New gene expression in dimethylsulfoxide-treated friend erythroleukemia cells

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0014.48271801110119-08$02.0010

Cell Research 130 (1980) 119-126

Experimental

NEW GENE

EXPRESSION FRIEND

ERYTHROLEUKEMIA

N. HANANIA, Institui

Gustave-Roussy.

IN DIMETHYLSULFOXIDE-TREATED CELLS

D. SHAOOL, C. PONCY and J. HAREL

Groupe de Recherches

No. 8 du CNRS. F-94800 Viliejuif,

France

SUMMARY Our previous studies had shown that a small amount of single-stranded DNA (ssDNA) separated from the bulk nuclear DNA of different animal cells by an improved method of hydroxylapatite chromatography (HAC) contains two distinct molecular fractions. The major fraction consists of non self-reassociating sequences that are reassociable to the unique component of bulk DNA and in great part hybridizable to homologous RNA. The minor fraction consists of self-reassociable sequences also reassociable to moderately repetitious bulk DNA. In the present work ssDNA from Friend leukemia cells induced to differentiate (ind FLC) by DMSO was compared with ssDNA from untreated control Friend cells (cant FLC). It was shown that the relative amount of ssDNA is greater in ind FLC than in cant FLC (1S-1.6 % and 1.2-l .3 % of the total cell DNA respectively after a second step of HAC purification). The ind FLC-ssDNA contained a greater proportion of self-reassociable sequences (33-35%) as compared with cant FLC-ssDNA (18-20%). Also the relative amounts of ssDNA hybridizable to cytoplasmic RNA from homologous cells was slightly but constantly higher in ind FLC-ssDNA (33-34%) than in cant FLC-ssDNA (29-30%). Cross hybridizations were carried out between highly radioactive ssDNA and cellular RNAs in great excess, whether total cytoplasmic RNAs or polyadenylated mRNAs. At saturation levels, the hybridized ssDNA fraction was separated from the non-hybridized fraction, and both fractions were rehybridized to RNA from ind FLC or cant FLC. The results indicated that about 10% of ind FLC-ssDNA appeared to be specific for DMSO-treated cells. This may correspond to the expression of 1000-2000 different cytoplasmic mRNAs mostly belonging to the low abundance class.

Two mechanisms are invoked for cell differentiation: (u) Changes in the transcriptional pattern with an enrichment of specific mRNAs coding for the differentiation specific proteins, as shown in many cell types such as oviduct cells synthesizing ovalbumin under estradiol control [l] or murine erythroleukemic cells induced to synthesize hemoglobin [2-7-j; (6) changes at a posttranscriptional stage with a differential turnover of cytoplasmic mRNA leading to a different distribution in the abundancy classes [8-l 11, polyadenylated mRNAs which accumulate in differentiating cells belonging to the most stable class of mRNAs [ 12-131. Cultured murine erythroid cells (Friend

leukemia cells) have been used as a model system for differentiation. When these cells are grown in suspension with a doubling time of 18-24 h and are treated with 2% DMSO, they undergo changes similar to those observed in normal erythroid differentiation [ 141: they synthesize hemoglobin and accumulate specific mRNAs including the globin mRNA [2, 41. Previous studies from our laboratory showed that a minor fraction of ssDNA, usually amounting to 1.5-2% of the total nuclear DNA is related to the transcriptional activity, since 30-35% of the ssDNA from chicken embryonic cells [ 151,chicken leukemic cells [ 161, human RD cells [17], Exp Cd

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et al.

and mouse SV-3T3 cells [18] could be hybridized to total cytoplasmic RNA or purified polyadenylated RNA from homologous cells. The present work indicates that changes in the transcriptional activity of Friend cells treated with an inducer of erythropoietic differentiation, can be demonstrated at the level of ssDNA.

