J. Cell Sci. 2, 23-32 (1967) 23

Printed in Great Britain

THE REACTIVATION OF THE RED CELL

NUCLEUS

H.HARRISThe Sir William Dunn School of Pathology, University of Oxford

SUMMARY

When the nucleus of a mature hen erythrocyte is introduced into the cytoplasm of a HeLacell it resumes the synthesis of RNA and DNA. This reactivation of the red cell nucleus in theheterokaryon is associated with a marked increase in its volume. There is a direct relationshipbetween the volume of the nucleus and the amount of RNA which it makes. The nuclear en-largement is not a consequence of increased RNA synthesis, or of DNA synthesis: enlargementis the primary event, and the increase in RNA synthesis is determined by it. The possibility isconsidered that changes in nuclear volume may regulate not only the amount of RNA made inthe nucleus but also the areas of chromatin on which it is made.

INTRODUCTION

It has been shown that when, under the influence of inactivated Sendai virus, thenucleus of a mature hen erythrocyte is introduced into the cytoplasm of a HeLa cell,the previously dormant erythrocyte nucleus may be induced to resume the synthesisof RNA and DNA (Harris, 1965; Harris, Watkins, Ford & Schoefl, 1966). In thepresent paper this reactivation of the red cell nucleus is examined more closely; andsome evidence is presented concerning the mechanism by which it is brought about.

MATERIALS AND METHODS

Formation and maintenance of HeLa-hen erythrocyte heterokaryons

HeLa-hen erythrocyte heterokaryons were produced by treating the HeLa cells andthe red cells with Sendai virus inactivated by ultraviolet light. The techniques usedwere essentially as previously described (Harris et al. 1966). By a process of trial anderror it was found that a high yield of heterokaryons could regularly be obtained bytreating 2-5 x io6 HeLa cells and 4x10 ' erythrocytes with 500 haemagglutinatingunits of inactivated virus. All the experiments described in the present paper weremade on heterokaryons produced with this dose of virus and these cell numbers:between 20 and 30 % of all cells in the culture contained at least one red cell nucleus.The heterokaryons were maintained on coverslips as described by Harris et al. (1966).

Cytological examination of cultures

The coverslips were removed as required, fixed in methanol and stained either withMay-Grunwald-Giemsa, as described by Harris et al. (1966), with Weigert's ironhaematoxylin or with a Feulgen stain (Culling, 1963).

2 4 H. Harris

Radioactive precursors

Undine-s^H], specific activity 13 C/mM, and thymidine-6[8H], specific activity13 C/mM, were obtained from the Radiochemical Centre, Amersham. They wereused at a concentration of 10 /iC/ml of medium.

Autoradiography

The present study involved measurement of the tritium content of individual redcell nuclei by means of grain counts in autoradiographs. An attempt was thereforemade to minimize the errors arising from variation in the geometry of the fixed cellsby flattening these as far as possible before application of the autoradiographic emul-sion. The cells were flattened during fixation in methanol by the technique of Gaillard& Schaberg (1964); a modification of their apparatus was used, which will be de-scribed elsewhere. This technique is very effective in flattening the cells and producesmuch less cell disruption than conventional' squash' preparations. The fixed prepara-tions were extracted and the autoradiographs prepared as described in Harris et al.(1966). Exposures were adjusted to give a suitable density of grains for counting.

The two sources of error in comparing the amount of tritium in different nuclei byautoradiography are variation in the vertical thickness of the individual nuclei andvariation in the thickness of the layer of cytoplasm overlying the nuclei. The measure-ments of Perry, Errera, Hell & Diirwald (1961) show that in unflattened preparations

0-2/i

OlS/i

0-2/i.

Fig. 1. For explanation see text.

the average vertical thickness of the nuclei of HeLa cells growing as a monolayer is2 fi; the average thickness of the layer of cytoplasm overlying the nuclei is 0-2 /<• andthe average thickness of the cytoplasm surrounding the nuclei is 1-4/*. Consider thetwo extreme cases illustrated in Fig. iA and B.

