Nucleolar transcription during plant mitosis

Experimental Cell Research 102 (1976) 3 1l-3 16

NUCLEOLAR

TRANSCRIPTION PLANT

DURING

MITOSIS

In Situ Assay for RNA Polymerase Activity G. MORCILLO,

C. DE LA TORRE and G. GIMfiNEZ-MARTIN

Departamento de Citologia, Institute de Biologia Celular (CSIC), Madrid-6, Spain

SUMMARY Allium cepa L. root meristems, after a brief fixing in cold methanol, retain endogenous RNA polymerase activity. This is shown by autoradiography of meristems squashed after being incubated with the four ribonucleotides, UTP being 3H-labelled. The pattern of labelling after this assay is similar to that shown after in vivo incorporation of [$H]cytidine. Nucleolar transcription appears to be as important in prophase as in interphase and under our conditions no mitotic cells present significant RNA polymerase activity outside the nucleolus. The nucleolar transcription is timed in relation to chromosome and nucleolar cycles. It stops at the very end of prophase, shortly before the last nucleolar remnants disappear. Reinitiation of RNA polymerase activity is found in organizer regions of nucleolar chromosomes, where it first appears in mid-telophase. This work also supplies a new evidence in favour that prenucleolar bodies are not synthesized in telophase since they appear before any transcription reinitiates.

Persistent activity of endogenous RNA [12] or in chromatin [2, 111 or in nucleus polymerase has been detected in fixed cells. [13, 191. Frozen sections of root meristems also The studies by Moore [16] of incorporation of r3H]UMP in the presence of the outer show endogenous RNA polymerase activity ribonucleotides in fixed mouse testes and when incubated with the ribonucleotides, those of Moore & Ringertz [17] in human UTP being 3H-labelled [7]. In this paper, the in situ RNA polymerase fibroblasts showed that endogenous RNA polymerase activity remained in such tis- assay is performed in slightly fixed merisues after a short cold-methanol fixing and stems to obtain the time course of tranthat the incorporation of the labelled pre- scription on mitotic cells ofAllium cepa L. cursor allows cytological detection of en- root meristems and it is compared with the zyme activity. The sensitivity of nucleo- in vivo incorporation of [3H]cytidine. By superimposing the transcriptional plasmic label to cu-amanitin and that of nucleolar label to actinomycin D during this pattern on the matched chromosomal and nucleolar cycles, the end and the starting reaction, was consistent with the distribution of the extranucleolar and nucleolar points of nucleolar transcription are timed RNA polymerases [ 171.The assay is essen- in relation to both mitotic and nucleolar tially similar to the in vitro test for RNA phases. polymerase activity either in isolated DNA Exp

Cell

Res

102 (1976)

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et al.

Fig. 1. [3H]Cytidine incorporation

,,.,i-

MATERIAL

/,

AND METHODS

Ailium cepa L. bulbs were grown in the dark at 25”+

05°C in tap water, which was renewed every 24 h and aerated continuously by bubbling air.

Endogenous RNA polymerase

assay

The terminal 0.5 mm of each root apex was discarded and the next 2 mm used since they contain a nearly pure meristematic zone. They were placed in 95% methanol at 0°C for 1 min. Then, they were washed twice with distilled water at WC, for 5 min. Six or seven meristems were incubated for 20 min at 37°C in 50 ~1 reaction mixture. It contained 300 nmoles each of ATP, CTP, CTP and 24 nmoles of 13H]UTP (15.0 Ci/mM) in 0.5 ml of 0.1 M Tris HCI pH 7.9 buffer containing 0.15 M sucrose, 12 mM mercaptoethanol and 12 mM MgCI, [16]. Afterwards, the reaction mixture was replaced by the buffer containing 1 mM cold UTP as the only nucleotide and kept at 37°C for 5 min. The roots were then washed twice, now at room temperature, with this UTP-containing buffer, and kept in it for a total of 10 min. Subsequently the roots were washed with 10 mM sodium pyrophosphate for 5 min. The roots were then posttixed in 3 : 1 ethanol/acetic acid containing 10 mM sodium pyrophosphate. The Exp Cell Res 102 (1976)