MATERIALS

AND METHODS

Cell culture and induction cells with DMSO

of Friend

Friend cells (line 745 obtained from Dr C. Friend) were grown in suspension in Eagle’s medium supplemented with 10% fetal calf serum (FCS), at an initial density of 0.1 x lO”/ml. The cells were induced by adding 2% DMSO, at a slightly higher initial density (0. 15x106/ml). Under these conditions, hemoglobin synthesis, as detected by benzidine staining of cells, began after about 48 h which corresponded to about two cell generations. The cell number remained constant after 3-4 days, when the cell density reached about 2x 106/ml and BO-85% of the cells were benzidine positive.

Labelling

procedures

The induced cells, grown for 24-30 h, in the previously described conditions were labelled by twice adding 5 &i/ml of [3H]thymidine (spec. act 51 Ci/ mM) for 24 h, at 3PC.

Isolation

of ssDNA

Cell nuclei were purified by using non-ionic detergent [19], resuspended in 0.01 M NaCl, 0.01 M Tris-HCl, pH 8.4, 0.002 M EDTA (NTE) and lysed by incubating for 30 min at 37°C in the presence of 0.1% sodium lauryl sulphate (SLS, Merck) and 100 pglml pronase. The concentration of SLS was increased to 1% and the nuclei were reincubated for 30 min. After addition of sodium perchlorate to 1 M, the DNA was extracted three times with chloroform isoamylalcohol (1 : 24) precipitated with ethanol and redissolved in NTE. Pancreatic ribonuclease (20 pglml) and RNase T, (10 U/ml) were added and the solution was incubated for 30 min at 37°C. The DNA was re-extracted, precipitated with ethanol, redissolved and dialvsed against the same buffer. The single-stranded *and double-stranded DNA fractions (ssDNA, dsDNA) were separated by an improved method of hydroxylapatite chromatography (HAC) [20], using phosphate buffer, pH 7.85. For hybridization experiments, ssDNA was further purified by a second HAC. Exp Cdl Res 130 (1980)

Preparation

ofcytoplasmic

RNAs

Total cytoplasmic RNA was extracted from induced (3 days after addition of DMSO) and non-induced cells bv a previously reoorted method 1151.In brief. the cells were washed and lysed in the presence of 0.1% Nonidet P-40 in 0.01 M Tris-HCl oH 7.4. 0.2 M KCl, 0.02 M MgCl,, 0.25 M sucrose (TKM). After two successive centrifugations (10 min at 3000 rpm and 20 min at 12000 rpm) the supernatant was made 2 M LiCl and kept at least 24 h at 4°C. The precipitated RNA was pelleted and dissolved in 0.15 M NaCl, 0.01 M Tris-HCl, pH 7.4, 0.002 M EDTA. Pronase (100 pg/ml) and SLS (0.1%) were added and the mixture was incubated 30 min at 37°C. After adjusting to 1% SLS and reincubating 15 min at 37°C RNA was extracted three times with a mixture (V/V) of uhenol and chloroform-isoamvlalcohol, and precipitated overnight in 2 M LiCl to remove traces of DNA, dissolved again and finally reprecipitated in ethanol and kept at -20°C.

Preparation ofcytoplasmic containing mRNA

poly(A)-

Total cytoplasmic RNA was subjected to oligo (dT)cellulose chromatography [21]. The RNA was redissolved in high-salt buffer (0.5 M NaCl, 10 mM TrisHCl oH 7.6. 0.1% SLS) and nassed three times over an oiigo (dT)-cellulose column at room temperature. After rinsing with four column volumes of buffer, the polyadenylated RNA was recovered by elution with low-salt buffer (without NaCl). After a second chromatography cycle, the poly (A)+ pool was concentrated by precipitating in 0.5 M NaCl with 2.5 vol of ethanol. ,

Preparation

n

of 1251-labelled ssDNA

The ssDNA from induced and non-induced cells isolated by hydroxylapatite chromatography was alkalitreated (0.1 M NaOH for 18 h at 3PC), neutralized and dialysed against NTE. lodination of ssDNA was carried out by a modification of the procedure of Commerford [22]. The specific activity of the ssDNA was about 10’ cpm-’ Fg-‘.

