Nucleolus-like bodies in micronuclei of cultured Xenopus cells

All

Experimental

Cell Research 120 (1979) 295-306

NUCLEOLUS-LIKE CULTURED STEPHANIE

PrInted in Sweden Copyright @ 1979 by Academic Press. Inc. rights of reproduction in any form reserved 0014.4827/79/060295-12$0?.00/0

BODIES

IN MICRONUCLEI

XENOPUS

GORDON PHILLIPS’

OF

CELLS

and DAVID M. PHILLIPS2

‘Department of Human Genetics and Development, College of Physicians and Surgeons, Columbia University, New York, NY 10032, and 2The Population Council, The Rockefeller University, New York, NY 10021, USA

SUMMARY Two of the 36 chromosomes in Xenopus laevis are known to carry nucleolar organizer loci. Partitioning of the chromosomes of cultured, early-passage Xenopus cells among variable numbers of micronuclei could be induced by extended colcemid treatment. A large, obvious nucleolus occurred in a maximum of 4 micronuclei per colcemid-induced tetraploid cell. The large, deeplystained nucleoli incorporated [3H]uridine and appeared by electron microscopy to have typical nucleolar morphology with tibrillar and granular areas disposed in nucleolonema. In situ hybridization to radioactive ribosomal RNA (rRNA) resulted in heavy labelling of nucleoli in no more than 4 micronuclei per cell. The other micronuclei generally contained small bodies (blobs) which stained for RNA and protein as well as with ammoniacal silver. In the electron microscope, these appeared as round, dense bodies resembling nucleoli segregated by actinomycin D treatment. Nucleoplasmic RNA synthesis occurred in all micronuclei regardless of whether they contained definitive nucleoli. These observations suggest that micronuclei which formed large, typical, RNA-synthesizing nucleoli contained nucleolar organizer chromosomes, while the other micronuclei, which contained nucleolus-like “blobs” probably lacked nucleolar organizer loci. It is possible that the nucleolus-like bodies may have been aggregates of previously synthesized nucleolar RNA and protein trapped in micronuclei after mitosis.

In an earlier study, we analyzed nucleolus be enlightening to carry out similar experiformation in multinucleated Chinese ham- ments in a system where the number of nuster cells [ 11. Multinucleation was induced cleolar organizer chromosomes had been by extended colcemid treatment during determined by other means. Therefore, we which mitotic cells, containing a 4C amount undertook to induce micronucleus formaof DNA, reverted anomalously to inter- tion in cells of Xenopus laevis, in which it phase, partitioning chromosomes among a has been well established that there are two variable number of micronuclei. We found nucleolar organizer chromosomes per dipthat most micronuclei contained bodies loid cell [2, 3, 41. We report here that nuwhich appeared by several criteria to be nu- cleolus-like bodies occurred in most microcleoli, and we interpreted this to mean that nuclei of multinucleated Xenopus cells even many Chinese hamster chromosomes had though most micronuclei must have lacked nucleolar organizer loci. Since our ap- a nucleolar organizer chromosome. Howproach to the enumeration of nucleolar or- ever, morphological and functional features ganizer sites was novel and the results of some of the nucleolus-like bodies sugsomewhat surprising, we thought it might gested that they may have been material agExp CellRes IZO(IY7Y)

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gregated post-mitotically from nucleolar RNA and protein synthesized before mitosis and conserved in the colcemidarrested mitotic cells. MATERIALS

