Failure of reactivation of chick erythrocytes after fusion with temperature-sensitive mutants of mammalian cells arrested in G1

Experimental

FAILURE

Cell Research 113 (1978) 359-367

OF REACTIVATION

FUSION

WITH

Department

ERYTHROCYTES

TEMPERATURE-SENSITIVE

MAMMALIAN YOSHIHIRO

OF CHICK

TSUTSUI,

of Pathology

CELLS

ARRESTED

MUTANTS

AFTER OF

IN Cl

SHAW DING CHANG and RENATO BASERGA

and Fels Research Institute, Temple University Philadelphia, PA 19140, USA

School of Medicine,

SUMMARY Two temperature-sensitive (ts) mutants of mammalian cell lines (AF8 and cs4D3) that arrest in Cl * at the nonpermissive temperature were fused with chick erythrocytes and the induction of DNA synthesis was studied in the resulting heterokaryons. While both AF8 and cs4D3 could induce DNA synthesis in chick nuclei at the permissive temperature, they both failed to do so when arrested in Gl at the nonpermissive temperature. When S phase AF8 cells were fused with chick erythrocytes, chick nuclei were reactivated even if the heterokaryons were incubated at the temperature nonpermissive for AF8. A third ts mutant, tslll, that is blocked in cytokmesis but continues to synthesize DNA, reactivated chick nuclei at both permissive and nonpermissive temperature. It is concluded that chick erythrocyte reactivation depends on the presence of S phase-specific factors.

When chick erythrocytes are fused with growing mammalian cells, the chick nuclei in the resulting heterokaryons are reactivated [ 1, 2 and see review in 31. When the chick nucleus is reactivated, it undergoes a series of morphological and chemical changes that have been listed and discussed in detail by Ringertz & Bolund [3]. These changes include migration of nucleoplasmic and nucleolar proteins from the mammalian cell into the chick nucleus [4, 5, 61, and the induction of DNA synthesis in the chick nucleus [7, 81. Recently, Dubbs & Kit [9] reported their studies on a temperature-sensitive (ts) mutant of a Chinese hamster cell line (K12 cells) that arrest in Gl at the nonpermissive temperature. When fused with chick erythrocytes, K12 cells failed to induce DNA synthesis in chick nuclei if the heterokaryons were incubated at the temperature 24-781812

nonpermissive for the ts mutant. As a general approach to an understanding of the biochemistry of the cell cycle, we have extended these investigations to other cell cycle-specific ts mutants under different growth conditions. MATERIALS

AND METHODS

Cell cultures The AF8 cell line is a ts mutant of BHK 21/13 cells isolated by Basilic0 and co-workers flO1. At the nonpermissive temperature (39”-4O“C), AF8-cells arrest in Gl [lo, 111at a point roughly located 8 h before the beginning of S. The cs4D3 cell line is a cold-sensitive mutant of CHO cells described by Crane & Thomas [12]. When shifted to the nonpermissive temperature (32”-33°C). cs4D3 cells also arrest in Gl. from which ihey can be stimulated to enter S by simpiy incubating them at 39°C f121. tslll is a ts mutant of Chinese hamster tibrobiasis, originally isolated and described by Hatzfeld & Buttin f131. In this line. cvtokinesis is blocked at 39°C while macromolecular synthesis (including DNA synthesis) continues at a steady rate. This mutant, therefore, arrests at the nonpermissive temperature, after S. The tsAF8 cells were grown in

360

Tsutsui, Chang and Baserga

Dulbecco’s high glucose medium (Gibco) plus 10% calf serum (Gibco) and antibiotics, as previously described [1 11. The cs4D3 cells were grown in Basal Medium Eagle’s (TIME) (Microbiological Assoc. Co.), supplemented with nonessential amino acids as described by Crane & Thomas [12], and with 10% fetal calf serum (Gibco). tslll cells were grown in monolayers, like AF8 cells, in Dulbecco’s medium and 10% serum.