Nucleic acid hybridization The hvbridization mixture contained the RNA sample inb.4 M NaCl, 10 mM Tris HCl, pH 7.4, 5 mM EDTA and iZ51- or [3H]thymidine-labelled ssDNA. Aliquots were distributed in plastic microtubes, recovered with liquid uarafftn and incubated at 66°C for various times. The proportions of ssDNA taken in hvbrid form were determined by treating aliquots with S-1nuclease and measuring the acid-prespitable radioactivity collected on Whatman tibreglass paper GF/B in a liquid scintillation spectrometer for-3H or in a Gamma 4000 Beckman counter for iz51. In some experiments, RNA-DNA hybrids were treated with pancreatic RNase (25 &ml) and RNase T, (10 U/ ml) for 30 min at room temperature, before centrifugation in cesium sulphate gradient [ 171.

DNA transcription in differentiated Friend cells

121

of ssDNA exhibited a polydisperse sedimentation profile, with fractions up to 30 S or more (not shown). Self-reassociation ofssDNA and crosshybridization betbt’een ssDNA.from induced and non-induced cells and hybridization to bulk DNA As shown in fig. 1, only 18-20% of ssDNA from non-induced cells -reassociated at a C, t of approx. 1000 mol 1-l S, whereas C,t (mol. s/e) 35% of the complementary chains of Fin. 1. Reassociation kinetics of ~‘Z511ssDNAfrom DMSO-induced and non-induced ~ells~ [1251]~~DNA ssDNA from induced cells reassociated from control cells was annealed to a 6 500-fold excess with itself and 25 % with ssDNA from nonof 0, unlabelled ssDNA from control cells; 0, unlabelled ssDNA from DMSO-induced cells. Under the induced cells. Besides the greatest part of same conditions, [12JI]~~DNA from induced cells was Friend cell [3H]ssDNA could be hybridized annealed to unlabelled ssDNA from A. control cells: to non-repetitious bulk DNA (results not a, unlabelled ssDNA from induced cells. The fraction hybridized was determined by treating half of each shown similar to those obtained with all sample with Sl nuclease and counting the acid-oreother cell species studied [16, 17, 18, 28, cipiiable radioactivity in a scintillation spectrometer. Time zero values (l-2.5 % of ssDNA resistant to Sl 341).

I

nuclease) were deduced.

Hybridization between 3H-labelled ssDNA and cytoplasmic mRNAs RESULTS 3H-labelled DNA from induced cells was Isolation and characterization fractionated into ssDNA and dsDNA and of ssDNA micro-quantities of these fractions were hySingle-stranded DNA was isolated from the bridized to large excess of total cytoplasmic nuclear DNA of mouse cells, by the im- RNA from non-induced cells or from huproved method of hydroxylapatite chro- man HEp cells used as control. Hybridizamatography previously characterized [ 181. tion yields were calculated by measuring It amounted to l-2% of the total nuclear the fraction of radioactive ssDNA resistant DNA, behaved like denatured DNA when to Sl nuclease digestion. Non-incubated analysed by hydroxylapatite chromatog- control DNA samples were similarly proraphy, CsCl density gradient centrifugation, cessed (time zero). Hybridization kinetics or digestion with Sl nuclease. In ten dif- illustrated by fig. 2 showed that about 30ferent preparations, the amount of ssDNA 35 % of the input ssDNA hybridized to total isolated from induced Friend cells was cytoplasmic RNA from induced cells (not always higher than that isolated from con- shown) or non-induced cells, as compared trol Friend cells (1.5-1.6% and 1.2-1.3% with less than 10% for bulk dsDNA and respectively after the second HAC). When 4-5% for the unique dsDNA component centrifuged in alkaline sucrose gradient, (representing 60-70% of the total dsDNA) ssDNA sedimented at 10-12 S (1.2 Kb) isolated as described by Kleiman et al. whereas the peak of bulk DNA was at 18- [231. 20 S (6-8 Kb). However, a noticeable part The ssDNA from induced cells was hyExp Cell Res 130 (1980)

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~anania et al.

