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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hnF cet cyttd cannot ot B4 6end ltunrc ptlan<br />
abG we.e airdried and expos€d lo prcllashed X'OMAT AR (Kodak) X_<br />
Ray fih botw66n tlvo Cronex Lighling PlusiM i.rensitving scr@s al _<br />
7o'c. Blols wore quantnaled by subjecling $€m lo a p_emEson<br />
analyzer (Beta scope 603, B€iagei) The valu€3 w€re expressed<br />
relativolo ribosomal RNA after co sclion io. non-sp€cilic binding by<br />
puc19. The r€sulls ol rhese transciptional run_on analyses {see Fig.<br />
22) were tound to be hiqhly va.iabre and wore nol us6iul lor anarvsis.<br />
The rbosomalFNAwas iound lo b€ Pa.ticubnv v4 abl€ du.ing the limg<br />
cou6e (dala not shown). Ihorsloro, lh€s6 erp€ m6nlsw€r€ penormed<br />
with RNAse protection assay, dsscnb€d in the lolloring section<br />
bo|arion oi tolalcellut3r FNA trom msmmalian cells<br />
Totalcellllar BNA was isolaled from synch.onizsd stabl€ Hela cels al<br />
various t me points after release lrom he synchroniza on piocedlre bv<br />
established procedures (choimcynskiAnd Sac.hi 1987). cell cultures<br />
(co.taining approxlmalery 3.7x1or cells/ml) we.e translered lo 50 mL<br />
plaslic rubes 6nd centrauged 4'c at 1500 rpm lor s mi'utes cell<br />
per els w€r€ wash€d lwico In phosphal€ b!n6r saline (l 50 mM NaCl l0<br />
mM sodium phosphare pH 6.8), and €rlpollets w€r6.€susp€nded in 4<br />
ml ol guanidinilm soluion (5 M guanidinium, 0-75 M Na cirate pH 7 0,<br />
0.36 M p mercaploelhanol). The slisponsion was hor'iogenlzed !si'g<br />
rhe Po yrron, wh ch<br />
nucl€i and genomic oNA subseqlently'<br />
'ragments<br />
1/loth volume ol 20% sarcosyl was added and 500 ,J ol lhe ninure<br />
was alquoled in 2 ml sl.aigh cent iluge tubes (Ph€nit) The samples<br />
105
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