Serum stimulation

of resting cells

tsAF8 cells and cs4D3 cells were grown on 22 mm square coverslips in 100 mm Petri dishes. To obtain populations of quiescent tsAF8 cells the growth medium was removed when the cultures were about 60 % confluent, the cells were washed twice with Hanks’ solution and then incubated in fresh medium plus 0.5 % serum. After 48 h of serum deprivation the cells were stimulated by changing the medium to 10% serum. In one experiment, tsAF8 cells were made quiescent by isoleucine deprivation [ 141. Subconfluent cultures were kept in isoleucine-deficient medium for 66 h (the last 2 h at 40°C). The cells were then fused (see below) with chick erythrocytes and plated in normal medium plus 10% serum. cs4D3 cells were made quiescent by shift-down, when about 60% confluent, to the nonpermissive temperature of 33°C without changing the medium. After 65 h at 33°C the medium (containing 10% serum) was changed and the cells were stimulated by shifting up to the permissive temperature of 39°C.

Preparation

of heterokaryons

Cell fusion between the ts mutants and chick erythrocytes was carried out either in suspension or in monolayers in the presence of UV-inactivated Sendai virus (Microbiological Assoc., Walkersville, Md; titer 1: 16000). Chick erythrocytes were obtained from loday-old chick embryos [15]. Allantoic vessels were damaged to bleed into the allantoic fluid. The allantoic fluid was centrifuged at 1000 rpm for 5 min in an International Centrifuge. The pelleted red blood cells were washed three times with Dulbecco’s MEM and were resuspended in the same solution at a concentration of 5~ lOr/ml. The suspension method of fusion was performed basically according to the technique described bv Croce et al. 1161.Serum starved tsAF8 cells were detached from the Petri dishes with 0.25 % Trvnsin in Ma*+ and Ca2+-freeHanks’ solution at 37”C, was’hed three times with Dulbecco’s MEM and resuspended in the same solution at a concentration of 3-5x 10scells/ml. A 1 ml of tsAF8 cell suspension was mixed with 1 ml chick ervthrocyte suspension. The mixture was spun and resuspended in 1 ml Dulbecco’s MEM, kept on ice for 5 min and then 500 U of inactivated Sendai virus was added. The suspension was kept on ice for 20 min with occasional gentle shaking, then incubated in a water bath at 37°C for 30 min with constant shaking. After incubation, the cell SUSpension was spun and resuspended in medium containing the desired concentration of serum. The cells were plated in coverslip-containing 30 mm Petri dishes. Exp Cell Res 113 (1978)

For the monolayer method, fusions were carried out as described by Appels et al. [17]. Cells grown on coverslips in 100 mm Petri dishes were washed three times with Dulbecco’s MEM. 6 ml of MEM were then layered over the coverslips, and the cells were chilled at 4°C for 15 min. 1600 units of inactivated Sendai virus were added by drops directly over the coverslips. After gentle shaking, 1 ml of chick erythrocyte syspension (5x 10’ cells) were added over the coverslips in the same way as Sendai virus. The cells were chilled at 4°C for 15 min with occasional gentle shaking and then removed to a 37°C incubator for 30 min. After incubation the cells were washed gently and finally incubated in fresh medium containing the desired concentration of serum.

Autoradiography Cells were continuously exposed to r3H]thymidine 0.2 &i/ml, beginning after cell fusion. Autoradiography and analysis of autoradiographs were carried out as described by Baserga & Malamud [18]. At least 500 cells or 500 heterokaryons were counted for each experimental point. *

Immunojluorescence

procedure

Hamster liver nonhistone protein-DNA complex (NHP-DNA) and hamster liver nucleoli were prepared as previously described [19, 20, 211. Immunization schedule and preparation of antisera against the NHPDNA complex and nucleoli were also described previously [22]. After fixation of cells with ethanolacetone (1 : 1). the coverslins were divided into rectangles by marking with a glass pen. Immunofluorescent staining using the anti NHP-DNA complex and antinucleolar antisera was performed as previously described 120. 221. For auantitative determinations all heterokaryons which had fluorescent erythrocyte nuclei were first counted in a marked rectangular area by immunofluorescent microscopy. Then the number of heterokaryons in the same area was counted by phase contrast microscopy.

