Proc. Nat. Acad. Sci. USA Vol. 69, No. 1, pp. 256263, January 1972 Neurotransmitter Synthesis by Neuroblastoma Clones (neuroblast difTerentiation/cell culture/choline acetyltransferase/acetylcholinesterase/ tyrosine hydroxylase/axons-dendrites) TAKEHIKO XhIANO, ELLIOTT RICHELSON, AND hIhRSH;ILL NIRESISERG L&orator), of Biochemical Genetics, Nrrtionnl Hemt and Lung Institute, Ndotd Institutes of Henlth, Bethesdn, JIxylantl 20014 ABSTRACT Neuroblastoma clones were examined for choline acetyltransferase (EC 2.3.1.6), tyrosine hydroxyl- ase (EC 1.14.3.a), acetylcholinesterase (EC 3.1.1.?), and also for neurite formation. One clone does not form axons or dendrites. Three types of clones were found with respect to neurotransmitter synthesis: cholinergic, adrenergic, and clones that do not synthesize acetylcholine or cate- cbols. All clones contain acetylcholinesterase. These re- sults show that genes determining neurotransmitter species can be expressed in dividing cells, that the parental programs of gene expression are inherited, and that dividing cells can be programmed w-it11 respect to their ability to communicate with other cells. Elegant biological studies have yielded much information pertaining to the problem of how neural circuits form as the nervous system is assembled. However, virtually nothing is known about the molecular mechanisms for synnl)se forma- tion. The problem ultimately must be defiued in terms of the genetic I)rogTarn for generatin g different cell t,ypes and the steps that determine the specificity of neurons iu forming functional synapses. The ueuroblastoma system established by hugusti-Tocco and Sato (1) provides an unusual opportunity to esplore steps in neuron differentiation and function. The cells multiply iapidly in vitro, yet exhibit many properties characteristic of differentiated neurons (2-7). In this report, the properties of additional clones derived from the mouse neuroblastoma are described. Three cell types! cholinergica cells, adrenergic cells, and cells that do not synthesize acetylcholine or catechol- amines, were detected. METHODS AND MATERIALS Cells. Mouse neuroblastoma C-1300 cells were grown as described (7). Some clones were obtained in two stages: tist, a well-isolated colony of cells in agar was picked and then cloned by isolation of a singIe cell with a stainless-steel cylinder. In other cases, cells were added to petri dishes con- taining broken coverslips; each glass shard with a single cell was then transferred to a separate dish. Chromosomes were analyzed by incubation of cells in logarithmic growth for 6-12 hr with 15-300 ,hl colcemide (Ndesacetyl-N-methyl-colchicine) obtained from Ciba; chromosomes were spread by the method of Merchant, Kahn, and Murphy (8). Choline Acetyltransjerase (EC 2.3.1.6) Assay. Cell mono- layers were washed 3 times with an isotonic salt solution; then cells and protein were harvested by scraping and washing with 10 mM potassium phosphate buffer (pH 6.8)- 1 m;\I EDTZ1 (I)otn.Alm~ salt). The recovered suspension was sonicatetl for 5 min at 3"C, divided into sni:dl portious, and stored in a vapor-phase liquid-uitrogeu freezer. Choliile acetyltrausfernse activity was assayed by a method modified (manuscript iu preparation) from that of Schrier and Shuster (9). Each reaction contaiued the follon-iup comJ)ouents in a final volume of 0.05 ml, esrept n-here noted: 50 n1.