2

in meristem cells after in vivo incubation. Label is mostly concentrated on nucleolar zones of both interphase and prophase. Orcein stain. X3 Ooo. Fig. 2. Autoradiography after the in situ assay for endogenous RNA polymerase activity showing incorporation on the bleached nuclear zone corresponding to the nucleolus. x7 000.

fixer was changed three or four times during the first half an hour, and finally the roots were kept overnight in 3 : 1 ethanol/acetic acid. Some roots were washed with cold 5 % TCA.

In vivo incorporation

of [3H]cytidine

Other roots still attached to the bulbs were subjected to a 20 min pulse of [3H]cytidine at a concentration of 10 &i/ml and of a specific activity of 25 Cilmmol. Then, the roots were immediately fixed in 3 : 1 ethanol/acetic acid.

RNAse assay Intact roots and root squashes were incubated in a 1 mg/ml RNAse at 37°C for 60 and 30 min respectively. Then they were washed in ethanol/acetic acid.

Staining and autoradiography 0.1 N HCl-acetic orcein was used to stain nuclei. The silver impregnation technique was used for staining nucleoli in unlabeled preparations [6]. Labelled preparations were covered with Kodak AR-10 stripping film, and after 3 weeks’ exposure they were developed in D-19 developer and fixed in Kodak ultrarapid acid fixer.

Nucleolar

transcription

during plant mitosis

3 13

Fig. 3. Metaphase showing no incorporation after the assay for RNA polymerase activity, indicating metaphase chromosomes do not carry out any transcription. The interphase cell at the bottom of the figure shows the normal incorporation pattern characterized by a high incorporation in unstained nucleolar zones. X4 000. Fig. 4. Telophase showing incorporation on a pair of symmetric zones in both sister nuclei. These zones must represent the pair of nucleolar organizer regions. ~6 000.

RESULTS AND DISCUSSION The incorporation pattern after in vivo incubation with r3H]cytidine in meristems is that shown by fig. 1, where interphase and prophase nuclei show preferential grain incorporation on nucleolar zones. Root meristems subjected to the assay for endogenous RNA polymerase, show the image displayed in figs 2, 3 and 4. Hence, the assay for RNA polymerase evidences that nucleolar transcription follows a pattern which seems to correspond to the in vivo situation. In both cases labelling intensity seems always to be greater on nucleolar than on nucleoplasmic zones, as Das [5] showed for Nigella and Allium itself. However, it seems that extranucleolar transcription is slightly higher after the in vivo incorporation of r3H]cytidine than in the RNA polymerase assay. Certainly the Mg2+ in the reaction mixture seems to favour the activity of the nucleolar RNA polymerase [13, 17, 191.

The endogenous RNA polymerase assay is specially valuable for cells where in vivo incorporation of [3H]uridine shows labelling kinetics different from that expected for an RNA precursor. The digression of uridine through different metabolic pathways towards DNA has been previously reported in plant [ 1, 15-Jand animal cells [3]. To circumvent this difficulty, labelled cytidine has sometimes been applied, although the incorporation of cytidine is only useful for detecting transcription in non-replicating cells. The radioactivity incorporated in the nucleolus after the RNA polymerase assay remained either after washing with 5% TCA at 4°C or after the 0.1 N HCl acetic orcein. RNAse digestion of either the intact roots or root squashes take out over 85% of labelling. Hence, the labelling should correspond to RNA since it is acid-insoluble and RNA sensitive. Exp Cell Res 102 (1976)

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Table 1. Percentage of unlabelled and labelled cells in interphase and in the different mitotic phases, after in situ [3H]UTP incorporation assay on Allium cepa root meristems Each value represents the mean percentage of over 2 000 cells Unlabelled 1.75 0.42 1.32