RESULTS Induction of DNA synthesis in chick nuclei after fusion with tsAF8 cells

After 48 h of serum deprivation using 0.5 % serum tsAF8 cells were quiescent (less than 5% cells were labeled by [3H]thymidine). These quiescent tsAF8 cells were fused with chick erythrocytes by the monolayer method, and the fused cultures were incubated in fresh medium plus 10% serum and [3H]TdR. As shown in fig. 1, in heterokaryons incubated at the permissive temperature (34”C), about 80% of the chick

Cell cycle ts mutants

361

nuclei and of the tsAF8 nuclei were labeled 36 h after fusion. When chick nuclei in heterokaryons were labeled, tsAF8 nuclei in the same heterokaryons were always la-

beled. On the other hand, in heterokaryons incubated at the nonpermissive temperature (in 10% serum), about 20% of either tsAF8 or chick nuclei were labeled 36 h after fusion. Similar results were obtained when tsAF8 cells and chick erythrocytes were fused by the suspension method (not shown), except that the percentage of tsAF8 cells entering DNA synthesis was only 43% with an even lower number of labeled chick nuclei (see also fig. 4). Using the suspension method the efficiency of fusion was about 30% of the total tsAF8 cells. However, the cytotoxic effect of Sendai virus was marked, since in control experiments 90% of tsAFg cells were labeled at the permissive temperature 36 h after serum stimulation and mock-fusion without Sendai virus. In monolayer fusion, although the efficiency of fusion was about 15% of total tsAF8 cells, the cytotoxic effect of Sendai virus on tsAF8 cells in terms of induction of DNA synthesis was low (fig. 1). In addition the percent heterokaryons in which tsAF8 nuclei were labeled but chick nuclei were not labeled, was significantly lower than with the suspension method.

Fig. 2. Immunofluorescence microphotographs of heterokaryons between tsAF8 cells and chick erythrocytes after treatment with anti-hamster NHP-DNA

complex antiserum (A) and with anti-hamster nucleolar antiserum (I#). The heterokaryons were cultured at the nonpermissive temperature for 32 h after fusion.

6

12

18

24

30

36

Fig. I. Abscissa: after fusion time (hours); ordinate: % of heterokaryons with labeled nuclei. Induction of DNA synthesis in chick nuclei at both permissive (34°C) and nonnermissive temnerature (40°C) after fusion with tsAFg cells by the monolayer method. tsAF8 cells were made auiescent in 0.5% serum for 48 h, then fused with chick erythrocytes. Atier fusion the cells were plated on coverslips in 30 mm Petri dishes and cultured in 10% serum medium containing 0.2 &i/ml of [3H]thymidine. Per cent of heterokaryons in which tsAF8 nuclei are labeled at 34°C (0), at 40°C (A); per cent of heterokaryons in which both tsAF8 nuclei and chick nuclei are labeled at 34°C (O), at 40°C (A).

Exp Cdl Res 113 (IY78J

362

Tsutsui, Chang and Baserga

50

30

20.

10 !

0II3

I

/

40

4

I

after fusion. Fig. 3A shows that chick nuclei began to react (by immunofluorescence) with antisera against hamster chromosomal proteins about 12 h after fusion at both permissive and nonpermissive temperatures. Fig. 3B shows that chick nuclei also began to react with antisera against hamster nucleolar proteins about 20 h after fusion at both permissive and nonpermissive temperature, although the percentage of positive chick nuclei was slightly less at the nonpermissive temperature.

