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

kir F: cettctttd contal at H4 cen. fnrcutptt,
Mateiats adt Mott'ods
lrom vector sequences Folbwng ethanol preciplr:lo' the p€nels
'ragm€.t
were dissolved in 40 r, TE bufigr, and 10 ,rl ot 5 x dy€ was add€d pnor ro
Samples were loaded omo a 4% (3Ol) polyac.vlamide gel contanrng 1
x TBE butle.. Gels were prepa€d by mi{ng 10 ml ol 10 x TBE, 10 ml 40%
(30:1) acryiam d€, and 80 ml ot HrO) Tho solution was subjecl€d to a vaclum
( de.gassed') lo remove oxvgen which inhiblE rhe polvmeizaio' reaclion
Tho gel was polymgrized by adding 50 rrl ol TEMED (NN'N'Nr
lelramelhy'erhylenediamine) and 5OO d ol 10% ammonium persulphale Th€
soluiion was gently swided (while avoiding fo.mation ol an-bubbles) and he
solution was pour€d in a ca$ considing ol 24 cJn X 12 cn precision_cut g|ass_
prares (Hoele4 and allowed to polvrneizs lor ons hour' Ihe gelwas p'e'r!n
for al ea$ 30 mlnules. The gels containlng tM probe sampleswere run lor 2 5
hours rhe Localon ol ihe corect DNA band was idenli'ied by
aubradiography ot th6 wel gel, and th€ €orr€sponding gel slic€ wes isoralBd
using a scalpel. The ONA in rho gel slice was recovered bv el€clrophoresis
inro dialysis bags and ihe DNA was recowr€d wilh ethanol ptecipita on
The second step involvod pr€psra on of nuclea' prot€ins Nlclear
exvacls were prepa.ed accordi.g 1o lhe Dignam pr@€dure (Dignam d a''
19s3) lslng HoLa 53 cels (d€.sily 7-9x10! cels/ml) as d€scribed pr€viouslv
(vanwiine. elal., 1991b,c), bd magn6sium_salt was.€placed i' allbur'€rs by
1 mM EGTA,0.2 mM EDTA, O 75 mM sp€.hidine add015 mM spermlne and
a broad sped.um proiease inhibitor cocktarl was used {PMSF' leupeptn'
63