In A, one of the red cell nuclei is assumed to have the thickness of a HeLa nucleus(2 /i) and the other is one-eighth as thick (0-25 /i). The overlying cytoplasmic layer has

Reactivation of the red cell nucleus 25

the same thickness over both nuclei (0-2 fi). In comparing the amount of radioactivityin the two nuclei, the factor introduced by overlying cytoplasm can be neglected, sinceit is the same for both nuclei, and error is introduced only by difference in the thick-ness of the nuclei. The work of Maurer & Primbsch (1964) shows that only about50 % of the radioactivity from a nucleus 2 /i thick is registered in the autoradiograph;but 98 % of the radioactivity from a nucleus 0-25 ji thick is registered. In this case,therefore, a comparison of the amount of radioactivity in the two nuclei is subject to asystematic error in which the score for the larger nucleus is about 50 % too low relativeto the smaller nucleus. In B, the two nuclei have the same dimensions as in A, but differin the amount of overlying cytoplasm. This layer is 0-2 fi over the larger nucleus, but1-15 /i over the smaller, which is assumed to lie at the bottom of an average thickness ofcytoplasm. In this case the error due to the difference in the thickness of the nuclei isthe same as in A, that is, 50 % too low for the larger nucleus. From the data of Maurer& Primbsch (1964) it can be deduced that with an overlying layer of cytoplasm 0-2 fithick about 85 % of the radioactivity from the underlying nucleus is registered. Witha cytoplasmic layer 1 • 15 fi thick only about 25 % is registered. The overlying cytoplasmintroduces a systematic error in which the score for the smaller nucleus is about 70 %too low relative to the larger nucleus. The errors due to overlying cytoplasm and tovariation in thickness of the nucleus are thus in opposite directions and tend to canceleach other. The net error for case B, taking both these factors into consideration, isthat the score for the smaller nucleus is about 40% too low relative to the larger.

The errors in cells which have been flattened are, of course, smaller than thosecalculated for the two extreme cases A and B, since both variation in the thickness ofthe nuclei and in the thickness of the overlying cytoplasm are reduced. However, thetwo extreme cases are relevant because they indicate the maximum range of errorwhich can be expected from geometrical considerations alone. If the difference inaverage grain count between two groups of nuclei clearly exceeds this maximum rangeof error, one can conclude that the two groups of nuclei contain different amounts ofradioactivity.

Comparative measurements of the dimensions of red cell nuclei in heterokaryons

Photomicrographs were taken with the focus of the objective adjusted so that eachnucleus occupied its maximum area in a horizontal plane. The photographs wereprinted at the same magnification on uniform paper, and each nucleus was then cutout and weighed. This weight was taken as a measure of the maximum horizontalcross-sectional area of the nucleus. In flattened preparations this parameter could betaken as an index of changes in nuclear volume, although the volume changes weregreater than the changes in maximum cross-sectional area, since the larger nuclei,even in flattened preparations, had, on average, a greater vertical thickness than thesmaller nuclei. No attempt was made to measure the vertical thickness of the nucleiaccurately or to account for irregularities in shape, so that only a rough approxima-tion of the volume changes could be made.

26 H. Harris

Irradiation of red cells with ultraviolet light

One ml of Hanks's solution (Hanks, 1948) containing 4 x io7 erythrocytes wasspread over the surface of a Petri dish 9 cm in diameter. This film of cells was exposedto ultraviolet light emanating from a Philips 15-W 18-in. germicidal tube, type'T.U.V.'. The Petri dish was tilted and rotated continuously during irradiation. Thecells were subjected to a flux of approximately 10* ergs/mm2.

OBSERVATIONS

Morphological changes in the red cell nuclei in heterokaryons

Fig. 4 shows a smear of hen erythrocytes in which the nuclei have been stainedwith Weigert's iron haematoxylin. Essentially the same picture is obtained with aFeulgen stain. The nuclei are usually elliptical, with long and short axes of about4-5 n and 2-3 n, respectively. The nuclei show a number of discrete, deeply stainingareas (about 7 per nucleus, on average), which have been termed ' nuclear bodies' byDavies (1961). These bodies are dense aggregates of DNA and protein (Davies, 1961).Fig. 5 shows a dikaryon containing one HeLa nucleus and one red cell nucleus. Thered cell nucleus has about the same dimensions as it has in the intact erythrocyte andthe nuclear bodies are visible. A few hours after the heterokaryons have been formedthe red cell nuclei begin to enlarge. The time at which enlargement begins varies fromcell to cell and, to some extent, from experiment to experiment. Usually between20 and 40 % of the red cell nuclei show some degree of enlargement 24 h afterformation of the heterokaryons. By the second day, this figure has risen to about90 %, and by the third day unenlarged red cell nuclei are rarely seen. It appears thatvirtually all red cell nuclei in heterokaryons eventually undergo enlargement, but thatthere is a lag which ranges from a few hours to 2 or even 3 days before enlargementbegins.