t

Effect of low serum on induction of DNA synthesis and migration of chromosomal proteins in chick nuclei of heterokaryons Serum-deprived tsAF8 cells were fused with chick erythrocytes by the suspension method, then plated in either 0.5 % or 10% serum at 34°C. As shown in fig. 4A, in Fig. 3. Abscissa: after fusion time (hours); ordinate: heterokaryons in 0.5 % serum only 6% of % of heterokaryons with fluorescent chick nuclei. Uptake of chromosomal and nucleolar hamster pro- the chick nuclei were labeled by r3H]thymiteins by chick nuclei after fusion with tsAF8 cells. dine 30 h after fusion, while in 10% serum, Same experimental conditions as in fig. 1 except fusion was carried out by the suspension method. 35% of the chick nuclei in heterokaryons (A) Heterokaryons incubated at 34°C (O), or at 40°C (A), and treated with anti-hamster NHP-DNA com- were labeled. The percentage of labeled plex antiserum; (II) heterokaryons incubated at 34°C AF8 nuclei was essentially the same as the (O), or at 40°C (A) and treated with anti-hamster percentage of labeled chick nuclei (not nucleolar antiserum. shown). Fig. 4B shows that chick nuclei of heterokaryons in 0.5 % serum reacted Uptake of chromosomal and nucleolar with antihamster NHP-DNA complex antiproteins of tsAF8 cells by chick nuclei serum almost the same as chick nuclei of in heterokaryons heterokaryons in 10% serum. Both anti-hamster NHP-DNA complex and anti-hamster nucleolar antisera reacted by Effect of serum stimulation of tsAF8 immunofluorescence with nuclei and nu- cells before fusion on the induction of cleoli of tsAF8 cells, respectively (fig. 2A, DNA synthesis in chick nuclei B). The same antisera did not react with When resting tsAF8 cells are stimulated nuclei of unfused chick erythrocytes (not at the permissive temperature and then shown). Fig. 2 (A, B) shows that chick nu- shifted-up to the nonpermissive temperaclei in heterokaryons with tsAF8 reacted ture at various intervals after stimulation, with both antihamster NHP-DNA complex the fraction of cells entering DNA synthesis (A) and anti-hamster nucleolar antisera varies with the time interval between stimueven at nonpermissive temperature 32 h lation and shift-up. These studies have been Exp CdRrs

113 (1978)

Cell cycle ts mutants

363

Table 1. Fraction of tsAF8 cells capable of entering the S phase when the cells are shifted to the nonpermissive temperature at various times after stimulation Time at permissive temperature (hours)

Fraction of cells in DNA synthesis

0

8

6

15

10

29 43

12 20 36

100 100

Serum-deprived AF8 cells were stimulated with 10% serum at the permissive temperature (34°C) and shifted to 40°C at the times indicated in the first column. The experiment was terminated at 36 h. For convenience, the fraction of cells is expressed as % of the maximum number of cells stimulated, which is 9095 % of the total population.

c ,:

cultured in 10% serum at the nonpermissive (fig. 5A, B) or permissive temperatures (fig. Fin. 4. Abscissa: after fusion with tsAF8 time (hours): 5C, D) for 12 h and 27 h after fusion in ordinate: % of heterokaryons with labeled chick the presence of [3H]thymidine. The control nuclei. (A) Induction of DNA synthesis in chick nuclei after experiment is the one in panel D. It shows fusion with tsAF8 cells which had been kent in 0.5% serum for 48 h before fusion. After fusion, the hetero- that if the heterokaryons, after fusion, are karyons were incubated in either 0, 0.5% serum me- incubated in 10% serum at the permissive dium or in 0, 10% serum medium. (B) Uptake of temperature for 27 h, tsAF8 nuclei enter hamster proteins by chick nuclei after fusion with tsAF8 cells in the same conditions as in A. The or- DNA synthesis and chick nuclei are redinate gives the percentage of fluorescent chick nuclei activated, regardless of the time of stimula(see figs 2 and 3). tion prior to fusion. The crucial experiment is shown in panel B. When the heteroreported in detail in another paper (Rossini karyons, after fusion, are incubated at the & Baserga, in press), and are simply sum- nonpermissive temperature for 27 h, the marized for convenience in table 1. Al- percentage of labeled tsAF8 nuclei and of though tsAF8 cells enter S between 20- reactivated chick nuclei is a function of the 30 h after stimulation, a shift-up at 12 h time of stimulation prior to fusion, as could allows almost 50% of the cells to enter be predicted from the results of table 1. DNA synthesis. On the basis of these Thus, if unstimulated AF8 are fused with data serum-deprived tsAF8 cells were stim- chick erythrocytes and the heterokaryons ulated by changing the medium to 10% incubated at 4O”C, very few tsAF8 cells serum at the permissive temperature for 0, enter DNA synthesis and the number of re6, 12, 18 and 24 h prior to fusion, then activated chick nuclei is correspondingly fused with chick erythrocytes by the mono- low. As the hours of stimulation prior to layer method. The fused cells were then fusion increase, the fraction of tsAF8 cells 0

10

20

30

Exp Cdl Rrs 113 (19781

364

Tsutsui, Chang and Baserga

100 A I

C

i 100 B

rL

n

n

n

D ”

karyons seems to be delayed when the heterokaryons are kept at the nonpermissive temperature, since the per cent of heterokaryons in which tsAF8 nuclei were labeled, but chick nuclei were not labeled (difference between open and closed columns), was significantly higher at the nonnermissive temuerature than at the permissive temperature.