AND METHODS

Xenopus cultures were initiated from tissues of young adult Xenopus laevis. The toads were anesthetized with MS 222 (tricaine methanesulphonate; Sandoz Inc., Hanover, NJ.) and the carcasses were immersed briefly in 1% lysol followed by 9.5% ethanol before dissection. Tissues were washed in Hanks’ balanced salt solution diluted with water to 65 % of the standard concentration before mincing with scissors. Tissue fragments were cultured in a medium made up of 60% Ll5 (Leibowitz) medium (Gibco, Grand Island, N.Y.), 10% heat-inactivated fetal calf serum (FCS), and 30% distilled water [.5]. Successful outgrowth was obtained from heart, ovary and kidney. Heart and ovary cultures had tibroblastic morphology; kidney cultures were epithelioid in appearance. Cells were subcultured by trypsinization with 0.2% trypsin (Difco 1 : 250) in 0.65% Hanks’ balanced salt solution and maintained in tightly capped plastic flasks at room temperature. To induce micronucleus formation, cells were placed into 35 mm plastic Petri dishes with or without (for electron microscopy) coverslips. After several days of growth at 25°C cells were incubated with colcemid, 0.2 pLg/ml, for 20-30 h, then washed free of colcemid with 65% strength Hanks’ balanced salt solution. The detached cells floating in the medium were also washed and returned to the Petri dish. The medium was replaced and cultures were incubated an additional 8 h to allow post-mitotic, possibly multinucleated, cells to re-attach to the coverslip and spread. For autoradiography, 10 &i/ml [3H]uridine, 29.4 Ci/mM (Schwarz-Mann) was added to the culture and cells were fixed 6 h later. Radioautographs were exposed 24 h. For autoradiography, cells were fixed in 3 parts ethanol: 1 part glacial acetic acid, extracted with 5% trichloroacetic acid (TCA) at 4°C for 15 min, rinsed thoroughly in water, and air-dried. Coverslips mounted on slides were dipped in Kodak NTB 2 emulsion and developed in D19 developer at 18°C for 2 min. Radioautographs were stained through the emulsion with azure B at 4°C for 8 min. After radioautographs were photographed, silver grains were removed by treating slides 3 min with 7.5% K,Fe(CN)B followed by 4 min in 20% Na2SZ03 and 3 rinses of water. Cells were then restained with Azure B and rephotographed. Cells fixed with ethanol : acetic acid were stained for RNA with Azure B [6] or for total protein with fast green at pH 2 [7]. For silver staining [8], 50% silver nitrate was dropped on a slide and the coverslip with fixed, air-dried cells was placed, faced down, on top. The slide was exposed 10 min to a 150W photo floodlight 15-20 cm above it, then rinsed in distilled water. Three drops of 3% formalin adjusted to pH 4.5 with 1 M sodium acetate and formic acid were dropped onto the coverslip along with 3 drops of 30% silver nitrate Exp Cell Res I20 (1979)

in 40% ammonium hydroxide. When the cells were golden brown (30-60 set), the coverslip was rinsed in Kodak Stop Bath, rinsed in distilled water, and dried. In situ hybridization was carried out in the laboratory of Dr Ann S. Henderson according to the procedure of Gall & Pardue [9]. Fixed, air-dried coverslip preparations were rehydrated in 2~ SSC (SSC corresponds to 0.15 M sodium chloride with 0.015 M sodium citrate), treated with 100 pg/ml RNase 1 h at 37”C, washed thoroughly in 2~ SSC, dehydrated in an ethanol series, and air-dried. DNA was denatured by treating coverslips 90 set with 0.07 N NaOH, plunging into cold 70% ethanol, dehydrating in 95% ethanol, and air-drying. The coverslips were incubated on a slide with lZSI-humanrRNA [lo] from cultured Wills II cells in 3 x SSC with 50 % formamide pH 7 for 18 h at 45°C. They were then washed with 3X SSC in 50% formamide followed by 2~ SSC. They were treated with 100 pg/RNase for I5 min at 37°C to reduce nonspecific cytoplasmic binding, dehydrated through a series of ethanols, air-dried, and radioautographed as above. Exposure time was 24 weeks. Radioautographs were stained with 0.05% fast green at pH 2 for 10 min and photographed. For electron microscopy, cells grown on Lux or Falcon plastic dishes were fixed in situ with 2 % glutaraldehyde in 50% Hanks’ balanced salt solution containing 0.05 M collidine, pH 7.4, for 1-24 h. After a brief rinse in 0.2 M collidine, cells were post-fixed in 1% collidine-buffered 0~0, and dehydrated in alcohol. Absolute alcohol was replaced by a thin layer of Epon and the dishes immediately put in a 60°C oven for polymerization. After polymerization and separation of the Epon disc from the Petri dish, cells to be sectioned were marked with a Zeiss cell marker.

RESULTS For these experiments, it was important to have an estimate of the extent of diploidy in Xenopus cultures under the conditions used, since cells of higher ploidy would have more than two nucleolar organizer chromosomes per cell. We observed that in primary, first and second passages, about 99% of the cells had one or two nucleoli. This suggested that most cells remained diploid in the first few passages. However, by the sixth passage, more than 20% of cells in some cultures had more than two nucleoli. This suggested that tetraploidy or aneuploidy could occur with a high frequency after cells had been in culture for some time. Therefore, cells from first and second passages were used for analysis.

