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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aziz F: call cr'cl. cqttnt at <strong>H4</strong> Gqe 7ldns.detian<br />
mulant <strong>H4</strong> promoter/CAT tusion construcl- To engineer fte cAT repoler<br />
gene in ifo ol rhe mulant promot€r sequsnces, lhe inlemedialo conslruds<br />
were dig€st€d wilh Psl I, dephosphorylaied as d€scribed below and puriied<br />
Th€ llneariz€d inrermediate plasmids w€r€ lr€aled with calf lnlestnal<br />
phosphataso lo remove lhe 5 teminal phosphale group. This step reduces<br />
self ligation dlring the lgauon reaction and w€s Pedormed as described<br />
prevjously (Maniatis el a/., 1982). In biet, lhe working butfer lor the snzym€<br />
conbins 500 mM Tis Hcl (pH 8.5), i mM EDTA and 1 mM spenidlne, and<br />
's<br />
prcparcd irom a 10 x bufter obtained lrom lhe supplisr (Boehringer<br />
Mannheim). Ihe desied ONA fraqmenl to be d€PhosPhorylated was mrxed<br />
wilh 0.01 unil per pmol 5'ends at 37'C tor 30 minules. After the li.st<br />
inclbaton, a second allqlol of th€ 6nzyme was added a.d lhe sample<br />
rncubaled tor an additional30 minutes at 50'c. The reaction was stopped by<br />
adding 0.1 [4 EGTA (elhyleneglyclfbis(p-aminoethyletre.) N,N,N',N:telra<br />
acetic acid) and lhe enzyme was h€at- nactivaled tor 30 mlnlies at 65'c. Ths<br />
DNA was €nraded twics wnh an equal volume of phenol/chlorctom/isoamyl<br />
atcoholi'25:24:1vlv/v)(saru€led wirh 10 mM Trls/Hcl, pH 8.0 and 1.0 mM<br />
EDTA) afd recove.qd by ethanolprecalat on.<br />
The nsert lragm€nt was prepafed by digssling pFOloEcAT wlh Psl I<br />
ro rerease lhe 1 7 kb Pstl/Psrr kagmenl spanning lhe chlo.amphenicol<br />
36
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