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

azi. F: cetl cttcL ctu.t or Ha Gtu lftMtip.ion
prob€ (1x106 cpmlml) was added. The m€mbrane was incubarodinthe
presenc€ ol prob€ tor 24 hours with agiratjon_ Th€ membrane was
removed lrom fte cylinder and washed iwice ar 65.C in solltion i (1
mM EOTA, 40 mM NEhpoa pH 7.2:5% SOS) Ior 30 to 60 m nll€s
whlle shaking, iotlowsd by two washes in sotllton lt {1 l.ntv EDTA, 40
mM NaIHPO4 pH 7.2 and j % sos) tor 30 to 60 minltes at 6s,C. rrs
moist membrane was wrapped in ptastic toit (Saran wGp) and oxposed
to x,Ray oMAT-AF fih, and ptaced at _70.c with an inlensifying
screen overoighl.
The titm was devotoped and the membran€ was subjected ro rh€ Bela
scope 603 anaiyzer (Betagen, MA) over.ighl. The intensily oi tie
genomic DNA iragmed spanning the tnregrared hisrone H4 CAT
reporter gene plasmid w6s used as a m€asure tor the copy nlmber of
he ntegrared constructs (s6e Ftg. 17). Th€ copy nlmber vaiues
obtain€d this way were us€d lo catcLrtal€ the tevet ot expression por
inbgrated copy ot each H4 CAT consrtuci. (See Fig. 18 )
synchronizarbn ol srabte HsL, c€[ unes
- Suspenslon cutiureot HeLa S3 C€[ lin€s wnh dablyintograred
F/ozen siocks oi stable Heta Sg monotayer cetis were lhawed in a 37 . C
water barh. cers wers grown sl a d€nsity ot 3.ox1o5 c€ts/m|n SMEM
(Joklik-rnodltod minimLrm esse.tiat medtum) supptemen€d wrrh 5%
carr serum, 2?q holse serum, l0O unirs/mt peniciJrin 1oo /rglml
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