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

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o lu r60 120 80 Con3lruct binding: Stabtg cell llnos ot <strong>H4</strong> promotsr CAT conslructs HINF-tl blndlng slto mutants toa wl usP-16 ltPr-r7 tGM.18 P+lrI-lO+ P-l)l-lDt P-/lU r/Dr 69
ktz F. cen ctct6 ctuat at tu 6e rftN.tipti@ stepromycin and 2 mtM oi L-glllamine c€Lls wefe maintalned in suspension llasks, with 6 capacity ot twice lhs volume of lhe cultur6 As lhe c€ll lines were initjally gtown on plates, it was necessary ro adapt |he siable calllinssto growth ii srlspension by slowly adiusllng mediLlm conditions. Inil ally, c€lls wers Prov d€d with h gh concenlrations ol letal call serum a.d L-glutamin€ and lhen gradually shiffed to comdeb medium as lollows. The lirst two days, lhe cells were led win5 7% lelal cali serum, 2 r,rlv L-glllamin€ p€nicilin and sreflomvci. as desdb€d above, and a 1 x mixture ol non_essential amino acids. The cells wer€ checked daily under the microscoP and fed al regular dailv inlervals with pre-warmed lresh m€dia. After two davs, !]e celrs rere ted wilh s[,lE[4,5% l€ral ca]t serum,2o,6 calf serlrrn, 2 mM L-grulamne, peniclllin/strepromycin as abov€ wilhout non-€ssenlial amino acids Subseque.tly, the cells were shilted to SMEM supplemented with 2% fetal call serum and 5% call serlm. Alter two additional daily feedings with lhe latter medilm, lhe cells wsre led wilh SMEM supplemenl€d with 1% letal cai s€r!m, 5% cal' se.um, and 1% horse donor serum Finally, cells were sh:ned ro SMEM supplemenled with 5% cll serum, 2% donor horse serlm, and 2 mM !-G[nam ne Pe.ici]lin/slfeptomyon The c€Lls were grown at leasl 4 to 5 days in comp eie med a GLycerol stocks were made by spinning the cells down at 1500 lpm in the lEc rotor and lhe cell pellet was resuspended in sbrage media (70% SMEM,20%l€lalcali s€rum lovo glvcerol) Ce ls were stored in 4 5 ml cryotubes al a d€nsity oi 1x107 cels/ml (4 5x107 cels per lube) and 90
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\ ?^p 6r.1 CELL CYCLE CONTROL OF HU
Page 3 and 4:
In tho narn€ of Alah, olt G6cloua
Page 5 and 6:
Acknowl6dg6msnts Contents Abstract
Page 7 and 8:
Selection ol cell linss contalning
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ACKNOWLEDGEMENTS
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I would also lkg ro rhank Dr. Pafic
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ABSTRACT
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i.leraclion elem€nt, sil6 ll, Thr
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8. Colony hybridizalion sc.eening o
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INTRODUCTION
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t t F: con cda c@tot d H. G@ r@Mbn
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Azh F: C6n Ca/cro C@t.t ot H. G.n.
Page 25 and 26:
Aziz f: Celi Ctcle Cotutol ol H4 Ae
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= 2. q t6l tl9t E L:I la-lf titl I.
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En F. Ce Cycl. Cantd d h. Gft ra.sc
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,r1z F Cett cy.te connot ol H4 Gene
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A2i2F: Cettcdt cotubtot e@IrNiotid
Page 35 and 36:
Ntz F: Ce Cwte Contd d 114 Gm tansc
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t- s- i;> f z x + n q e o, { T T z
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AzzF cett Crct. ContolatH4Gen ldn*t
Page 41 and 42:
a2i2 F cdt c$t. connd d H4 Gm Tdnsc
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o rl$orrE GEIE FEEI{IXU =; FrN -!r
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!e 3lhllrllon c.I cycl. control aa
Page 47 and 48:
A.E F: Cet CEl. Cotuot ot H4 t3.M f
Page 49 and 50:
Crct C@nd d H. GNf,'/MAi@ '2izE:Ce
Page 51 and 52:
9C| -= = , o:li?eiP :< :::? il il 7
Page 53 and 54:
s.tudi. dhcrm or do"rn! -l /4 I I I
Page 55 and 56:
aziz F: cel crd. cotuot d 81 cd. fn
Page 57 and 58: a2lz F: celt cyd. c.ntol d H4 G@. f
Page 59 and 60: A2iz F Cett cycta contat al Hl Gene
Page 61 and 62: E2 F. cen crct. Connat al H4 c*e Ta
Page 63 and 64: Colony hybridization 4a aa a a
Page 65 and 66: ,dd f . c.t cfi|e cotud d H4 cd'e f
Page 67 and 68: Restriction Analysis Of Mutants e'b
Page 69 and 70: ,d, F: ccl crc!. cqfrol ot H10d fdt
Page 71 and 72: AtIzF: Cd CJ.L Cdtuotol BaGq:€lat
Page 73 and 74: ,.8 F: 6t W @tlol o, rk 0.,t rn',df
Page 75 and 76: ldD F: Cett CrL Cotuot at t14 G* Iw
Page 77 and 78: A2E F. cdt crcto Cantat at Ha Gqa r
Page 79 and 80: Aziz F: Cdl Ced. Cd*ol ol H4 Gse ra
Page 81 and 82: o E o !!tH I rtog 5l €s c_g - t-
Page 83 and 84: kir F: cettctttd contal at H4 cen.