The extent of enlargement is illustrated in Figs. 6-8, which show several stages ofthe process. The largest red cell nuclei seen in heterokaryons have a maximum cross-sectional area about 10 times that of the unenlarged red cell nuclei, indicating an evengreater change in nuclear volume. There is no dramatic change in the volume of theHeLa nuclei. The earliest morphological changes associated with the enlargementof the red cell nuclei are to be seen in the 'nuclear bodies'. These begin to stain lessdeeply with chromatin stains and become more diffuse (Fig. 6). They disappearaltogether at an early stage of enlargement (Fig. 7). The nucleus as a whole also stainsless deeply with chromatin stains as it enlarges. These changes clearly indicate a pro-gressive dispersion of chromatin which was initially present in a highly condensed state.Structures that could, on morphological grounds, be called nucleoli do not appear untilthe last stages of enlargement, and they are not always obvious even in maximallyenlarged nuclei. Frequently the HeLa nuclei enter mitosis while the red cell nucleiremain in interphase. When this occurs in a dikaryon containing one HeLa and oneerythrocyte nucleus, the erythrocyte nucleus, still in interphase, may pass to one of thedaughter cells during cell division. When two or more nuclei in the heterokaryon enter

Reactivation of the red cell nucleus 27

mitosis simultaneously they commonly fuse together: this process results in progres-sive nuclear fusion within the multinucleate cell, as previously described (Harris et al.1966). In some cases a dikaryon containing one HeLa and one erythrocyte nucleusseparates without mitosis into two mononucleate cells (Fig. 8). This process gives riseto a new type of diploid cell in which the nucleus is derived from a hen erythrocyteand the cytoplasm from a HeLa cell (Fig. 9).

The effect of temperature on nuclear enlargement

Some coverslips bearing HeLa-erythrocyte heterokaryons were maintained at 37 °Cfor 24 h after cell fusion. By this time about 40 % of all erythrocyte nuclei showed atleast some degree of enlargement. The coverslips were then divided into two groups:one was incubated for a further 24 h at 37 °C and the other was incubated for 24 h at26 °C. At 26 °C HeLa cells remain alive for some days and continue to synthesize bothRNA and DNA, although at much reduced rates. In the cultures maintained at 37 °Cfor 48 h more than 90 % of the erythrocyte nuclei were enlarged; in those which weremaintained at 26 °C for the second 24 h this figure did not exceed 50 %. It thus ap-pears that enlargement of the red cell nucleus depends, in a general way, on a highlevel of metabolic activity in the cell; when this level is not high enough, as at 26 °C,nuclear enlargement does not take place. This may explain the variable duration of thelag period which precedes nuclear enlargement. The formation of a heterokaryonunder the influence of a relatively high dose of inactivated virus is a traumatic eventfor the cells concerned, and obviously some time must elapse before a normal levelof metabolic activity is re-established. There is little doubt that the durationof the recovery period varies from cell to cell, and it is possible that this variationmay be responsible, at least in part, for the variation in the time of onset of nuclearenlargement.

Relationship between nuclear enlargement and RNA synthesis

Twenty-four-hour cultures containing heterokaryons were exposed to tritiateduridine for 20 min and then fixed. The cells were flattened by the procedure of Gail-lard & Schaberg (1964), and the fixed preparations were extracted in the appropriateway prior to autoradiography (Harris et al. 1966). The autoradiographs, after ex-posure for a suitable time, were systematically scanned and the number of grains overeach red cell nucleus counted. Under the conditions of labelling used in these experi-ments, exposure to a radioactive RNA precursor for 20 min produced negligiblelabelling of cytoplasmic RNA. Each nucleus was photographed after the grain counthad been made, and its maximum cross-sectional area in the horizontal plane deter-mined as described under Materials and Methods. Fig. 2 shows the relationshipbetween the maximum cross-sectional areas of the nuclei (an index of their volume)and the number of grains in the emulsion overlying them. It is clear that unenlargedred cell nuclei are not labelled and that there is a direct relationship between the sizeof enlarged red cell nuclei and the number of overlying grains. The difference in theaverage grain count per nucleus between the largest and the smallest group of en-larged nuclei (39-5 and 6-8 grains per nucleus, respectively) exceeds by a factor of