Rot (mol. s/I I

Fig. 2. Hybridization of rH]ssDNA from induced cells to cellular RNAs. Samples (2000 cpm each) e, rH]ssDNA; or 0, bulk rH]dsDNA from induced cells were hybridize to 50~-fold excess amounts of unlabelled total RNA from non-induced cells. As control. other samples of rH]ssDNA from induced Friend cells were hybridized to the same excess amounts of RNA from A, human cells (epidermoid carcinoma). The fraction hybridized was determined by the use of S 1 nuclease.

from non-induced cells. After reaching saturation levels, the hybridized sequences (h-ssDNA) were separated from the nonhybridized ones (nh-ssDNA) by equilibrium centrifugation in Cs*SO, gradient. Most of the ssDNA-RNA hybrids sedimented as a band with a mean buoyant density approx. 1.55 g/cm3. As indicated in fig. 3, fractions corresponding to h-ssDNA, and those corresponding to nh-ssDNA were pooled, alkali-degraded and rehybridized to total cytoplasmic RNA and to polyadenylated mRNAs from respectively induced and non-induced cells. Re-hybridization of “h-ssDNA” and “nh-SSDNA” sequences to total cytoplasmic RNA and to polyadenylated mRNAs from induced and non-induced CdlS

bridized to an excess of total cytoplasmic RNAs from non-induced cells. Vice versa, the ssDNA from control cells was hybridized to an excess of cytoplasmic RNA

Fraction

no.

Fig. 3. Density gradient eentrifugation of DNA-RNA hybrids. The solution containing rH]ssDNA from induced Friend cells hybridized to RNA from control cells was treated with RNAses, brought to I % Sarkosyl. mixed with an equal volume of saturated caesium sulphate to yield a starting density of 1.51 g/cm3 and centrifuged for 65 h at 20°C in a 50 Ti rotor (Spinco). Fractions were collected and aliquots were assayed for TCA-precipitable radioactivity.

The re-hybridization kinetics of h-ssDNA from induced cell with RNAs from induced and non-induced cells, were quite similar (fig. 4); the saturation levels were respectively 75 and 80%. In contrast, for nhssDNA, the proportion of re-hybridized sequences was about twice as great with cytoplasmic RNAs from induced cells as for RNA from control Friend cells. This seems to indicate that about 10% of the unique ssDNA sequences expressed in differentiated erythroleukemic cells, are not expressed in the non-differentiated cells. When the control sort of reaction was carried out, the same small amounts of nh-ssDNA from control Friend cells (isolated after the first hybridization with RNA from control cells) could be re-hybridized to a similar excess of cytoplasmic RNA from both induced and non-induced cells (see the lowest curve in fig. 4). The results obtained with total cytoplasmic RNAs were confirmed with polyadenylated mRNAs. The purified poly-

DNA t~~~~s~ripti~~in different~ffted Friend cells

R.t (mol. s/e)

4. Re-h~bridi~tion of the me-hvbridized and Nan-prehybri~jzed fractions of ssDNA to cytoplasmic RNA from differentiated or control cells. [%]ssDNA from differentiated cells was prehyb~dized to a large excess of total cytoplasmic RNA from non-induced cells. The hybridized (h-ssDNA) and non-hybridized (nh-ssDNA) fractions were isolated by Cs2S04 gradient centrifugation. ‘h-ssDNA’ was rehybridized to a 80000-fold excess of unlabeiled cytoplasmic RNA from 0. control cells and from 0. DMSO-induced cells. nh-ssDNA was rehybridized to the same excess of unlabelled cytoplasmic RNA from A, control cells and from A, DMSO-induced cells. As control, [VIssDNA from non-differentiated cells was pre-hybridized to a large excess of cvtoolasmic RNA from the same cells. The non-hybridized fraction isolated. as already described was rehvbridized to a 106-fold excess of unlabelled cytoplasmic RNA from n , control cells and El. differentiated cells.