1

Fusion of chick erythrocytes with isoleucine-deprived tsAF8 cells In these experiments, tsAF8 were blocked in isoleucine-deficient medium as described Fig. 5. Abscissa: after serum stimulation prior to in Methods and Materials. They were then fusion time (hours); ordinate: % of heterokaryons with labeled nuclei. fused with chick erythrocytes, and the Induction of DNA synthesis in chick nuclei after fusion with tsAF8 cells and incubation at either non- heterokaryons were plated in regular mepermissive (40°C) for 12 h (A) or 27 h (B), or at dium plus 5% serum. Under these condipermissive temperature (34°C) for 12 h (C) or 27 h (0). Serum-denrived tsAF8 cells stimulated for 0. 6. tions by 24 h after release of the block, 12, 18 and 24 h prior to fusion. Heterokaryons were 90-94% of tsAF8 cells are labeled by r3H]then incubated in 10% serum and continuously labeled TdR, at either permissive or nonpermissive with r3H]thymidine (0.2 &i/ml) after fusion. Open columns are per cent of heterokaryons in which tsAF8 temperatures, confirming the observation nuclei are labeled. Closed columns are per cent of of Burstin et al. [lo] that isoleucine-deheterokaryons in which both tsAF8 nuclei and chick nuclei are labeled. prived tsAF8, when the block is released, can enter S even at the nonpermissive temperature. Fig. 6 shows that chick nuclei are that have passed the shift-up point (table 1) reactivated when fused with ileu-blocked increases, a large number of tsAF8 cells tsAF8 cells, even when the block is reenter DNA synthesis, and a proportional leased at the temperature nonpermissive for number of chick nuclei are reactivated. tsAF8. Panels A and C are shown to stress the fact that 12 h of incubation, after fusion, are not sufficient for a complete display of DNA synthesis in tsAF8 (and, therefore, of reactivation of chick nuclei). It might be concluded from the results shown in fig. 5 that chick nuclei can be induced to synthesize DNA in heterokaryons even at the nonL/L 2: 4.5 permissive temperature when they were fused with tsAF8 cells, which were already Fig. 6. Abscissa: time after fusion with tsAF8 (hours); % of labeled chick nuclei. in S phase, or had been allowed to pass the ordinate: tsAF8 were blocked by isoleucine deficiency (see shift-up point before fusion and, therefore, Methods) and then fused with chick ervthrocvtes. The heterokaryons were incubated in reguiar medium plus to enter S in subsequent hours. However, 5% serum at either 33°C (O-O) or 40°C (O-O) in reactivation of chick nuclei in hetero- the presence of r3H]thymidine (0.2 &i/ml). Exp Cell RPS 113 (1978)

Cell cycle ts mutants 100

80 [

60

-

10

-

20

-

0

6

12

IS

24

30

36

time after fusion (hours); ordinate: % of heterokaryons with labeled nuclei. Induction of DNA synthesis in chick nuclei at both permissive (39.W) and nonpermissive temperatures (33°C) after fusion with cs4D3 cells. cs4D3 cells had been at the nonpermissive temperature for 65 h before fusion. Per cent of heterokaryons in which cs4D3 nuclei were labeled at 39.X (0), or 33°C (0); per cent of heterokaryons in which both cs4D3 and chick nuclei were labeled at 39.5”C (O), or 33°C (A).

365

monolayer method, and the fused cells were incubated in 10% serum at either 33” or 39S”C. As shown in table 2, both tslll and chick nuclei in heterokaryons were stimulated to synthesize DNA by serum. There was very little difference whether the heterokaryons were incubated at either permissive or nonpermissive temperatures. This is in agreement with the report of Hatzfeld & Buttin [13] that tslll cells, at the nonpermissive temperature, are blocked in cytokinesis.