28 H.Harris

about 3 the maximum error that can be expected from variations in geometry: thedifference in grain count obviously reflects a difference in tritium content. The in-creased labelling of the larger nuclei could, in principle, be due either to increasedsynthesis of RNA or to changes in the speed with which the exogenous radioactiveprecursor enters the intranuclear RNA precursor pool. The latter possibility is, how-ever, effectively eliminated by the large body of evidence which indicates that radio-active nucleosides enter the nuclear RNA almost without lag, apparently by-passing

6 0 r

50

3

g 40ua•3 30

20

10

Av.68

0 1 2 3 4 5 6 7 8 9 10 11 12Area of nucleus (arbitrary units)

Fig. 2. The relationship between the maximum cross-sectional areas of erythrocytenuclei in heterokaryons and the number of grains overlying these nuclei in autoradio-graphs. The cells were exposed for 20 min to tritiated uridine. The arrow indicatesthe mean cross-sectional area of unenlarged erythrocyte nuclei.

the main intracellular RNA precursor pools: incorporation of radioactive nucleosidesinto nuclear RNA under the present conditions of labelling is virtually linear for thefirst hour (Harris, 1959; Watts, 1964). Exposure of the heterokaryons to tritiateduridine for periods of 10, 20, 30 and 40 min confirmed that this was also the case forred cell nuclei. It can therefore be concluded that the increased tritium content of thelarger red cell nuclei is an expression of increased RNA synthesis: the larger the redcell nucleus the more RNA it makes.

While this experiment clearly shows that nuclear enlargement and increased RNAsynthesis are intimately linked, it does not decide whether the nucleus makes moreRNA because it enlarges, or whether it enlarges because it makes more RNA. Isenlargement the primary event which determines the increased RNA synthesis, or isenlargement the consequence of increased synthesis and intranuclear accumulation ofRNA? This question was resolved by the following experiment.

Reactivation of the red cell nucleus 29

The effect of ultraviolet irradiation on nuclear enlargement

The red cells were subjected to a flux of 10* ergs/mm2 of ultraviolet light before theheterokaryons were formed. After 24 and 48 h cultivation the preparations were fixedand the maximum cross-sectional areas of the red cell nuclei in the heterokaryons were

•S 6

x, 2

uo

o3C

1ii2 3 4 S 6 7 8 9 10

Area of nucleus (arbitrary units)

4VVV/'/v// V

2 3 4 5 6 7 8 9

Area of nucleus (arbitrary units)

10

Fig. 3. A, Histogram showing the range of enlargement of hen erythrocyte nuclei inheterokaryons. B, Histogram showing the range of enlargement of hen erythrocytenuclei which had been irradiated with ultraviolet light before being incorporated intoheterokaryons. The arrows indicate the mean cross-sectional areas of unenlargederythrocyte nuclei.

measured. These measurements were compared with those of controls in which the redcells had not been irradiated with ultraviolet light. For reasons which are at presentobscure, the lag which preceded the onset of nuclear enlargement was more prolongedin the red cell nuclei which had been irradiated, but once nuclear enlargement beganit occurred to the same extent in both irradiated and control red cell nuclei. Fig. 3 Ashows the distribution of maximum cross-sectional areas of enlarged red cell nuclei

30 H. Harris

in a control preparation 24 h after the formation of the heterokaryons. Fig. 3 B showsthis distribution for the red cell nuclei which had been irradiated with ultraviolet light.The same results were obtained at 48 h. Twenty-four-hour cultures of these two groupsof cells were incubated for 20 min in medium containing tritiated uridine and werethen subjected to autoradiography. The fixed preparations were all exposed to the auto-radiographic emulsion for the same length of time. The mean number of grains over-lying enlarged red cell nuclei in the control preparation was 9-8 (standard error of themean i-o); the mean number overlying enlarged red cell nuclei which had beenirradiated with ultraviolet light was 1-5 (standard error of the mean 0-5). It is thusclear that red cell nuclei in which RNA synthesis has been drastically reduced byultraviolet light nonetheless undergo enlargement to the same degree as unirradiatednuclei. Nuclear enlargement cannot therefore be a secondary effect resulting from in-creased synthesis and accumulation of RNA in the nucleus. Enlargement must be theprimary event and the increase in RNA synthesis a consequence of it.

