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

ND F Cen atct6 cannot ot B4 6ene rrctctlptian
37'c (wam foom). cell pellats w€re washed lwic€ with 10 ml cold
'soronic bufi€r which is (125 mM KCl.30 mM T.is/HCl. pH 79 5 mM
MqCl2, and 10 mM !-mercaprcethanol). From lhis point on cens we.e
kepl cod througholt the romalnder ot lhs proc€dlre. Cells w€re
centiluged at looo rpm and r€susP6nded in 10 ml cold hvpotonrc
('swelling buller': 10 mM kcl,30 mM T.is HCI pH 7 9' 5 mM MqCl2 10
mM B m€.capioelhanol) and allow€d to swelltor 10 ' 15 minubs Cells
were dou.ce homogenized wilh a tight Pesrle lo. 10 - 15 sl'okes; a
smallaliquot w€s stained with 2% trypan blue und€r |h6 mrcroscope lo
checl ii cells w€re lysed). The lysato was transl€r€d lo th€ 15 ml lubes
and cenr.ituged at 15oo lpm lor 5 ro 8 mind€s in lh€ lEc rotor' The
slpernalanl was drainod carefully, and the nucleiwere resuspendeo n
storage buter (50 mt\4, (NH.), SO1' 30 mM Tris/HCl pH 80 5 mM
MgCl,,1 mM MnCl,,lOmM t-morcapro€|hanol 25% v/v qlvc€roD The
nuclei were coumed and divided inlo aliq@ls ol approximalelv lxlot
nucle/1oopl. Nlcei wer€ imm6dietely l.ozen i. Liquid nitogen and
stored at -85 C {See
TranscriPiion reaclion
F g.21).
Assessmenl ol transcription rates in isolared nuclei was accomplished
accordng 1o 1he procedure by (Gr€enbeQ and zlt1, 1944) Fozen
a iquob ot 1t1O? n!cl6i/1oo /rl wer€ lhawed our on lc€ a'd qlickry
added to 15 ml cofex tubes conlaining 1oO ,,1 o, 2 t reaction buffer (10
mM Tns/HCl, pH A.O, 5 mM M9Cl2 03 M KCI). which was
9a