Page 85 and 86: arE F: CeI Crct Cotuatat 114 G40 fa
Page 87 and 88: c A B e'l' t i _ f '', \J..$.'
Page 89 and 90: Nz F: Cel Ctd. Ctuol at h4 Cq. faBd
Page 91 and 92: Aziz F: Celt Cyct. Contd d H4 6@e l
Page 93 and 94: azE F. celt cy.b contol al H4 Gene
Page 95 and 96: A Geneticin (G4'18) rssistanl HeLa
Page 97 and 98: NizF: Cen qcb cttuat al tu G@ ft|tl
Page 99 and 100: M F: Cd W W.l h. 4.. 7'.t'll'Pn'd F
Page 101 and 102: 'dit F: ce qcle cotuot ol Ha Gm ra@
Page 103 and 104: M. F: Ce|| Crd. Cqnrot ot Ha Gerc T
Page 105 and 106: azi. F: cetl cttcL ctu.t or Ha Gtu
Page 107: kiz F: c.lt
Page 111 and 112: Azi2 F: Cen Cvcte Contot .t H4 Gere
Page 113 and 114: I 200000 1mmo DNA Synth.3l3 In Sync
Page 115 and 116: High mitotic index alter relsase tr
Page 117 and 118: ND F Cen atct6 cannot ot B4 6ene rr
Page 119 and 120: NE F: Cell C6t cotuat .t H4 cffi fh
Page 121 and 122: A22 F: Cett cytl. Connd d H4 64. rn
Page 123 and 124: hnF cet cyttd cannot ot B4 6end ltu
Page 125 and 126: Nuclear Run-on TranscriPlion wr Fol
Page 127 and 128: NlzF Cett cr.t. canuat oI H4 Gene f
Page 129 and 130: RNA isolated f rom cell synchrony H
Page 131 and 132: A2iz F: c.u cwL contat al Ha GeN f@
Page 133 and 134: RESULTS
Page 135 and 136: trb F: cett clcL cotuot al H4 Gen.
Page 137 and 138: M2F:Co Crrt6 Cdrtot ot H4G.n. fn.pi
Page 139 and 140: HiNF-tvl HiNF-P HiNF-D random DNA p
Page 141 and 142: andom DNA poly G/C competitor oligo
Page 143 and 144: a2i2 F: ceu ctcte contol al H4 Gene
Page 145 and 146: aziz F: c.ll crd. cdtd 0t H4 Gene t
Page 147 and 148: compeutor oligonucleolides: Site ll
Page 149 and 150: A1i2 F: Ceu a:rcte cotuot ot H1 G@
Page 151 and 152: andom DNA poly G/C competilor oligo
Page 153 and 154: EEF:Ce Olcta Cont t ol114 Gene l@ns
Page 155 and 156: , F: cal qcb cotfrtl d rh 9.n ftuaf
Page 157 and 158: kL E d N. Cdfr.t ol k e.rE ltlr&tu
Page 159 and 160:
Mt F: Cett C\.1e Catud cn Ha Ger. I
Page 161 and 162:
A.*F: Cen q.h cdtuotot Ha 6an ranfl
Page 163 and 164:
3!j{99!caaw arir c",. r_*rooo FI9 3
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I -a2b F: cel cyde c@xol d B1 aa@ h
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-= GAPDH mRNA = = 012 66 hours afle
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Fq *' I E t li! lil tlr ilt Frlr li
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(! (! t- (Y) F o 'F (E d z l t52 +
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E Comparison ol TgT9 ratios lor cAT
Page 175 and 176:
Azd F: Cd Ctcle Ca\ttol ot ltt Gtuf
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:tE &I qiE{, ccaoi oftaa Cer tir fi
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kEE@q.l.co''o,a,tl,od,',rutg/bt!,.
Page 181 and 182:
ibEc|,Otd.c!,'t,dlHao'tllep,rcl Fg.
Page 183 and 184:
DrscussroN
Page 185 and 186:
I azz F Con CYb Cnror ot H4 G@e ntn
Page 187 and 188:
,@2 F. Cdt CEle Cotud .l U1 G.N fan
Page 189 and 190:
l NE P C.l qE/€ Cotud d H4 Gw f@f
Page 191 and 192:
aznF:couct l. cdtold H16@f@tioti@ s
Page 193 and 194:
ari2 F: c.t] cyctd contnt ot H1 Ger
Page 195 and 196:
Atiz F. Cdl cycl6co.ttol d H4 Gene
Page 197 and 198:
l A2izF C.ttctct (onntt ot a. calo
Page 199 and 200:
azi. F: cei c\cb cotuot d H4 G@ fon
Page 201 and 202:
NizF Can a$t. c@tot ot H4 G.n fftNc
Page 203 and 204:
Atiz F: Catl Cvcte contot ol H4 6.n
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Aztz F a.tt ctcte conr ot .t Hl 6en
Page 207 and 208:
,rit 9: Ceu Cycle C.htot ot Ha C.n6
Page 209 and 210:
tai. F: ceu cycte cantat ot ,r1 Gen
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