Xenopus nucleolus-like bodies

5 I, 2. Azure B-stained Xenopus cells containing colcemid-induced micronuclei. Only one or two nuclei contain large structures stained for RNA. Small bodies which sometimes appear to be bipartite with light and dark regions occur in other micronuclei. Figs 3, 4. Colcemid-induced micronuclei in Xenopus Figs

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6 fibroblasts stained with fast green for total protein. Almost all of the nuclei contain deeply-staining proteinrich nucleolus-like bodies. Figs 5, 6. Multinucleated cells stained with ammoniacal silver. Deeply stained nucleolus-like bodies occur in all micronuclei. Figs 1-6, X 1500.

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Table 1. Number of nuclei of multinucleated No. of nuclei/ cell 1 2 3 4 5 6 i 9 10 11 12 13 14 15 16 18 21

cells in which well-formed

No. of cells scored”

No. of nuclei containing obvious nucleoli 1

2

3

4

200 67 59 39 35

10 26 19 13 7

190 22 20 20 20

19 13 3 ::

7 3

31 28 28 27 29 11 14

79 9 4 12 4 2 2 A

13 17 13 18 10 7 8

2’ I 2 1

1 1

i 0 1 0

22 5 4 5 00 31 0 1 1 00 00

1 1 2 0 0 0

5

6

0 0

0

0 0 0 0 0 0 0 0 0 0 00000000 0 I: x 0 0 0 0 0

nucleoli are visible

7

8

9

10

11

12+

0 00 0 0

0 0 0 0 0

x 0 0

0 0 0

0 0

0

8 0 0 0

0 0 0 0 0

8 0 0 0

8 0

II 0 0 0

: 0 0 0

00

a These numbers do not reflect the relative numbers of multinucleated cells with particular numbers of micronuclei since a special attempt was made to find cells with high numbers of micronuclei.

Lengthy periods of colcemid treatment have contained a full chromosomal complewere required to accumulate significant ment. This supposition was supported by numbers of mitotic cells since Xenopus cul- the observation that both nuclei of binutures at 25°C grew quite slowly. (A doubling cleate cells often contained two nucleoli. time of 30-40 h has been reported [ll], but Multinucleated cells with as many as 21 our cells seemed to be growing more slowly micronuclei were observed, but cells with than this.) After 20 h in colcemid, many fewer nuclei were more common. Since the rounded cells were present which were diploid number in Xenopus laevis is 36, this loosely attached to the glass or floating. meant that most micronuclei contained Some of these were mitotic and were able to more than one chromosome. Regardless of return to interphase when the colcemid was the number of micronuclei in a cell, most or washed out. all of the micronuclei generally appeared to Cultures fixed 4-10 h after colcemid re- contain nucleolus-like bodies. These bodies stained for RNA with Azure B (figs 1,2) and moval had varying frequencies of multinucleated cells. These presumably resulted for protein with fast green (figs 3, 4). They also stained strongly with ammoniacal silwhen chromosomes of colcemid-arrested metaphase cells did not move to the poles before the cells reverted to interphase, and nuclear membranes formed around scat- Fig. 7. (a) Colcemid-induced micronuclei of a Xenopus tered small groups of chromosomes. By far cell. The two nuclei at the left contain structures which look like true nucleoli. Other micronuclei contain the most frequent type of multinucleated nucleolus-like bodies which are similar in appearance cells seen were binucleate. These may have to structures in nuclei of cells which have been with RNA synthesis inhibitors. Nucleolus and been formed from cells which did not com- treated nucleolus-like body are enlarged below. (a) X6000; plete cytokinesis; thus, each nucleus might (b, c) x24000. Exp CdRes IZO(1979~