\1 potassium JhOsJhate buffer (pII 6.8), 200 m;\I EaCI, 1 mM EDTA (potassium salt), 2.5 mAI choline iodide, 0.57, Triton X-100 (Packard), 2.2 m1I ["Clncetyl Co.4 (10 Ci/mol), 0.1 mM neostigmine methylsulfate, aud O-O.5 mg of homogenate protein. Each reaction was incubated at 37oC for 10 miu; then 0.5 ml of H20 at 3'C n-as added aud the diluted reartioll aud 2 subsequent l.O-ml washes were p:lhsed through a 0.5 X 5 cm column of Bio-Rad AC l-X8 rehin (Cl- form, 100-200 mesh). Each eluate was collected ill a scintillation vial; 10 ml of ,scintillntion solution [lo00 g Triton X-100-2 liters of toluene16.5 ml Liquifluor (Sen England xuclear Co.)] was added and radioactivity NW determined. The counting efficiency for 14C n-a5 8&9Ogi,. Duplicate or triplicate homogenates were prepared nud each was assa>-ed for choline acet;vltrausferase activity at 4 con- centrations of protein. The rate of reaction n-as proportional to enzyme concentration withiil the range 5-350 pm01 of [W]acetylcholine formed per 10 min. Assay reproducibilit> with replicate homogenates wac * 15yc,. Each value reported is the average of values obtained with 2-3 homogenates. `4C-Labeled reaction products in column eluates were characterized by paper chromatography or electrophoresis. Reactions were modified so that the specific activity of the [`4C]acetyl CoA was 4&50 Ciimol, and choline chloride rather than choline iodide was used. Solutions containiup W-labeled products (25-30 ~1 of a column eluate); 0.2 pmol of unlabeled acetylcholine, and 0.2 hmol of unlabeled acetylcarnitine were subjected to ascending paper chroma- tography for 16-24 hr with 1-propauol-0.1 IY acetic2 acid 3: 1. Chromatograms were dried and sprayed with the Dragendorf reagent (18) to visualize acetylcholine 01 acetylcarnitine. The chromatogram was cut into 1.0 X 0.5 cm segments, and the radioactivity of each was determined with a scintillatiou counter. Acetylcholinesterase (EC 3.1 .1.7) Assay. The enzyme was assayed as described by Blume et al. (7). Tyrosine Hydroxylase (E'C 1 .l&?.a) Assay. Cell monolayers were washed, harvested, and sonicated as described above, 258 PI-oc. Nat. Scud. Sci. C:Sli 69 (197.2) Neurotransmitter Synthesis by Neuroblastoma 259 except that cells and protein were harvested in 0.1 M potas- sium phosphate buffer, pH 6.2. Tyrosine hydrosylnse activity was assayed by a modifica- tion of the methods described by Sagatsu, Levitt, and Udenfriend (lo), and by Shiman, hkino, and Kaufman (11). Each reaction contained the following components in a final volume of 0.05 ml: 0.1 XI potassium phosphate buffer (pH 6.2), 0.5 mJI L-[3,5-di 3H]tyrosine (12 Ci/mol, from Ain~ersham-Searle), 0.3 mM 6,7-dimethyl-2-amino4hydroxy- j,6,7,%tetrahydropteridine (Calbiochem), 0.25 mM X'ADPH (sodium salt), about 27 ,ug of sheep-liver dihydropteridine reductase protein [purified through the second ammonium sulfate-precipitation step of Kaufman (12)], and O-O.5 mg of homogenate protein. Reactions were incubated at 34oC for 10 min, and were stopped by the addition of 0.5 ml of 0.17 lu' acetic acid at 3oC and assayed as described by h'agatsu etal. (10). 93% of the 3Hf released from L-[3,5JH]tyrosine lvas recovered in the column eluate; appropriate corrections were applied to reported values. -In internal standard of `H OH t,hen was added to each sample and pudioactivity again was determined. The counting eficiency for 3H was about 30$&. The rate of reaction was proportional to the concentration of homogenate protein for values reported. Duplicate homoge- nates were prepared and each was assayed for tyrosine hydrosylase at four protein c*onceutrations; rel)roducibility was +25%. Average values are rel)orted. Protein was assayed by a modification of the method of Lowr!- (13). Cllnmctcriraiion oj the 311-labeled product of the Tyrosine IIydro.rylase Rcacfion. The tyrosioe hydrosylu~e reaction (8011 tailled 0.1 nib1 ~~-~~ro~~io-~t~-li~`Iros~~be~~z~los~~ami~~e, an ilrllibit,or of aronlatic L-:Lmillo acid dec:~rbosylase, in addition to the coml)oIlel~ts described above. 3H-Labeled products formed during incubation were adsorbed to alumina and sel)aratetl from [3H]tyroAne as described by Sagatsu et al. (L-I), rscept that "H-labeled products were eluted with 0.2 S H(`I. Recovery of 3,~-dil~~tlros~~~he,~!