Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...
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,dd f . c.t cfi|e cotud d <strong>H4</strong> cd'e f@daton<br />
2 sr 10 X readron bunor (suPPli6d by N.w England Blorabs; 10 X Bulrer<br />
3 or 10 x butfer 4 was us€d; 1 x 8ufisr 3 contains 50 mM Tris_HCl, pH<br />
79, lOmMMgCl, IOO mM NaCl, 1mMOTI| l XBufi€r4 contains 20<br />
mM Tr s'ac6tar€, pH 7,9,10mM megngsium acetal€,50 mM polassium<br />
acetate, 1 mM DTT), Th€ fsacton volumewas b.ought up to 20rlwllh<br />
distiled wal6r. Feactions w6rs InitlaGd by addilion ol 10 units ol<br />
enzyrne (added in 2 ,rl a iquots lrom diluled €nzyme stocks) Fsaclions<br />
were rncubated al 37 c lor 2 . 4 hours, The samples we.e lradionaled<br />
by 1% agarose eleclropho.Esas. lollow€d by erhidium b.omide $aining,<br />
and eEmination using long-wave uhr6viol6l light p10 nm 6lter) (see<br />
Fig.9)<br />
DNA €eq!enc*anarFl6<br />
Each mulanl co.strlcls was sublsct€d to s€quence analvsis oi lhe<br />
reglon m lhe <strong>H4</strong> promorer suiiollnd ng Fl4 Sil€ llin bolh lhe sense and<br />
antisense stands. Ths p.ocedur€ was pedom€d to connrm pfesence<br />
ol the deslg.ated mLrlatlon, and abs€nca of possible mutations<br />
genera@d during lhe Kl€now medialed cloning prod€dlre. sequence-<br />
analyses wer€ pedomed by uslng a commsrcially avaibbb oNA<br />
sequencmg kn (sequenass v€rsion 20i usB carabgue number<br />
70770). As described by rh€ manufactursr, app.oximalelv 4_5 pg ol<br />
clean DNA (lreated wth FNas€ or CsCl preparation) was h€aled with<br />
one primer (ehhe. the foNard primsr or rcv€rso primor) at 95'C lor 5<br />
minutes. Sequenase 5 x reaction bufi3r was added and tubes were<br />
45
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