Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...

NE F: Cell C6t cotuat .t H4 cffi fhNtiotlan
complem€nled with unlab€lled and labetled nucleolds triphosphales [2
mt!,t dATP, I mM dCTP, 1 mM dGTP and 100 pciol 9PUTP (10 rlol a
10 llcilll stock-solullon with a sp€cilic aclivty ol 3000 cilmrnos)j.
Nlcl€as€-lree glass tubes and solutons were ls€d thfoughout the
prccadurc. The samplos (olal roadion volume ol 200 /]r) werg
indbared al 3o'c for 30 minutes with intermit€nt shaklng Alier
incubaring the sampies lor 30 minut€s at 30'c, 600 !l ol nNase free
DNase | (Promega) in high s€ t bufie. (0.5 M NeCl, 0 05 M rr/gc 2 0 002
M caot and O.o1 rM T.islHCl pH 74) was added. The samples were
r4uspendod by.epetitiv€ mixing wlh a Pasleurpipelte
which lacilitates
lhe breakdovin oI nuclei and DNA, end lhe susp€nsons were
incubated al30'C for 10- 15 mlnjJl€s. To each oilhe r€acion m xtures
2oO /rlol protenase K (2omg/ml) n proteinase Kbufier (5%SDS 0 5 rv
Tris pH 7.4, 0.125 M EOTA) was added, and tubes were incubared ar
37'C ior 30 m:nules wnh gen e shaking at regular in€tuals. Reactions
were stoPPed by addlng 300 !l(.e. 1/10 oI the odginalvollme) ol2 M
NaoAc pN 50 lo a linal concentralon 02 1\,l The volume oi lhe
samples was ilrther increased by addi.g 2 ml ol 10 mlr,4 T s/Hcl pH
8.0 and 1 mM EOTA Reaclions were extlacled several iimes with an
equalvolume ol phenol eqsilibraled with 0 2 M ol NsOAc (pH 5 0) and
10 mM EOTA. The radiolabelled lranscriplion reaclions were
ranslerr€d lo 30 ml cor€x nrbes and nlc eic ac ds were precipilal€d n
ihe prosence or 75 /19 glycogen (Boehringer l'lannheh) and 25
lolumes ot 95% mld elhanol. The sampies {€re kept at 20'c
101