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ver (figs 5, 6). Usually in one or two micronuclei per cell, the nucleolus-like bodies were particularly large, irregularly shaped, and deeply stained for RNA, resembling nucleoli of mononucleate cells. The nucleoluslike bodies of the rest of the micronuclei were usually small, round bodies which stained faintly with Azure B. Often several of these occurred in one micronucleus. Sometimes they had a segregated appearance, that is, they had a rim of material which stained more intensely with Azure B than the core. Within one micronucleus, all nucleolus-like bodies were of the same type: either they were large, irregular and deeply staining, or they were small, round, lightly staining and perhaps visibly segregated. Multinucleated cells with different numbers of nuclei were scored for how many of their micronuclei contained the large, deeply-staining type of nucleolus-like body. As can be seen in table 1, regardless of the number of micronuclei present, the maximum number of micronuclei with large, deeply-staining nucleolus-like bodies was four; there were more cells with only one or two nucleolus-containing micronuclei than with three or four nucleoluscontaining micronuclei. No cells were found in which more than four micronuclei contained the large type of nucleolus-like bodies. The above observations suggested that the large, irregular, deeply-staining nucleolus-like bodies might have occurred only in micronuclei which contained nucleolar organizer chromosomes. Diploid cells in which sister chromatids did not separate prior to micronucleus formation might be expected to have up to two micronuclei containing nucleolar organizer chromosomes. In cases where sister chromatids did separate or where cells were tetraploid, up to four micronuclei containing nucleolar orExpCdRes

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ganizer chromosomes could presumably be formed. In electron micrographs, micronuclei contained what we judge to be either “true” nucleoli or they contained nucleolus-like bodies. A maximum of two micronuclei per cell contained definitive nucleoli. Morphologically, “true” nucleoli were irregular in shape and contained a large granular and smaller dense fibrous component (figs 7 and 9). The granules were typically disposed in nucleonema-like arrays, as has been described in nucleoli of many cell types. The dense fibrous regions were small and irregular. Sometimes there were several fibrous regions per nucleolus (figs 7-9). Nucleolus-like bodies were very different in morphology from the structures judged to be “true” nucleoli. Nucleolus-like bodies were small, spherical, electron-dense structures composed of fibrous material often with a granular cap on one side. In general these structures resembled nucleoli of cells treated with inhibitors of RNA synthesis such as actinomycin D [12, 13, 141. Nuclei which had typical-appearing nucleoli did not contain nucleolus-like bodies and nuclei with nucleolus-like bodies did not contain typical-appearing nucleoli. In situ hybridization was performed on multinucleated cells using 1251-labelledhuman rRNA. Clear sites of labelling were generally seen in nuclei of one or two micronuclei per cell (figs l&13); some cases with up to four labelled micronuclei were seen. The background was such that one could not definitely rule out hybridization to sites in other micronuclei, but such labelling, if it existed, was certainly not at a level comFig. 8. Micronuclei in Xenopus cell showing that one

micronucleus contains a nucleolus. Nucleolus-like bodies have been transected in some of the other micronuclei. (b, c) Enlargements of a (6) nucleolus; (c) nucleolus-like body. (a) x3500; (b, c) x24000.

Xenopus nucleolus-like

20-791807

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Figs 10-13. Radioautographs of multinucleated Xenopus cells hybridized in situ to rz51-humanrRNA. Two

micronuclei per cell are heavily labelled, indicating that they contain rRNA cistrons . x 800.

parable to the one or two major hybridization sites most commonly observed. This suggested that at least the bulk of the rRNA cistrons were localized within the nucleoli of fewer than four micronuclei. RNA synthesis was analyzed autoradiographically in multinucleated cells which had been previously stained with Azure B and photographed. In radioautographs of

cells incubated 6 h with [3H]uridine, all micronuclei were labelled, regardless of the number of micronuclei per cell or the presence or absence of typical-appearing nucleoli in the micronucleus (figs 14-16). Thus, it was evident that a nucleolar organizer need not be present in a micronucleus for nucleoplasmic RNA synthesis to occur. Labelling of nucleoli in one or two of the micronuclei was usually detectable, but nucleoplasmic incorporation resulted in such heavy labelling relative to nucleolar incorporation that nucleolar incorporation was often obscured. The high nucleoplasmic labelling together with the small size of most of the nucleolus-like bodies and

Fig. 9. (a) Cell in which the largest micronucleus may

not have a nucleolar organizer chromosome since it contains nucleolus-like bodies rather than typicalappearing nucleoli. An apparently “true” nucleolus is seen in a smaller micronucleus. (b, c) Enlargements of the “true” nucleolus and nucleolus-like bodies. (a) x7500; (b, c) x37000.

