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

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DNA sequencing of <strong>H4</strong> promoter construcls wt FO1O8 m MSP-16 m MPM-17 m lcM-18 a :l= " 16 ""1 PUC 19 lorward primer/<strong>H4</strong> Dromoter sense-stGnd ]'
ldD F: Cett CrL Cotuot at t14 G* Iwion@ Preparation ol glycerol stock badlorlal strairu lor slorage sacterial cullurcs 0O mD Mr€ prepared by inodla on tom a singl6 colony or trom a previous glycerol stock (25 _ 50 pl) and ovsrnighl incubalion at 37'C wlh shaking. Ihe nexl morning 500 rlol g.own bacteria were mxed {fth 5oo pl ol sterile 8o% glyc€rol or 1oo % glycerol in cryotubes. The suspefslons wer€ mxed by inv€rting th€ tubes mulliplgllmes and stored al _ 70,c. To ensure the clrr€cl idsntily ot plasmld DNAS grown Jrom thess bacrerial stocks, construcls wer€ analyzed by both lestricron enzyme analyses and seque.ce.analysis ol large scale Plasmid preparalions derived lromlheslocks large scare prepa.ation ol Plasmid oNA by alkallne lvsis Large scale prepration of plasrnid DNA was initial€d by prepanng a loml ove.night cultwe which was grown io late log phase to an optical density or O.O al A@. The culll]re was lransloned to 5OO ml TB medium Oeriiic Brcth: Ausubeler a/., 1987); the lquid culure was s€t !p in such a way that the arto liquid ratio was appronmately 3:1 in each culture llask (reqardless ol size) Cuhu.es were lncubated at 37'C ov€rnght in a shaking incubator with 10 mg/nlampicilin. Bacteria we.€ hatuesled by spinning samples in a sorvall GS3 roror at 4'C ior 10 minutes at 5000 rpm. Bacler alpellets wers dissolved in ao d ol ST€ solution (0.1 M NaCl, 10 mM T s HCl, pH aO, 1 mM EDIA PH 8.0). Tubes we€ centrillrged lo corlect 55
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\ ?^p 6r.1 CELL CYCLE CONTROL OF HU
Page 3 and 4:
In tho narn€ of Alah, olt G6cloua
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Acknowl6dg6msnts Contents Abstract
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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
Page 19 and 20:
INTRODUCTION
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t t F: con cda c@tot d H. G@ r@Mbn
Page 23 and 24: Azh F: C6n Ca/cro C@t.t ot H. G.n.
Page 25 and 26: Aziz f: Celi Ctcle Cotutol ol H4 Ae
Page 27 and 28: = 2. q t6l tl9t E L:I la-lf titl I.
Page 29 and 30: En F. Ce Cycl. Cantd d h. Gft ra.sc
Page 31 and 32: ,r1z F Cett cy.te connot ol H4 Gene
Page 33 and 34: A2i2F: Cettcdt cotubtot e@IrNiotid
Page 35 and 36: Ntz F: Ce Cwte Contd d 114 Gm tansc
Page 37 and 38: t- s- i;> f z x + n q e o, { T T z
Page 39 and 40: AzzF cett Crct. ContolatH4Gen ldn*t
Page 41 and 42: a2i2 F cdt c$t. connd d H4 Gm Tdnsc
Page 43 and 44: o rl$orrE GEIE FEEI{IXU =; FrN -!r
Page 45 and 46: !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: ,.8 F: 6t W @tlol o, rk 0.,t rn',df
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 and 108: kiz F: c.lt
Page 109 and 110: ktz F. cen ctct6 ctuat at tu 6e rft
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
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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
Page 165 and 166:
I -a2b F: cel cyde c@xol d B1 aa@ h
Page 167 and 168:
-= GAPDH mRNA = = 012 66 hours afle
Page 169 and 170:
Fq *' I E t li! lil tlr ilt Frlr li
Page 171 and 172:
(! (! t- (Y) F o 'F (E d z l t52 +
Page 173 and 174:
E Comparison ol TgT9 ratios lor cAT
Page 175 and 176:
Azd F: Cd Ctcle Ca\ttol ot ltt Gtuf
Page 177 and 178:
:tE &I qiE{, ccaoi oftaa Cer tir fi
Page 179 and 180:
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
Page 205 and 206:
Aztz F a.tt ctcte conr ot .t Hl 6en
Page 207 and 208:
,rit 9: Ceu Cycle C.htot ot Ha C.n6
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tai. F: ceu cycte cantat ot ,r1 Gen
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