize shoots, for nuclear fractions did not meet the criteria for purity and yields obtained with the original tissue or plant. An early method which isolates a nuclear-chromatin fraction (Bonner, J., et al., Methods Enzymol. 1213:3-65, 1968) yielded few intact nuclei despite high yields of nucleic acids and chromatin proteins, and thus allowed the possibility of cytoplasmic contamination. Grinding of maize shoots with a mortar and pestle in a solution containing glycerol, sucrose, KCI, and tris buffer, pH 7.0 (Stern, H., Methods Enzymol. 12B:100-112, 1968), gave a clean preparation of intact nuclei, but the yields were low and the time required was excessive. The popular method of Y. M. Chen et al. (Plant Physiol. 56:78-82, 1975) employs homogenization of tissue in a solution including glycerol, sucrose, KCI, MgCl2 in MES buffer (pH 6.0), followed by centrifugation through dense buffered sucrose. This technique resulted in extremely low yields of maize shoot nuclei. The method of R. H. Hamilton et al. (Anal. Biochem. 49:48-57, 1972) involves rinsing the tissue in ether for 30 sec., followed by homogenization in a Waring blendor in sucrose, MgCl2, and tris, pH 7.6. High yields of a nuclear fraction were obtained from maize, but the nuclei were severely disrupted.
Various homogenization and filtration techniques were tried with each of the above methods. In general, certain trends were consistently observed with maize seedlings, regardless of the media and method used. The Waring blendor caused severe fragmentation of nuclei in all situations. Homogenization via the Sorvall Omni-Mixer increased the number of intact nuclei in the preparation. Best results were consistently obtained by the use of the VirTis "45" homogenizer. Filtration of the resulting slurry was best accomplished by the use of cheesecloth and miracloth (Chen et al., 1975) rather than mesh or screens; this gave high yields with less contamination.
Methods employing gum arabic in the isolation media gave improved results with maize shoots. A recent method (Stout, J. T. and C. K. Hurley, Methods Cell Biol. 16:87-96, 1977) gave higher yields of chromatin material, but fragmented nuclei and contaminating debris were abundant. The method of K. J. Tautvydas (Plant Physiol. 47:499-503, 1971) gave superior results when reduced-volume gum arabic gradients were used with the original 14-hour incubation period; high nuclear yields were obtained and low cytoplasmic contamination was observed. Activities of endogenous RNA polymerases remained intact (Nebiolo, C. M. and V. Ulrich, Fed. Proc. 41:1294, 1982).
To avoid the proteolysis of nuclear proteins, however, a shorter incubation period was desired. We have developed a modification of the Tautvydas method which is optimized for etiolated maize shoots (to be published). Briefly, 72-hr. shoots are harvested and placed on ice; all operations are performed at 0-4 C. Proteolysis of histones and nonhistone proteins is prevented by the inclusion of 1 mM phenylmethylsulfonyl fluoride and 1 µg/ml soybean trypsin inhibitor (TI) in all solutions. 5.5 gm of tissue are minced and rinsed with diethyl ether for only 1-3 sec. (prolonged exposure causes severe nuclear damage). The tissue is vacuum infiltrated (4 min., twice) in 20 ml of Buffer A, or 4% gum arabic (w/v) in Buffer B: 0.15 M sucrose, 4 mM magnesium acetate, 5 mM 2-mercaptoethanol, 0.1% octanol (v/v), and 5 mM MES (2-[N-morpholinol ethanesulfonic acid), pH 6.05. Following storage in the dark for 3 hr., the suspension is sheared for 8 sec. at medium speed with a VirTis "45" homogenizer and filtered through 4 layers of cheesecloth and one of miracloth.
The filtrate is layered over 4 step-gradients, each constructed of 12 ml layers of 12%, 10%, and 8% gum arabic in buffer B. These are centrifuged for 12 min. at 900 g in a swinging bucket rotor. The cellular debris trapped in the upper layers of the gradient is aspirated, and the nuclear pellets are homogenized in 24 ml of Buffer A by one stroke of a glass-teflon tissue grinder (.13-.18 mm clearance). The suspension is layered over 3 additional gradients and centrifuged as before; the final pellets are resuspended in 30 ml of Buffer B without TI. If desired, 3 lots of tissue (5.5 gm. each) can be processed simultaneously, each staggered by only 10 min. to prevent autolysis. Aliquots can be routinely stained with aceto-orcein to monitor purity and quantified on a hemacytometer. This procedure yields 5-7 X 107 nuclei from 17.5 gm of the FRMO17 X FRN28 strain of maize. Examination by EM showed intact nuclei with little cytoplasmic contamination. Subsequent chromatin isolation and analysis of DNA, RNA, histone, and nonhistone content, as well as digestion kinetics with DNase II, show these nuclei to be quite suitable for a variety of biochemical studies.
S. E. Palmer and V. Ulrich
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