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Figs

14-16. Multinucleated cells from cultures incubated 6 h with [3H]uridine. Radioautographs exposed 24 h. All micronuclei exhibit nucleoplasmic RNA synthesis, regardless of whether a well-formed nucleolus is present. Heavy labelhng is seen over

definitive nucleoli. Figs 14a, 15~1,and 16~ show cells stained with Azure B after removal of silver grains from the corresponding radioautographs shown in figs 14b, 15b and 16b, x800.

the limited resolution of radioautographic technique made it impossible to determine whether the small nucleolus-like bodies were labelled.

DISCUSSION Most often, one or two of the micronuclei induced in a cultured Xenopus cell contained bodies which, by a variety of criteria,

Exp Cell Res 120 f/979)

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appeared to be true, functional nucleoli. Oc- ganizers [18, 191. They did, however, stain casionally, cells were observed in which ap- for RNA and protein and with ammoniacal parently normal nucleoli were present in silver, which preferentially stains nucleolar three or four micronuclei. The criteria used proteins in cytological preparations by an unknown mechanism [8]. Thus, it appears to characterize these bodies as functional nucleoli were stainability for RNA and pro- that these bodies may be analogous to the tein, stainability with ammoniacal silver, nucleolus-like “blobs” which form in corn fine structure, content of rDNA as demon- microspores [20] and Xenopus embryos [21] strated by in situ hybridization, and ability which lack nucleolar organizer chromoto synthesize RNA. The data are consistent somes. Apparently similar bodies were obwith the expectation that functional nucleoli served by Das [22] in micronuclei of onion could form only in micronuclei containing a root tips. Despite the apparent absence of nucleolar organizer chromosome. The col- ribosomal genes, the micronuclei containcemid-induced micronucleated cells were ing nucleolus-like bodies were active in obligate tetraploids since they did not un- RNA synthesis. This is to be expected from dergo cytokinesis. Therefore, they must the observation that non-ribosomal species have had four nucleolar organizer chromo- of RNA are synthesized in anucleolate somes. If sister centromeres separated be- Xenopus embryos [22]. Nucleolar ribosomal precursor RNAs are fore micronuclei formed, then nucleolar organizers might have been distributed to four reportedly stable during mitosis [23, 241. It different micronuclei. However, more often has been suggested that this RNA resides this probably did not happen since most on the metaphase chromosomes during mimultinucleated cells had normal looking nu- tosis and is sloughed at telophase to be incleoli in no more than two of their micro- corporated as part of nucleoli when they renuclei. Similar work has been done on hap- form at that time [25, 26, 271. Similarly, nuloid cell lines of Rana pipiens which ap- cleolar proteins are thought to be stable pear to have only one nucleolar organizer through mitosis [28]. Thus, formation of nuchromosome per cell. In this case, col- cleolus-like aggregations of RNA and procemid-induced multinucleated cells would tein in micronuclei lacking nucleolar orhave been obligate diploids. A maximum of ganizers could be explained if nucleolar two micronuclei in these cells contained RNA and protein synthesized before mitypically staining nucleoli, while the rest of tosis were associated with mitotic chromothe micronuclei had small nucleolus-like somes and trapped in micronuclei as they bodies (Freed, J, Mezger-Freed, L & Phil- formed in colcemid-blocked cells. Then, as lips, S, unpublished observations). nucleolar material was sloughed at teloIt was expected that most micronuclei of phase it could be reorganized into numultinucleated Xenopus cells would not re- cleolus-like bodies, even in micronuclei ceive a nucleolus organizer chromosome. lacking nucleolar organizer chromosomes. Nevertheless, these micronuclei apparently In our previous work on multinucleated formed nucleolus-like structures. These Chinese hamster cells, we found apparently roundish nucleolus-like bodies had an ultra- functional nucleoli in many micronuclei [ 11. structure typical of nucleoli in which RNA The situation in Xenopus cells where most synthesis had been inhibited [15, 16, 171or cells had true nucleoli in no more than two, which formed in nuclei lacking nucleolar or- or at most four, micronuclei is in clear disExp CdRes 120(1979)