The possibility must also be considered that enlargement of the nucleus is aconsequence of DNA synthesis. This is, on the face of it, rather improbable, sincethe extent of nuclear enlargement greatly exceeds the doubling that might be expectedfrom reduplication of the DNA. This idea was formally dismissed by examining theincorporation of tritiated thymidine into red cell nuclei which had been irradiated withultraviolet light before being incorporated into heterokaryons. In control preparationsa i-h exposure to tritiated thymidine 24 h after formation of the heterokaryonslabelled approximately 80 % of all enlarged red cell nuclei. In heterokaryons in whichthe red cell nuclei had previously been irradiated with ultraviolet light this figurewas only 15 %. Moreover, with equal exposure times, the mean grain count over thoseirradiated nuclei which were labelled was only 5, whereas at least 80% of labelledunirradiated nuclei had more than 50 overlying grains per nucleus. (The length ofexposure of the autoradiographs was not calculated to reveal the low levels of labellingrecently described by Rasmussen & Painter (1966) following ultraviolet irradiation.)The extent of nuclear enlargement was, however, comparable in the two groups ofnuclei, as shown in Figs. 3 A, B. It is therefore clear that enlargement of the nuclei isnot a consequence of DNA synthesis.

DISCUSSION

The obvious conclusion to be drawn from the present experiments is that thesynthesis of RNA in the red cell nucleus is governed by changes in nuclear volume.The HeLa cytoplasm appears to turn the red cell nucleus on by causing it to enlargeand open up the condensed chromatin within it. The amount of RNA made is closelylinked to the degree of enlargement. It is not at present possible to decide whetherenlargement of the nucleus is a consequence of increased dispersion of the chromatinor whether it is the enlargement which permits this dispersion to take place. Davies(1961) has shown that in the nuclear membrane of the mature erythrocyte there arediscontinuities which permit the free passage of haemoglobin. If these discontinuitiespersist in the heterokaryon, the enlargement of the red cell nucleus can hardly have

Reactivation of the red cell nucleus 31

an osmotic basis: the volume changes might then be determined by direct interactionof the chromatin with charged ions, as described by Davies & Spencer (1962). Thismechanism does not, however, provide a convenient explanation for the lag periodwhich precedes nuclear enlargement, or for the effect of temperature on the process.It is possible that during this lag extensive changes take place in the erythrocytenuclear membrane, so that it comes to present a formidable ion barrier, as has beendescribed by Loewenstein & Kanno (1963) for the nuclear membrane of Drosophilasalivary gland cells.

Is it possible that changes in nuclear volume might regulate not only the amount ofRNA synthesized in the nucleus, but also the areas of DNA on which it is synthesized?Two pieces of evidence speak for this possibility. Mittwoch, Lele & Webster (1965)have shown that the condensation of the X chromosome in female human cells isconnected with variations in nuclear volume: cells in which the X chromosome is notcondensed have larger nuclei than the average, and nuclei which are smaller than theaverage show not only a condensed X chromosome but other areas of condensedchromatin as well. One may suppose that as the volume of the nucleus decreases theX chromosome undergoes condensation, and as the nucleus becomes still smalleradditional areas of chromatin condense. At the other extreme, the behaviour of the' nuclear bodies' in the present experiments suggests that when the dormant red cellnuclei enlarge, the opening up of the condensed chromatin is also not a random pro-cess : all the nuclear bodies disappear very early in the process of enlargement, whereasone might expect, if the opening up of the chromatin were not co-ordinated in anyway, that some of these bodies would persist through most stages of enlargement. It isthus possible to envisage a system of genetic regulation in which changes in nuclearvolume are associated with the opening up or closing down of specific areas of chroma-tin in an ordered sequence. By controlling the state of dispersion or condensation ofthe chromatin the cytoplasm could thus regulate not only the amount of RNA madein the nucleus but also the areas of DNA on which it was made. One feature of amechanism of this sort is that it does not require cytoplasmic signals of a high order ofspecificity. The specificity could reside in the structure of the chromatin itself and inthe manner in which it was disposed within the nucleus: essentially simple cyto-plasmic signals controlling the size of the nucleus could evoke highly specific patternsof chromosomal condensation and dispersion.

