A greater degree of purity of nuclei isolated from conidial cells of N. crassa is
obtained by combining the procedures described by Reich and Tsuda
(1961 Biochem. Biophys. Acta 53: 574 ) and Munkres et al.
(1966 Neurospora Newsl.9: 14) for mycelial cells with some modifications
made in our laboratory. The strain used was wild type 74A and al I operations are
conducted in the cold at 0-4'C . The conidial mass is squeezed dry between
Whatman blotting papers and then ground gently with twice its volume of acid-washed
sea sand (prepared by powdering the commercially obtained sea sand in a Wiley Mill
and passing through a 60-mesh screen ) until a smooth paste is obtained.
About five volumes of sucrose-EDTA (0.5M sucrose, 1mM NaEDTA, 0. 01M Tris HCl, pH 6.5)
is added gradually, stirred into a thick paste, and filtered through four layers of
silk cloth. The filtrate is centrifuged at 2000 x 9 for 25 minutes in a refrigerated
centrifuge. This crude nuclear pellet is then suspended in a solution containing
0.5 M sucrose, 2 mM EDTA, and 5 mM CaC12 at pH 6.5 and centrifuged at low speed
(500 x g ) for two minutes. Two kinds of pellet were noticed. A hard pellet
was formed below the loose pellet. The nuclei contained in the loose pellet
were dispersed in their own supernatant and centrifuged again at 500 x g for
two minutes. This low-speed centrifugation was repeated until no more hard
pellet was noticed.
Comparatively pure nuclear pellet was obtained by passing
this final loose pellet through 1.70 M sucrose solution containing 1 mM EDTA.
This nuclear pellet was further cleaned from any cytoplasmic attachments by
suspending and stirring for two hours in 10 volumes of saline EDTA
(0.08 M NaCl, 0.02 M NaEDTA, pH 6. 2 ), sedimenting at 2000 x g and
resuspending in fresh solution of saline EDTA. In our process of chromatin
isolation from these nuclei, we use triton-X-100 (0.01 %) along with saline EDTA
in order to reduce the surface tension of nuclear membranes. The yield of nuclei
was low by this process but a consistent purity (as judged by electron microscopy )
was obtained, showing a 5:1 ( total protein: DNA) ratio.
Supported by NSF Grant No. Gy3894. - - -
Department of Botany, Howard University, Washington, D. C. 20001.
Return to the FGSC methods page
Last modified 9/04 KMC