~l~llat~it~e was iS'%. AII alq,rol)ri:lte correction was apl)Iied to values reported. The ["FI]~atecllol:lrllille~ then \verp characterized by paper a11d thiu-layer ~lirotii:~to#r:11)1~~ jvitll the following solvents: l-l)ut:ltlol-glacial acetic acid-Ifs0 12: 3:5; methylethyl- ketolle-formic: acid-Hz0 24: 1 :6; rtti\-[acetate-glacial acetic acitl-HZ0 15: 15: 10; and 1-butanol- 1 S acetic acid-ethanol 35: 10: 10. Thin-layer c~hromatogral)hy was performed with Eastman (`hromogr:nu Sheet 6065 (20 X 20 cm). Spots Lvere located after develolnnellt by sl)rayiug \vith ethylenediamine ferric~yailitle solution (15) to locate catechols. or with nin- lqdrin to locate tyrosille. RESULTS Cell types The specific activities of tyros;iIie hydrosylase, choline :IcrtgltraIisferase, :LII~ acet~l~llolitie~ter:~~e found with homog- enates of the neuroblustoma tumor grown in &o and different c+m:11 cell 1ine.s derived from this tumor are shown in Table 1. Values obtained \vith mouse L-cells, ;I fibroblsstic cell line, and mouse brain are also given for comparative purposes. The hpecifir activity of choline acetyltransferase from tumor was about 2's that of mouse brain. ~~cetvlrholine synthesis was detected with mozt caell extracts? includiI)g L-cells, and other est:~blishecl rell 1ine.i not .ihown here: however, the rate of :Lcetyl(*tlolille >yIlthesis was <2% that of mouse brain (l-10 pmol of acetylcholine formed per min per mg of pro- tein). The neuroblastoma-tumor and mouse-brain specific activities of tyrosine hydroxylase were <3% that of adrenal medulla. Of the 21 clones derived from neuroblastoma C-1300 that were assayed for tyrosine hydroxylase and choline acetyl- transferase, 12 clones were inactive with respect to both enzymes; 6 clones were cholinergic with high choline acetyl- transferase activity; and 1 clone was adrenergic, with tyrosine hydroxylase specific activity about 200-fold higher than that of brain. Two cell lines were found with low activities of both tyro- sine hydroxylase and choline acetyltransferase, but further evi- dence is needed to distinguish between a cholinergic-adren- ergic cell type and a mixture of adrenergic cells and cholin- ergic cells. No cells were found with high activities of both tyrosine hydroxylase and choline acetyltransferase. These re- sults show that three, possibly four! classes of neuroblastoma cells with respect to neurohormone synthesis can be derived from neuroblastoma C-1300. The specific activity of acetylcholinesterase was high with all neuroblastoma clones tested. In addition, electrically excitable cells were found with each cholinergic, adrenergic, and inactive neuroblastoma clone tested. Homogenates were prepared from stationary-phase cells rather than from logarithmically growing cells because neuroblastoma C-1300 tyrosine hgdroxylase, choline acetyl- transferase and acetylcholinesterase activities respond to regulatory mechanisms, and are 30-: A-, and 25-fold higher, respectively, in nondiriding than in logarithmically multi- plying cells (7, 16, and unpublished data). Genetic heterogeneity was examined by determination of the modal number of chromosomes per cell. The mouse neuroblastoma arose xpontnneously in 1940; a modal value of 66-70 chromosomes was found by Levan in 1956 (17). Modal values of 59 and 118 were found with cholinergic clones, 104 and 192 with adrenergic clones, and 10&120 with in- active clones. It is clear that neuroblastoma cells contain more chromosomes than does a dil)loid mouse caell, ;~IN~ that clones differ from one another in their chromosome content. Xeurobl:istom:t calories without tyrosine h~~tlros~l:~se or choline ncetyltransferase may synthesize other transmitter- like coml)ounds. Histamine was detected in unclolled neuro- blastoma cells that had been subcultured frequently at a concentration of 17 pmol :mg ljrotein (unpublished data), a value similar to that of rat brain. However, glutamic acid decarbosylase was not detected with neuroblastoma extracts (unpublished data). Subclones -1 cholinergic ~leuroblastomn clone and two inactive clones were recloned, and the properties of the sublines then were studied (Table 2). 