Fip.

adenylated RNA preparations, chromatographed on oligo dT-cellulose were equivalent to 2-3 % and 6-896 of the cytoplasmic RNA from non-induced and induced cells, respectively. As shown in fig. 5, the saturation levels were very similar to those observed with total cytoplasmic RNA (about 70 and 75 %). On the other side, about 30% of the ssDNA fraction from induced cells, which could not be rehybridized (or very little) to polyadenylated RNA from control Friend cells, hybridized to polyadenylated RNA from induced cells, at Rot values about 3 000 mol 1-I s. DISPASSION During the last few years, different models for transcription, implying a local destabili-

123

R-t (mol. s/l)

Fin. 5. Rehvb~dization of the nrehvbridized and nonpr~hybr~diz~d fractions of ssDhA from diffe~ntiated Friend cells to wIvadenvlat~ mRNAs from differentiated and controt cd&s. The same hybridized(h-ssDNA) and non-hyb~d~zed (nh-ssDNA) fractions of ssDNA isolated from differentiated cells as described in fig. 4, were respectively re-hybridized to a lOOOO-ford excess of unlabelled polyadenylated mRNAs from 0, A, control cells and from 0, A, induced cells.

zation of the DNA duplex have been described [24-271. Recent studies from our laboratory showed that a minor fraction of ssDNA isolated from nuclear DNA of embryonic chicken cells [ 151,human cells [ 171, chicken leukemic celts [16] or mouse cells transformed by SV40 virus [18] is related to synthesis of numerous RNA species, including viral RNA [16, 181. Furthermore 70-75% of the ssDNA from chicken cells, either normal [28] or virus-transformed [ 161 and ssDNA from SV-3T3 mouse cells [18] was found to consist of non-self complementary DNA segments, which can be reassociated with the non-repetitious component of bulk DNA and about 40-4.5% of which is complementary to total cytoplasmic RNAs. The present work shows that the ssDNA from Friend erythroleukemic cells exhibits the same properties as ssDNA from ail cells studied so far. However, up to 35% of ssDNA from induced cells and only 1820 % of the ssDNA from control cells could be self-annealed. About 30% of ssDNA Exp Cell Res 130 (19801

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et al.

from induced cells hybridized to total cytoplasmic RNAs from control cells and a slightly higher saturation level was obtained with total cytoplasmic RNAs from induced cells (34 %). It might be questioned whether these values could be explained by contamination of cytoplasmic RNA by heterogenous nuclear RNA (hnRNA). Results of our current studies do not support this possibility. In brief, only 4-5% of the unique dsDNA component could be hybridized to cytoplasmic RNA, whereas 4-5 times greater amounts (20-22 %) hybridized to hnRNA. In contrast, the ssDNA saturation levels were approx. 30% with cytoplasmic RNA and 55% with hnRNA. The number of polyadenylated mRNAs present in the polysomes of Friend cells was calculated by others, either from the hybridization kinetics of their cDNA copies to cellular RNA [29], or from saturation of unique DNA [23]. The values were respectively in the order of lo4 by kinetic analysis and 2-3 times more by the saturation method. Using cDNA kinetics, Mauron & Spohr [30] have studied the effect of DMSO in another line of Friend cells (F4N). Their results suggested a transcriptional activation for the few predominant mRNAs which appear in terminally differentiated cells. More recently, Reeves & Cserjesi [31] studying another inducer of Friend cell differentiation, n-butyrate, have compared the results of hybridization reactions between bulk DNA and excess cellular RNA to the total number of detectable cell proteins. Their conclusion was that the molecular complexity of the cytoplasmic mRNA populations is 38% greater in n-butyratetreated cells than in non-treated cells, while no augmentation of this sort had been previously found in the case of DMSO. For example according to Minty et al. [32] all or nearly all polyadenylated mRNAs are comExp CellRes 130 (1980)

