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Cell Preparation
Cells were prepared by the agarose bead encapsulation method11 with minor
modifications. A 200 ml YEPD culture was grown at 30 C to an O.D.600
of 1.0 and centrifuged at 400500 x g for 5 minutes at 4 C. Following
removal of the supernatant, cells were resuspended in 10 ml of SE (75
mM NaCl, 25 mM Na2 EDTA, pH 8.0), recentrifuged, washed twice, and resuspended
in 4 ml SE. In a 45C water bath, cells were mixed with 5 ml 1% low melt
agarose (Bio-Rad Laboratories) in SE, prior to addition of 20 ml temperature-
equilibrated mineral oil (Squibb). The resultant emulsion was mixed vigorously
for 30 seconds before it was quickly poured into a beakerkept in an ice
bathcontaining 100 ml of ice-cold SE and a stir bar stirring at medium
speed. The mixture was stirred for several minutes before being transferred
to several graduated polypropylene tubes which were centrifuged as before
at room temperature. The mineral oil was removed and any beads not pelleted
were dispersed by repeated pipeting with a large-bore pipette.
After recentrifugation and removal of excess SE and beads that did not
pellet, the remaining pellets were combined into a single tube and centrifuged.
The supernatant was removed, the inside of the tube wiped with a tissue
to remove excess mineral oil, the volume brought to 20 ml with 25 mM Na2EDTA
and the final SDS concentration to 1%. After repeated pipeting with a
wide-bore pipette to break up clumps, the tube was taped to a platform
shaker and shaken for 10 minutes at room temperature. Following recentrifugation
and removal of the supernatant, 0.5 ml of 2-mercaptoethanol and 10 mg
of zymolyase-100T (Seikagaku Kogyo Co.) were added, the v
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