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

a2i2 F: cdl dcte conrrot o! H1 ctu fdh*tiotlan
tor normalizing the blors. Dudng this lhesis, hwas eshblished whether FNase
prolection sssays can be used lo measurs H4 gone Promoter adivily during
The srabre celrine conralnlng lh€ wildtype H4 prcrnotff (Fo108/cAT)
was synchronzed and hatuested RNA sampl€s at hourly lime-points lolowing
release lrom a dooble rhymidine bock, FNA samples were hybrdiz€d to
inrernally labelled fadiolabelled rlboprcbes speciic for CAT, hislone H4, and
GAPoH mFNAs, slbjecr ro RNAso digestion and qel eiecrrophoresis of the
dige$jon-products. 'rhs signal lor CAT mRNA represents a medule ot th6
extenr ol histone gene promoler aclivily, which is diredly relaled to histone H4
gen€ t/ansciption. The H4 mRNA leveldelectsd in our assays is lntluenced by
bolh kanscription oflhe gene and post-kanscriplional regulation oi tho mFNA.
Histono mRNA is slabitiz€d dudng eady S phase and desrabilizod at rho
completon of s phase. GAPDH mBNAs encode a consiitutive house-
keeping'enzyme involved in gycolysis, and can be used lor normalizallon of
The results ol a iypical analysis lo. the wildtype H4 p.omoter in
synchronized cells containing lhs H4-Foro8/caT construct (Fig. 31) shows
lhal CAT mRNAs are mdimal al 2 to 3 hr atler enlry ol cens inio S phaser
preclsely as prcdicted based on dired measuremenrs ot H4 g€.e lranscriplion
by run-on anaLysss. ln mnrrast, H4 mRNA conrifue lo .ise du€ lo post.
lranscrptional evenls thal lac lilate furlh€r accumu ation oi histone mRNA
levels (F q.32). However, GAPDH mFNAlevels romalnconstanl during th€ cel