Fig. 7. Abscissa:

Induction of DNA synthesis in chick nuclei fused with cs4D3 cells cs4D3 cells were made almost quiescent (less than 13% cells were labeled with [3H]thymidine) by shifting down to the nonpermissive temperature for 65 h. They were then fused with chick erythrocytes by the monolayer method. As shown in fig. 7, induction of DNA synthesis in chick nuclei of heterokaryons incubated at 39°C (permissive temperature) was high. The per cent of heterokaryons with labeled chick nuclei was 61% at 30 h and 72% at 36 h after fusion respectively. Induction of DNA synthesis in chick nuclei fused with tslll cells These cells were made partially quiescent by incubation in 0.2% serum at 39.5”C for 48 h. Under these conditions, the percentage of cells labeled by C3H]TdR over a period of 30 h was 17.2%. These cells were then fused with chick erythrocytes by the

DISCUSSION Dubbs & Kit [9] have shown that a ts mutant of Chinese hamster cells, K12, that arrests in Gl at the nonpermissive temperature [23, 241cannot reactivate chick erythrocytes at the nonpermissive temperature, although capable of doing so at the permissive temperature. The term reactivation, in this Discussion, refers to the induction of DNA synthesis in chick nuclei. We have confirmed their results with two other ts mutants, which arrest before S at the nonpermissive temperature, tsAF8 (from BHK Syrian hamster cells) and cs4D3 (from

Table 2. DNA synthesis in heterokaryons between tslll cells and chick erythrocytes % labeled nuclei in heterokaryons Time after fusion (hours)

Temperature (“0

ts111 nuclei

Chick nuclei

30 30

33 39.5

67 69.5

51.2 58.5

tsl 11 cells were made partially quiescent as described in the text. After fusion, the cultures were exposed to [aH]TdR (0.05 /.&i/ml) and autoradiographs made as described in Methods and Materials. Only nuclei of heterokaryons were counted. Two other experiments, in which the conditions for quiescence were slightly modified, gave essentially the same results. Exp Cell Res II3 (I 978)

366

Tsutsui, Chang and Baserga

Chinese hamster cells), the latter being cold-sensitive rather than ts. Kit and coworkers used as a control the parent cell line of K12 cells, and showed that a temperature of 41°C does not inhibit per se the reactivation of chick erythrocytes, if the donor cells is not ts. We have also confirmed this with BHK cells, but we have omitted these results, since we already have a built-in control in our data of fig. 5 (see below). It seems, therefore, that cells arrested in Gl cannot reactivate chick erythrocytes. This conclusion is further supported by the findings that chick nuclei are not reactivated when the heterokaryons are kept at permissive temperature in 0.5% serum, a concentration of serum which keeps AF8 cells quiescent [lo, 111.In fact, one can go a step further and state that chick nuclei are reactivated only if the ts cell is in S phase or capable to reach the S phase. This conclusion is supported by the following findings: (1) chick nuclei are reactivated when fused with tslll cells, even when the heterokaryons are incubated at the nonpermissive temperature. tsl 11 cells continue to synthesize DNA at 39.5”C and are ts only for cytokinesis; and (2) much more important, only when tsAF8 cells can enter S, are chick nuclei reactivated (fig. 5B, D). The data of fig. 5 must be analysed together with those of table 1, since there is a delay in the ts arrest of AF8 cells shifted to the nonpermissive temperature. Thus, if AF8 cells are stimulated at 34°C for 6 h, and then shifted-up, a fraction of cells reaches S phase, the percentage increasing with increasing periods of stimulation at the permissive temperature. This is reflected in the percentage of chick nuclei that are reactivated. Only when AF8 cells are fused with chick erythrocytes immediately after stimulation, does incubation

