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

ND F: C.ll C\cte Cdtuot ol l1a GM I@Eaoti@
translalional stan codon; lh€ mFNA cap sit€ is localed at nt _3o) (van Wjnen et
a/,, 1991). This conslrlci lvas frst dig€sted with th€ r€striction enzym€ Nae I
ioltowsd by ethanol prgopnaiion. Tho dig€sled DNA was then cut wilh Pst I to
relsas€ rhe wild lype H4 site ll seqlonc6 which spans nt_93 lo .3a
lsolatlon ol DNA lragmEnts
fte large Nael/Psll lragment (containing th€ remainder oi lhs H4
prcmor€r fused to puclg) was isolaled Lom 1% agaros€ gel a.d eluled in
dialysis rubng (moecular weighr cul-oil of 12,000 - 14,000). fte lLrbing was
prcparcd as descibed prcviously (rlranialis el ar, 1982)- The tubing was cd
inro piec€s of con!€nienr lengrh (10 - 20 cm) and boiled for 10 minutes in 2%
(w/v) sodium b calbonale and 1 mM EDTA (ethylons diamine tetra acetic acid)
solulon. The tlbes were wash6d ssveraltim€s wilh dslilled H20 and lhen
boilad agBin ior t0 minures in 1 mM EoTA (pH 8.0). The tubss wsre kept
storedar4'cin a large volum€ ot TE bufe. (10 mM Tis.HCl, 1 ml"{ EOTA, pH
8.0). Thrclghout ths procedLr€, lh€ dialysis tublng was hand ed wrh
Th€ srltsd oNA was purilied by passing the eluate through ElutiprM
min-collmns (Schlecher and Schuell) according to the ma.ufacturers
suggestions- In bri€i, Elulips were cut at the botton and wash€d with 2 ml high
salt bufer 0.0 M Nacl, 20 mM Tris-Hct, pH 7-3-7.5, 1.0 mM EDIA) using a
syfng€, subs€qlenrly the syinge was washed wilh 3-5 rnlof low sall blfier
(0.2M NaCl20 mM Tris-HCl, pH 7.3-7.5, 1.0 mM EO'A). A iiher was lixed lo
lhe Elutp ro r€move gel padicles and lhe DNA sampls was passed hrough
34