23 of the 26 subclones derived from cholinergic clone NS-20 contained active choline acetyl- transferase, and closely resembled the parent-cell type; however, 3 clones were found with relatively low activities of choline acetyltransferase. Three sublines derived from inactive clone 5-J. and 5 sublines derived from inactive clone N-15, rvere similar to the parent cells. However, 2 subclones of S-1s contained higher activities of choline acetyltransferase than the parent clone. These results show that most, but not all, subclones resemble the parental type. Clone S-1, \vith both tyrosine hydrosylase nncl choline 260 Cell Biology: .\mano et al. TABLE 1. Types oj neuroblastmn C-1 SO0 clones Sourre of homogenate Mouse L cells, clone AB Mouse brain Neuroblnstoma tumor, in uivo Cholinergic clones NWOY NS-20 NS-"6 X3-2.5 NS-18 NS-21 NS-16 Adrenergic clones Nl-106 NlI<-11.; Inactive clones N-IA-103 N-3 N-4 N-7 N-X N-9 N-10 N-11 N-12 N-13 N-18 13 other clones Uncermin (clonal homogeneity not est:~l)liihed ) N-l N-5 Choline Acetyl- AIodnl tiunrber Tyrosine acetyl- cholin- of hydroxylase t.ransferase &erase chromosomes ----pmol product. formed per min per mg of protein--- 2 440 5 s.50 69,000 0 10* 130,000 6G70t 920 47,000 0 490 48,000 59 1 437 47,000 116 1 12.5 55 ,000 59 1 76 80,000 59 0 5" 36,000 61 0 43 67,000 60 60 - * 980 G.1 179 ( 000 104 2.56, 000 102 0 2 l!,,OOO 101 9 4 174,000 4 0. 1 46,000 10.; 6 0 09, 000 2 2 42) 000 11 1 X3 , 000 1 4 Xi , 000 0 .i * 404, on0 0 4 346,000 0 6 130,000 2 `2 lo.;, 000 llrl .j* 70 22 23, non 106 23 1X 1.51 ,000 lo!) * Estimate based on determination of I%!-labeled products in column eluates. t Data of Levan, cited by IIauschkn (17). acetyltransferase, was recloned, and the enzyme activities of the sublines were determined (Table 3). Unlike the other neuro- blastoma clones, N-l cells do not extend axon:: or dendrites. The cell population was homogeneous initially. consisting of cells that were attached well to the surface of a petri dish an TABLE 2. Subclones of cholinergic and in&ire clones Cell line Choline acetyl- transferase Tyrosine hydroxylase Cholinergic clone NS-20 23 Subclones 3 Subclones Inactive clone N-4 3 Subclones Inactive clone N-18 5 Subclones 2 Subclones pmol of product formed per min per mg of protein loo-750* loo-930* 25-35 * 0.1 r 1 i 2 2 1: 2 1 * Estimate based on determinat,ion of W- labeled products in column eluates. usually formed short spikes (<30 *In), but were entireI> devoid of neurites 1062000 Mm in length. However, after the 10-20th subculture, some relatively large cells were observed with long, branched neuritea. S-1 then was recloned and 19 subclones and 10 colonie< were examined. Two S-1 subclones were found that, wew without neurites, tyrosine hydroxylase, or choline acetyl- transferase; however, acetylcholinesterase was present, and cells with electrically active membranes were found when OII? clone was examined. Fifteen adrenergic clones with neurites were found. Each clone studied contained tyrosine hydroxy- lase and acetylcholinesterase activities, but cells mere almort devoid of choline acetyltransferase activity. One clone (X1-106) with neurites contained 104 chromosomes per cell; other cell lines contained about 200 chromosomes pet cell. Several nonadrenergic clones with neurites were found: clone NlE-113 contained 205 chromosomes per cell. The-e results show that the N-l cell population is heterogeneous and contains adrenergic and nonadrenergic cells. No cholin- ergic or adrenergic-cholinergic cell type was found. Clonal morphology Four types of neuroblastoma clones, incubated in the abseme of serum to stimulate neurite extension, are shown in Fig. 1: proc. Nat. Acad. Sci. USA 69 (297.2) Neurotransmitter Synthesis by Neuroblastoma 261 TABLE 3. Axon-minus and adrenergic subclones of neuroblastoma clone N-1 Choline Acetyl- Modal number Tyrosine acetyl- cholin- of Subclones hydroxylase transferase esterase chromosomes l'arent clone N-l t ,%xon-dendrite minus subclones K l A-103t NlA-104t .