mon to DMSO-treated cells and to nontreated cells. However, even a small amount of sequences making up a few per cent by weight of the polyadenylated mRNA that could not be readily detected by the limited resolution of the hybridization methods utilized so far might represent a great number (up to 1000 or more) of different mRNAs at low abundance. The present work is based on previous evidence indicating that a great part, if not all, of the ssDNA isolated by us arises via selective single-strand breaks in the vicinity of DNA transcription sites, as already discussed [18, 281. In these conditions, changes in the transcriptional process, characteristic of cell differentiation, may be reflected at the level of ssDNA. Recent data obtained in our laboratory in collaboration with Leibovitch & Kruh [33-341 have demonstrated that this is the case for ssDNA isolated from Yaffe rat cells undergoing muscular differentiation. The present results show that a significant portion (about 10%) of the ssDNA from DMSO-treated Friend cells which is hybridizable to homologous RNA appears to be absent in ssDNA from non-induced Friend cells. This cannot be due to a difference in the self-reassociation percentages of these two ssDNA species, under the annealing conditions utilized. As previously shown [16, 18, 28, 341 ssDNA from various sources contained no very repetitive sequences and this was found true for ssDNA from Friend cells either DMSO-treated or not (see fig. 1). The highest Rot values which were attained in RNA-driven reactions (see fig. 2) corresponded to maximum Cot values of 0.6 for ssDNA alone, Cot values at which selfreassociation did not exceed l-2 % ([ 16, 18, 28, 341 see also the control curve obtained with human RNA in fig. 2). Furthermore there is no reason why in the rehybridiza-

DNA transcription in differentiated Friend cells tion assays (illustrated by figs 4 and 5) self-reassociation of ssDNA from induced Friend celis would be much greater in the presence of homologous RNAs than in the presence of RNA from control cells. Another possible explanation was that ssDNA from induced cells and ssDNA from control cells were essentially similar but a greater proportion of the former could be hybridized to homologous RNA simply because the DMSO treatment enhanced some leakage of nuclear RNA into the cytoplasmic phase, If it were so we would have found the same sort of difference when ssDNA from non-induced cells was first hybridized to homologous RNA and the non-annealed ssDNA fraction was rehybridized to the same RNA preparations (either from induced cells or from non-induced cells) which were utilized in the case of ssDNA from DMSO-treated cells. The results illustrated by fig. 4 (see the lower curve), confirmed in three separate assays, clearly demonstrated the absence of any significant difference. It can therefore be concluded that about 10% of ssDNA from DMSOtreated cells must represent coding DNA sequences that appear to be expressed in the induced cells and to remain silent in the non-induced cells. This difference can be tentatively estimated in terms of genetic information. The haploid mouse genome contains about 1.8x 1012D of DNA [35]. Therefore, 10% of the ssDNA from DMSOtreated cells, corresponding to 0.1.5-O.16% of the total nuclear DNA, would be equivalent to 2.7x lo9 Dal. Assuming that the average molecular weight of cytoplasmic mRNA is 6-7x lo5 Dal and that ssDNA sequences which originate mainly from the non-repetitious component of bulk nuclear DNA, are not repeated more than 24 times in each cellular genome, then 10~2000 new cytoplasmic mRNA species might be

125

activated under the effect of DMSO. We must consider these values as gross approximations, but this degree of difference between the control and the DMSO-treated cells seems to be reasonable. Two conclusions may be drawn from the present work. First, the detection of differences between two populations of coding sequences with a high degree of community was rendered possible by the use of this small ssDNA fraction highly enriched for transcribed DNA segments, Second, contrary to recent claims based on the use of less sensitive methods, the DMSO treatment of Friend cells appears to switch on the transcriptional activity of many new genes, possibly in relation to single-strand breakage of the DNA 1361.It is very likely that at least some of these newly expressed genes are responsible for Friend cell differentiation. This work was carried out with the skilful technical assistance of G. Frezouls and financial support of DGRST (AC Cancerogenese et Pharmacologic du Cancer, no. 78.7.2640).