at 40°C result in no DNA synthesis, either for AF8 or chick nuclei (see fig. 5B). Thus, it seems that only cells in S phase or capable to reach S have all the necessary information to reactivate chick nuclei after fusion. This finding is a confirmation and an extension to ts mutants of previous reports on the inability of non-dividing cells (such as rat myotubes or mouse macrophage) to induce DNA synthesis in chick nuclei (see review in [3]). Our experiments are also in agreement with the conclusions of Johnson & Mullinger [15] using HeLa cells fused with chick erythrocytes that “once DNA synthesis has been initiated by an S phase environment . . . it can continue in the heterokaryon even though the cellular environment is G2 or mitotic in type”. The present findings that tsAF8 cells released from an isoleucine block reactivate chick erythrocytes even at the nonpermissive temperature is in contrast to the demonstration that the same cells released from serum deprivation cannot reactivate chick nuclei at the nonpermissive temperature. It would seem to indicate that serum-deprived tsAF8 cells are in a different state from isoleucine-deprived AF8, a fact also suggested on purely kinetic grounds by the data of Burstin et al. [IO]. Finally, our experiments show that hamster proteins of either nucleoplasmic or nucleolar origin migrate into chick nuclei after fusion with hamster cells. A migration of nucleoplasmic and nucleolar proteins of human origin into chick nuclei after fusion with HeLa cells has been reported by Ringertz and co-workers [4, 5, 61. Our experiments show that such proteins (in our case, of hamster origin) migrate into chick nuclei even when the heterokaryons are incubated at the temperature nonpermissive for AF8, i.e., when no DNA synthesis occurs. This seems to lend strong support to

Cell cycle ts mutants the hypothesis of Ringertz & Savage [25] that the initiation of DNA synthesis in Gl nuclei of heterokaryons depends on the migration into Gl nuclei of protein factors specific for the S phase. In their absence, as in Gl arrested ts mutants, other proteins from the donor cell are insufficient for the reactivation of the chick nucleus. In conclusion, we have used ts mutants that arrest at the nonpermissive temperature in Gl to show that chick nuclei in heterokaryons are reactivated only when certain factors from an S phase cell are present. This method should be useful for an analysis of the cell cycle, in terms of informational content in different physiological states. This work was supported by USPHS Grant GM 22359 from the National Institute for General Medical Sciences.

REFERENCES 1. Harris, H, J cell sci 2 (1967) 23. 2. Harris, H, Sidebottom, E, Grace, D M & Bramwell, M E, J cell sci 4 (1969) 499. 3. Ringertz, N R & Bohmd, L, Int rev exp pathol 13 (1974) 83. 4. Ringertz, N R, Carlsson, S, Ege, T & Bolund, L, Proc natl acad sci US 68 (1971) 3228.

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5. Ege, T, Carlsson, S A & Ringertz, N R, Exp cell res 69 (1971) 472. 6. Carlsson, S A, Moore, G P M & Ringertz, N R, Exp cell res 76 (1973) 234. 7. Harris, H, Nature 206 (1965) 583. 8. Bolund, L, Ringertz, N R & Harris, H, J cell sci 4 (1%9) 71. 9. Dubbs, D R & Kit, S, Somatic cell genet 2 (1976) 11. 10. Burstin, S J, Meiss, H K & Basilica, C, J cell physiol84 (1974) 397. 11. Kane, A, Basilica, C & Baserga, R, Exp cell res 99 (1976) 165. 12. Crane, M St J & Thomas, D B, Nature 261 (1976) 205. 13. Hatzfeld, J & Buttin, G, Cell 5 (1975) 123. 14. Ley, K D & Tobey, R A, J cell biol47 (1970) 453. 15. Johnson, R T & Mullinger, AM, J cell sci 18(1975) 455. 16. Croce, C, Koprowski, H & Eagle, H, Proc natl acad sci US 69 (1972) 1953. 17. Appels, R, Bolund, L & Ringertz, N R, J mol biol 87 (1974) 339. 18. Baserga; R & Malamud, D, Autoradiography, p. 17. Hoeber. New York (1969). 19. Wakabayashi, K, Wang, S & Hnilica, L S, Bio2. chemistry 13 (1974) 1027. ’ Tsutsui, Y, Suzuki, I & Iwai, K, Exp cell res 101 (1976) 202. 21. Huang, C H & Baserga, R, Biochemistry IS (1976) 2829. 22. Tsutsui. Y, Chang. H L & Baserga, R. Cell biol intl reports l(1977) 301. 23. Smith. B J & Wiaelesworth. N M. J cell _nhvsiol82 . (1973)339. 24. - Ibid 84 (1974) 127. 25. Ringertz, N R & Savage, R E, Cell hybrids, p. 69. Academic Press, New York (1976). Received September 30, 1977 Accepted December 14, 1977

Exp Cell Res I 13 ( 1978)