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

jziz F: cell cycla cannat ot H4 Gen. f@sqFtq
smal/Nml lragment ot pFo108x) ot the human hisione H4 g6ne, This
canstuct was kindly provided by Ihomas J. Lasl.
ln vtro transcripllon reaction
Tho reagonts of lhs SP6 lM€xi ScripltM kii were lhawed a^d placed on
ice, The reagenis were added in RNase f.66 Eppsndorl lubes (1,5 ml con cal
size) in the tollowing ordsr: 2 !lol 10 x tansciplion bufler, 1 !l ot 200 ml',|
OTT, 1 ,rl ot 10 mM ATP, I /l 10 nM CTP, 1 /l 10 mM GTP, 1 lll RNaso
inhibitor, 1 /rlor linearized template DNA (1pg),3!1o10.05 mrvl UTP,5 rrlor Ic-
kpl UTP 800C /mmol a.d i rl ot SP6 BNA po ymeras !r The contenls of the
lubes were mixsd and lhe tubss wer6 incubaled at 37.C tor 30 minutes. Atr6r
lhe incubalion, 1 pl oi RNase{ree DNaset was added to €ach reaction and lhe
conlenls were mlxecr by ticking the tlbes. The samptes were briefly
centriluged and incubared at 37'c tor 15 minutes. Tlbes were rranstered to
r@ and the transcription reaction was passed lhrolgh nuctease-tree ouick
Spinrt collmns to pu.iry rhe radiotabetted p.obe tom unincorporated
radLoactive ibonucleolideriphosphales. The spgciic acuvity was derermined
by counting 1 rl in 4 ml ol scintittarion tluid using a sc'nriilarion counler,
approxmalely &(10.1o 11106 cluots were used per /eacrion ro pertom lhe
ribonuclease protection assay.
Hybrldizarlon ot F NA transcrlpts
Th6 li|sr st6p invotved the
and 5rrgolsample FNA. Th€ RNA sampres wsre isolated dlring diflorenr time
@urses usng synchronized Hela
co.precipitation ot probs Oxlo5/readion)
cells conraining stably integrared H4/CAT
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