REFERENCES 1. McKnight, G S. Pennequin, P & Schimke, R T, J biol them 250 (1975) 8105. 2. Friend, C, Scher, W, Holland, J G & Sato, T, Proc natl acad sci US 68 (1971) 378. 3. Kabat, D, Sherton, C C, Evans, L M, Bigley, R & Koler, R D, Cell 5 (1975) 331. 4. Ross, J, Ikawa, Y & Leder, P, Proc natl acad sci US 69 (1972) 3620. 5. Ross, J, Gielen, J, Packman, S, Ikawa, Y & Leder, P, J mol biol87 (1974) 697. 6. Orkin. S H & Swerdlow. P S. Proc natl acad sci US 74 (1977) 2475. 7. Lowenhaupt, K, Trent, C & Lingrel, J B, Dev biol 63 (1978) 441. Kafatos, F C, Curr top dev bio17 (1972) 125. t : Marks, P A & Rifkind, R A, Science I75 (1973) 955.

IO. Gilmour, R S, Harrison, P R, Windass, J D. AfTara, N A & Paul, J, Cell differ.3 (1974) 9. 11. Aviv, H, Voloch, Z, Bastes, R & Levv, S, Cell 8 (1976)495. 12. 3uckingham, M E, Caput, D, Cohen, A, Whalen,

R G & Gras, F, Proc natl acad sci US 71 (1974) 1466.

13. Lodish, H F & SmaJI, B, Cell 7 (1976) 59. 14. Harrison, P R, Nature 262 (1976) 353. 15. Tapiero, H, Leibovitch, S A, Shaool, D, Monier, M N & Harel, J, Nucleic acids res 3 (1976) 953. 16. Leibovitch, S A, Tapiero, H & Hare], J, Proc natl acad sci US 74 (1977) 3720. 17. Hanania, N, Shaool, D, Poncy, C, Tapiero, H & Ha&, J, Cell biol int rep 1 (1977) 309. 18. Shaooi, D, Hanania, N, Hare], J & May, E, J gen viral 43 ( 1979)57I _ 19. Franze-Fernandez, M T & Pogo, A O1 Cancer res 36 (1971) 3394. 20. Tapiero, H, Monier, M N, Shaool, D & Harel, J, Nucleic acids res 1 (1974) 309. 21. Aviv, H & Leder, ‘P, Proc natl acad sci US 69 (1972) 1408. 22. Woo, S L S, Rosen, J M, Liarakos, C D, Choi, Y C, Busch, H, Means, A R, O’Malley, B W & Robberson, D L, J biol them 250 (1975) 7027. 23. Kleiman, L, Birnie, G D, Young, B D & Paul, J, Biochemistry 16 (1977) 1218. 24. Vogt , V , Nature 223 ( 1969)854. 25. Bick, M D, Lee, X S & Thomas, C A Jr, J mol biol 71 (1972) 1.

Exp CdlRes 130 (1980)

26. Groner, Y, Monroy, M, Jacquet, M & Hurwitz, J, Proc natl acad sci US 72 (1975) 194. 27. Frenster, J H, Cancer res 36 (1976) 3394. 28. Leibovitch, S A & Harel, J, Nucleic acids res 5 (1978) 777. 29. Birnie, G D, MacPhail, E, Young, B D, Getz, M J & Paul, J, Cell differ 3 (1974) 22. 30. Mauron, A & Spohr, G, Nucleic acids res 5 (1978) 3013. 31. Reeves, R & Cserjesi. V, J biol them 254 (1979) 4283. 32. Mintv. A J. Birnie. G D & Paul.;,J. Exo. cell t-es 115 (1978)‘l. 33. Leibovitch. M P. Leibovitch. S A. Harel. J & Ktuh, J, Eur i biochem 97 (1979) 321. 34. Leibovitch, S A, Leibovitch, M P, Kruh, J & Harel. J. Em i biochem 97 (1979) 327. 35. Young, B D, Bimie, G D & Paul, J, Biochemistry 1.5(1976) 2823. 36. Scher, W & Friend, C, Cancer res 38 (1978) 841. Received February 19, 1980 Revised version received June 23, 1980 Accepted June 25, 1980

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