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

Alir F: Cel cycta conxot ot t14 G@. t@cdpuan M.tlnate and Mahads
%
lhe cellpellets as abov6, and the p€Iets w€.€ r€suspsued n I rnl o, soturion I
(5O mM gru@se,25 mM Ttis-cr pH 8.0,10mM EDT pH 8'O), 1 mt of ,reshty
prepared ryso2ym6 sotution (10 mg/m|n 10 mM ris_ct, pH 8.0) and 20 hr ot
soruliof ll (0.2 N N€oH and i% sDs)_ Tub€s wor6 invert€d manytimos lo mir
r.6 conrenls genly and incubared at room temp€ratu€ tor 5 minur6s.
slbsequendy. 15 mt ot ice.cotd sotdion t (sotlrion t is compossd oi 60 mt5
M potassium acetat6, 11.5 mtgtaciatacatic acid, and 2B_5 mt dis ||ed H2O)
was added and tLtbes were inv€ded sev€rat ltmes to mix rh€ slsFension.
sampres were incubaled on ice tor m minut€s and centnfuged tor30 minules
al 15000 rpm d 4rC in a J2-21 Beckman c€mriftrge using rh€ J-20 rotor.
Supernah.ts w€re sbved rhrough the ch€es6 ctoth and rranstetred to another
srerile tube. DNA was p.ecipitated with 0.6votum€ oi isopropa.otand washed
once wrh 70% erhanot.
oNA peltels wer€ djssotved in I mt ol fE-bufier, and exac y 9 gm o,
CsCl p€r 6 mr or ONA soturion was addod to tha samptes, The cssium slt was
ossorved at room remporarura and 1 mt ot €lhidium bromide sotltio. (10
mg/m ) was added lhe conrenls of rh6 tubss w€r€ lhen tan$t6fied nio qurck
ssalllbes Tlbes wer€ spun in a ]lTO ro!o! tor a minimum-period ot06hours
ar 48,000 rpm d 20,C, Tubes were removod whjt€ taking cars not to disturb
rhe cesum chlorid€ gradie.r The ptasmid oNA band was id€ntilied wilh short,
wave UV lght and putt€d our ot the lube using a syinge, Th6 DNA was
gxiracred s€vefal tim€s w h wateFsaturat€d blianot to r€mov€ €rhidium
56