~\tirenergic subclollrs N l-106 NIE-11.; NIE-124 N IE-12.5 NlK-116 NlIG122 NlE-110 N 1 K-126 N 1 K-1 14 NllLlI'I NIX-12X NLLl2i X11',-12:s NlE-111 NLlGlllr Slinus tyrosine hydroxylase sub(xlones N 1 L 11:3 NlE-117 ---pmol of product formed per min per mg of protein 70 22 23,000 106 0 2 19,000 101 1 5 60 980 3.50 330 2X "1.5 150 lz% 93 63 60 60 40 32 17 5* 179) 000 104 0. 1 256,000 192 0.9 17.;) 000 207 1.3 98 ) 000 0.5 109 ) 000 202 0.4 40,000 0 ,3 0.1 73 ) 000 202 74) 000 6 205 1 * 1Mrnate based 011 delarmitu~tion of I'C-1nheled products in column eluates. t All srlbc.lones were 1)ositive for neuriteh, excbept t.hose marked t. FIG. 1. Axon-dendrite formation by (A) cholinergic clone NS-20; (B) adrenergic clone NlE; (C) inactive clone N-18; uud (D) inactive clone NlA-103, which does not form axons or dendrites. Cells were incubated in growth medium without serum for five days to stimulate neurite formation. The scale shown in A applies to all panels, and corresponds to 10 pm. 262 Cell Biology: Xmano et a2. TABLE 4. Neuroblnstomn C-1300 rlones Cell hype Axon-minus Inactive Cholinergic Adrenergic Acetylcholin- esterase and excitable membranes + + + + Keurites - + + + Choline \Id:11 Ilullltwr acetyl- Tyrorine of tmnsferase hydroxylas~ I'tlro"lownlc'~ ____- -~- ~- ~~___~~ - 101 - 110 + - .?I!), 110 - 1 104. 200 cholinergic, adrenergic, inactive with neurites, and the clone lacking neurites. Cells from each clone adhered well to the surface of the petri dish, but only the axon-minus line (NlA-103) was devoid of long neurites. Xxon-minus cells and N-18 cells that form long neurites were mixed and cultivated in the same flasks for more than a week; however, no influence of one cell t,ype upon the other w-as detected. Product identification Neuroblast,oma K-1E tyrosine hydroxyl:tse activity n-as dependent upon a pteridine cofac*tor (6,7-dimethyl-2-:mlillo- 4-hydroxy-5,6,7,%tetrahydropteridine). Tritium rele:tse from L[3,5-3H]tyrosine agreed well (tvithin 5%) with [3H]di- hydrosyl~l~enylalanine formation. The labeled product of the tyrosine hydroxylase reaction formed in the presence of :LII inhibitor of aromatic amino-acid decarboxylsse was character- ized by paper and thin-layer chromatography with four solvents. Between 86 and 99% of the applied t,ritiated produrt was identical in chromatographic mobility with authentic8 dihydroxyphe~lylal:Lriitle. Clone X-1 cells, in the ahserlc*e of XII inhibitor of aromatic amino-acid decnrboxylase, synthesize 3,4-dil~ydroxyphenylalanine, 3,Cdihydroxvphenylethylamille. tiorepinephrine, 3,4-dihpdroxyphenylacetic acid, 3,4- dihydroxyphenylethylglycol, 3-methosytyrosine, 3-methosy- tyramine, and 3-methosy-4-hydroxyphenylacetic acid (un- published data). Catechols formed by clone C-1300 have been characterized also by Schubert, Humphreys, Baroni, and Cohn (2), and by -1nagnoste, Goldstein, and Broome (4). The l*C-labeled products of the choline acetyltransferase reaction were eluted from the column and routinely character- ized by paper chromatography. 84-99y0 of the labeled product formed with homogenates of cholinergic cells was identical in chromatographic mobility to authentic acetyl- choline. However, only 2-2570 of the labeled material formed with adrenergic or inactive homogenates was acetylcholine. The major contaminant was identified as [WJacetyl carnitine (unpublished data). DISCUSSION Clones of neuroblastoma C-1300 were esamined for two enzymes required for neurotransmitter synthesis, choline acetyltransferase and tyrosine hydroxylase, catalyzing acetyl- choline formation and the first step in norepinephrine syn- thesis, respectively. A summary of data is shown in Table 4. Three types of clones were found: (a) clones that form little or no acetylcholine or catechols; (b) cholinergic clones; and (c) adrenergic clones. One additional clone was found with relatively low cholinergic and adrenergic