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tinction to this, indicating that there are many more than two nucleolar organizer sites in diploid Chinese hamster cells. However, in light of our subsequentfindings that nucleolus-like bodies can reform in micronuclei lacking nucleolar organizers, we cannot be certain that all nucleolus-like bodies in Chinese hamster micronuclei contain nucleolar organizer genes. As it is so hard experimentally to assess the functionality of very small nucleolus-like bodies, especially in the presence of many functional nucleoli, such experiments do not tell us the maximum number of nucleolar organizer chromosomes in diploid Chinese hamster. Taken together, the data confirm the ideas of McClintock [20] that specific genetic loci are active in organizing preexisting material into functional nucleoli. In some cells, in addition to the presence of a nucleolar organizer locus, synthesis of rRNA seemsto be required to maintain the integrity of nucleoli; when RNA synthesis is inhibited, nucleoli fall apart [14] or fail to form after mitosis [24,29], whereas in other cell types nucleoli may reaggregate after mitosis in the absence of RNA synthesis, though nucleolar structure is aberrant under these conditions [14, 161. The different responses of nucleoli in different cell types could be due to differences in the degree to which non-ribosomal RNA synthesis is affected by the inhibitor used, differences in the rate of processing (and therefore, depletion) of existing nucleolar components, differences in the amount of nucleolar material carried in stable form through mitosis, or to other unknown differences in adhesiveness of nucleolar constituents. In general, it appears that the presence of a nucleolar organizer is required for aggregation of nucleolar constituents into a coherent whole; in some situations RNA synthesis may also be important in this process. Exp Cd Res 120 (1979)

This work was initiated under support from NSF grant BG-29214. The authors would like to thank Dr M. T. Yu for providing the [12SI]rRNA and Dr Ann S. Henderson for her help with in situ hybridization. The authors also gratefully acknowledge Dr Jerome Freed and Dr Liselotte Mezger-Freed for their expert instruction in amphibian cell culture technique.

REFERENCES 1. Phillips, S G & Phillips, D M, J cell biol40 (1969) 248. 2. Fischberg, J & Wallace, H, The cell nucleus (ed J S Mitchell) pp. 3&34. Butterworth, London (1960). 3. Brown, D D & Gurdon, J B, Proc natl acad sci US 51(1964) 139. Wallace, H & Birnstiel, M L, Biochim biophys acta 114(1966) 296. Balls, M & Rubin, L N, Exp cell res 43 (1966) 694. Flax, M H & Himes, M H, Physiol Zoo1 25 (1952) 297. Swift, H, The nucleic acids (ed E Chargaff & J N Davidson) vol. 2, p. 51. Academic Press, New York (1955). 8. Goodpasture, C B Bloom, S E, Chromosoma 53 (1975) 37. 9. Gall, J G & Pardue, M L, Methods in enzymology (ed L Grossman & K Moldave) vol. 210, pp. 47& 480. Academic Press, New York (1971). 10. Yu, M T, Johnson, L D, Vogelman, D & Henderson, A S, Exp cell res 86 (1974) 165. Godsell, P M, Exp cell res 87 (1974) 433. i:: Reynolds, R C, Montgomery, R O’B & Hughes, B, Cancer res 24 (1964) 1269. 13. Schoefl, G I, J ultrastruct res 10 (1964) 224. 14. Phillips, S G 8t Phillips, D M, J cell biol 49 (1971) 785. 15. Geuskens, M &z Bernhard, W, Exp cell res 44 (1%6) 579. 16. Phillips, D M & Phillips, S G, J cell biol 58 (1973) 54. 17. Smetana, K, Gyorkey, F, Gyorkey, P&Busch, H, Exp cell res 91 (1973) 143. 18. Swift, H & Stevens, B J, Nat1 cancer inst monogr 23 (1966) 145. 19. Hay, E D & Gurdon, J B, J cell sci 2 (1967) 151. 20. g4Clintock, B, Z Zellforsch mikr Anat 21 (1934)

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21. Elsdale, T R, Fischberg, M & Smith, S, Exp cell res 14 (1958) 642. Das, N K, J cell biol 15 (1962) 121. 22:: Fan, H & Penman, S, J mol bio159 (1971) 27. 24. Phillips, S G, J cell bio153 (1972) 611. 25. Heitz, E, Planta 12 (1931) 775. 26. Kleinfeld, R G & von Haam, E, J biochem biophys cytol6 ( 1959)393. 27. Stevens; B J; J cell bio124 (1965) 349. 28. Harris, H, Nature 190(1961) 1077. 29. Gimenez-Martln, G, de la Torre, C, FernandezGomez, M E & Gonzalez-Femandez, A, J cell biol 60 (1974) 502. Received September 7, 1978 Revised version received December 1, 1978 Accepted December 4, 1978