Prophase Metaphase Anaphase Telophase Interphase

Transcription

in mitosis

Labelled 6.45 -

nucleolar organizer regions of chromosomes (NOR). These facts agree with nucleolar formation being conducted by chromosomal NORs [lo, 141. Experiments with inhibitors seem to support the fact that reformation of nucleoli depends on simultaneous rDNA transcription [9, 241 even though Semeshin et al. [22] reached the opposite conclusion. Nucleolar

cycle

Other root meristems, growing under the same experimental conditions, were silver impregnated. Silver impregnation is a very sensitive staining procedure for some nucleolar protein [4, 251, mostly located in fibrillar moieties of nucleoli [8]. The frequency of cells in the different nucleolar phases was scored in relation to mitotic cells in other silver impregnated root meristems. Table 2 shows that nucleoli in disorganization, characterized by their lobed periphery, are mainly detected in prophase. Nucleolar remnants are detected in 11% of metaphases but none in anaphases. We have used the term nucleologenesis for that period characterized by the appearance of scattered nucleolar material in telophase nuclei [24]. This material has the same silver affinity as the tibrillar portion of mature nucleoli. By late telophase, two

Under the experimental conditions used, the relative frequencies of meristem cells in the different mitotic stages is summarized in table 1. The frequency of labelled mitotic cells has been determined in root meristems after the assay for endogenous RNA polymerase activity. Prophase. All prophases are labelled on nucleolar zones and the label intensity appears to be as high as in interphase (fig. 2). Hence, transcription must occur up to the end of the disorganization stage. This situation is quite different from that which occurs in meiotic prophase where nucleolar transcription gradually stops while the nucleolus modifies its structure both in plant Table 2. Percentage of meristem cells in the [ 181and in animal cells [23]. different stages of the nucleolar cycle, in Metaphase and anaphase. None showed relation to the mitotic phases incorporation on it (fig. 3). Each figure represents the mean percentage of over Telophase. The common observation in 6 000 cells telophase was to see [3H]UMP incorporaMitotic phases tion on bleached symmetrical and paired ProMetaAnazones of sister nuclei (fig. 4). The same is Nucleolar phases phase phase telophase found after [3H]cytidine labelling. This suggests that the two largest prenucleolar Disorganization 3.67 0.19 1.56 Y.50 bodies are the only functional regions in Not visible Reorganization 1.84 transcription, i.e. those associated with the Exp Cell Res 102 (1976)

Nucleolar

transcription

during plant mitosis

3 15

tivity. The superimposition of transcription on the sequential phases of mitotic and nucleolar cycles is specially valuable in timing the reinitiation of transcription in daughter nuclei in relation to nucleologenesis. The appearance of prenucleolar material on Fig. 5. Nucleolar transcription and the matching of telophase chromosomes is shown to prechromosome and nucleolar cycles in onion meristems cede any detectable transcription in the growing under steady conditions at 25°C. The length of any phase in this figure is proportional to the fre- forming nuclei. Hence prenucleolar bodies quency of meristem cells found in this particular are apparently neither the product nor the phase. Each arrow marks the relative position of each cycle, considering that the passage between dis- site of transcriptional activity at telophase. organization and non-visible phases of nucleoli In the same line, inhibitors have shown that occurs within metaphase, close to the end of prophase. Since only 11% of the metaphases showed a nu- their appearance is independent of simulcleolar remnant this consideration has been used as a taneous macromolecule synthesis [9,21,22, criterion for matching. 241. Furthermore, micronuclei lacking any All prophases showed nucleolar incorporation, while only 60% of the telophases showed detectable NOR always contain prenucleolar material incorporation. As the reorganization stage precedes the re-start of [4, 241, also indicating that their appearance transcriptional activity, the scheme shows that pre- is independent of any rDNA transcription. nucleolar bodies are independent of the reinitiation of The two phases in nucleologenesis (apnuclear transcription. pearance of prenucleolar bodies and their fusion in the forming nucleoli) appear to be larger nucleolar bodies per nucleus are seen common to most eukaryotic cells, since the in symmetrical positions in both sister use of autoantibodies to nucleolar comnuclei. Finally, the developed nucleoli re- ponents similary evidenced the appearance main while the prenucleolar bodies have of prenucleolar bodies but also their failure disappeared. Nucleologenesis is com- to fuse into nucleoli in actinomycin-treated animal cells [20]. pleted by then. The transcriptional pattern and the matching of chromosomal and nucleolar cycles