What we know about the condensation of chromatin in the nuclei of animal cellssuggests that it involves rather large areas of the DNA: whole chromosomes or majorportions of chromosomes. If this is true, then the control of genetic activity associatedwith changes in nuclear volume must be a rather coarse regulatory mechanism. It has,however, been suggested that regulatory mechanisms involving a much higher degreeof discrimination operate at the level of the gene. Indeed, it has been proposed thattranscription of the DNA at discrete genetic loci can be prevented by specific cyto-plasmic substances (repressors) which are able to recognize these loci and attach tothem (Jacob & Monod, 1961). There is at present no decisive evidence for the existenceof a mechanism of this sort; but if it does exist, its operation in animal cells must besubject to the constraints imposed by changes in nuclear volume: those areas of the

32 H. Harris

chxomatin in which the synthesis of RNA has been suppressed by the process ofcondensation cannot, presumably, be subject to any further regulation. The necessityto postulate such precise regulation at the genetic level is, of course, removed, if, asnow seems probable, regulation of the synthesis of specific proteins can take place inthe cytoplasm of the cell.

REFERENCES

CULLING, C. F. A. (1963). In Handbook of Histopathological Techniques, pp. 203, 212. London:Butterworths.

DAVIES, H. G. (1961). Structure in nucleated erythrocytes. J. biophys. biochem. Cytol. 9, 671-687.DAVIES, H. G. & SPENCER, M. (1962). The variation in the structure of erythrocyte nuclei with

fixation. J. Cell Biol. 14, 445-458.GAILLARD, J. L. J. & SCHABERG, A. (1964). A new spreading procedure for human chromosomes.

Expl Cell Res. 36, 415-417.HANKS, J. H. (1948). The longevity of chick tissue cultures without renewal of medium. J. cell.

comp. Physiol. 31, 235-260.HARRIS, H. (1959). Turnover of nuclear and cytoplasmic ribonucleic acid in two types of animal

cell, with some further observations on the nucleolus. Biochem. J. 73, 362-369.HARRIS, H. (1965). Behaviour of differentiated nuclei in heterokaryons of animal cells from

different species. Nature, Lond. 206, 583-588.HARRIS, H., WATKINS, J. F., FORD, C. E. & SCHOEFL, G. I. (1966). Artificial heterokaryons of

animal cells from different species. J. Cell Set. 1, 1-30.JACOB, F. & MONOD, J. (1961). Genetic regulatory mechanisms in the synthesis of proteins.

J. molec. Biol. 3, 318-356.LOEWENSTEIN, W. R. & KANNO, Y. (1963). The electrical conductance and potential across the

membrane of some cell nuclei. J. Cell Biol. 16, 421-425.MAURER, W. & PRIMBSCH, E. (1964). GrSsse der y?-Selbstabsorption bei der 3H-Autoradio-

graphie. Expl Cell Res. 33, 8-18.MITTWOCH, U., LELE, K. P. & WEBSTER, W. S. (1965). Relationship of Barr bodies, nuclear size

and deoryribonucleic acid value in cultured human cells. Nature, Lond. 205, 477-479.PERRY, R. P., ERRERA, M., HELL, A. & DURWALD, H. (1961). Kinetics of nucleoside incorpora-

tion into nuclear and cytoplasmic RNA. J. biophys. biochem. Cytol. n , 1-13.RASMUSSEN, R. E. & PAINTER, R. B. (1966). Radiation-stimulated DNA synthesis in cultured

mammalian cells. J. Cell Biol. 29, 11-19.WATTS, J. W. (1964). Turnover of nucleic acids in a multiplying animal cell. Biochem. J. 93,297-

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(Received 19 August 1966)

Fig. 4. A smear of hen erythrocytes in which the nuclei are stained with Weigert's ironhaematoxylin. Note the discrete areas which stain more deeply than the rest of thenucleus: the 'nuclear bodies'.Fig. 5. A dikaryon containing 1 HeLa nucleus and 1 hen erythrocyte nucleus. Thenuclear bodies are still visible in the erythrocyte nucleus.

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Fig. 6. A dikaryon containing i HeLa nucleus and i hen erythrocyte nucleus whichhas begun to enlarge. The nuclear bodies stain less deeply and are more diffuse.Fig. 7. A dikaryon containing 1 HeLa nucleus and 1 hen erythrocyte nucleus at afurther stage of enlargement. The nuclear bodies are no longer visible.

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8Fig. 8. A dikaryon containing 1 HeLa nucleus and 1 hen erythrocyte nucleus whichis approaching the limit of enlargement. The dikaryon is probably in the process ofdissociating into two separate cells.

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Fig. 9. A mononucleate cell in which the nucleus is derived from a hen erythrocyte.This type of cell arises when a dikaryon containing a HeLa nucleus and a henerythrocyte nucleus dissociates into two separate mononucleate cells.

H. HARRIS