activities, but we do not know whether this is a fourth cell type or a mixture of adrenergic and cholinergic cells. However, no cells were Tumors of rholiiiergic l1eurolls h:lve Ilot lieeli rrllortt~cl before. They proh:~bly afflict man, but IU:IJ. IIO~ II:IVV beeli recognized for lack of :I diagnostic test. Sillcnr few m:unn~:di:LII (*ells other thaii neurons h:lve high :rctivitiez of c%olillf~ acetyltrnilsferase, the enzyme nild ac~et~lclioli~~e (`a11 br IIWI as sperific diapllostic markers for clrolirlprgi~ ~l~llrol)l:~~tot~~:l~. Since acetyl~lioliiiesterase was found with a11 Iielirolil~~~;toill:i cell types, the riizyrne :llili:irei~tly is imt :i sl)ec.ific in:ttkc~r 01 cholinergic~ neiiroi1*. Xt least six tyliez of iw~ir011~ or c~orresponding tunlor- tll:it arise from the neur:ll crest c;in lie distinguished 011 the liwi- of tralrstnitter s).lttltrsis: ttio.ir synthesizing :I~et?.l(,lioliit~~: 3,4-dil~~dros~~,I~e~~~l:~i~~~~i~~e, 3,4-dil~~~lt~o~~~~~l~e~~~lett~~l~~~~~i~~~. Irorepilrelilirille, epinephrine, and sensory 116uroiis that do imt synthesize these con~pounds. The three @lies of Ileurcl- blastoma thuh esllibit proliertieh esprctetl of ileur:d-c~i.c~~t neurons. Mthough ~leu~ol,lnstorl~:I stem cells c:~~~:ll)le of gi\-ilrg rise to cliolitlergic, adrelrergir, and inactive ~11s \v(`I'~' Ilot found, it seems likely that su(`h cells tromdly exist. The available informatioil suggests that tlrrre are relati\.rl! fen- kinds of universal Ilellrotrallsmitters ill the 11ervou+ system of both vertebrates and invertebrater. Slost tleuronx probablv are greatly restricted with respect to the lIunllw1 I of kinds of lleurotrallarliitters that (*an be synthesized. Ke find cholinergic, adrenergic, and inactive rreurobl:cstom:~ cell types, but have not detected cells capable of syllthesizill: both acetylchotine and catechols at rapid rates. It seem. likely that the expression of a gene required for the s~nthesic of one neurotransmitter map restrict the espression of gene- for alternate neurotransmitters. For example, a pr0duc.t derived from the choline acetyltransferase gene directI\. 01 indirectly might restrict the expression of genes for tyrosilrt, hydroxylase, and vice versa. Simultaneous expression oi acetyltransferase and tyrosine hydroxylase genes might inactivate both genes, or might result in a balanced state of mutual inhibition; i.e., a cell with low cholinergica and adrrn- ergic activities. Whether a product of one neuron affects the expression of genes for transmitter synthesi:, of neiglrlloril~~ neurons is a problem for future study. Analysis of sublines derived from rlonal c-ells drttro~l- &rates that most, but not all, resemble the parent t~.l)c', The mechanisms underlying altered gene expression are 11nf known. Gene expression may be reversible or mutations ma! affect structural or regulatory genes. In any event, the IO+ of a step that commits a cell to one developmental patter11 may enable the cell or its descendants to differentiate along an alternate pathway. Thousands of cell generations have elapsed since tumol C-1300 originated, and extensive genetic heterogeneity in the Neurotransmitter Synthesis by Neuroblastoma 263 ,,ell populat,ion is expected. By a relatively simple experi- ,,,plltal approach7 it may be possible to obtain lines that esl)ress genes that are characteristic of many kinds of neurons i,~,)tn :\ single neural tumor cell; i.e., with two sequential &ctire I)rocwlures: first, selection for dedifferentinted cell* ,,,ight yield cell I)opulations enriched in stem cells; then .&&ion for differentiated cells might yield cells that follow :Iltrrn:lte programs of differentiation. \Ye cnonclude that genes determining the species of neuro- rl.:~~nitter synthesized can be expressed in dividkg cells, :,,,d t,hat the I)arentsl programs of gene expreaGou are inherited :rl~(l l)erl)etuated for hundreds of cell generations. 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