The arrows in fig. 5 represent the reference points which allow the matching of nucleolar transcription on both chromosomal and nucleolar cycles. The end of nucleolar disorganization was precisely fixed in metaphase by considering the fact that 11% of the metaphases still had some nucleolar remnant on them. The nucleolar transcription has been taken as occurring up to the end of prophase, but not in metaphase, in agreement with data in table 1. Then, nucleolar remnants in metaphase are not the place of any detectable transcriptional ac-

This work has been partially supported by the III Plan de Desarrollo of Spain and by a grant of the Foundation Rodriguez Pascual. We express our gratitude to Miss 0. Partearroyo for her technical assistance.

REFERENCES 1. Albertini, L, Caryologia 25 (1972) 495. 2. Bonner, J, Huang, R C & Maheshwari, N, Proc natl acad sci US 47 (1961) 1548. 3. Comings, D F, Exp cell r& 41 (1966) 677. 4. Das, N K, Exp cell res 26 (1962) 428. 5. -Science 140 (1963) 1231. 6. Femandez-Gomez. E. Stockert. J C. Looez-Saez,

J F & Gimenez-Mar&,

G, Stain technol44 (1%9)

48. 7. Fisher, D B, J cell bio139 (1968) 745. 8. Gimbnez-Martin, G & Stockert, J C, Z Zellforsch

107(1970) 551. 9. Gimenez-Martin,

G, de la Terre, C, FernandezGomez, M E & Gonzalez-Fernandez, A, J cell biol 60 (1974) 502.

10. Heitz, E, Planta 12 (1931) 775. ExpCellRes 102 (1976)

316

Morcillo

et al.

Il. Huang, R C, Maheshwari, N & Bonner, J, Biothem biophys res commun 3 (1960) 689. 12. Huang, R C & Bonner, J, Proc natl acad xi US 48 (1%2) 1216. 13. Maul, G G &Hamilton, T H, Proc natl acad sci US 57 (1967) 1371. 14. McClintock. B. Z Zellforsch mikrosk Anat 21 (1934) 294. ’ ’ 15. McQuade, H A & Atchison, A A, Caryologia 22 (1%9) 7. 16. Moore, G P M, Exp cell res 68 (1971) 426. 17. Moore, G P M & Ringertz, N R. Exr, cell res 76 (1973) 223. 18. Parchman, L Q & Lin, K C, Nature new biol 239 (1972) 235. 19. Pogo, A 0, Littau, V C, Allfrey, V G & Mirsky, A E, Proc natl acad sci US 57 (1%7) 743.

Exp Cell Res 102 (1976)

20. Ringertz, N R, Ege, T & Carlsson, S A, Biology of fibroblast (ed E Kubue & J Pikkarainen). Academic Press, London and New York (1973). 21. SacristBn-G&rate, A M & Stockert, J C, Experientia 30 (1974) 294. 22. Semeshin, V F, Sherudilo, A I & Belyaeva, E S, Exp cell res 93 (1975) 458. 23. Stefanini, M, de Martino, C, D’Agostino, A, Agrestini, A & Monesi, V, Exp cell res 86 (1974) 166. 24. Stockert, J C, FernBndez-Gbmez, M E, Gimtnez-Martin, G & L6pez-Sgez, J, Protoplasma 69 (1970) 265. 25. Tandler, C J, Exp cell res 17 (1959) 560. Received May 7, 1976 Accepted May 13, 1976