An electron microscope autoradiographic study of the site of initial synthesis of RNA in the nucleolus of Smittia

Q 1967 by Academic

276

Experimental

AN ELECTRON OF THE

MICROSCOPE

SITE OF INITIAL

SYNTHESIS

J. Institute

of Animal University

Cell Research 48, 2Y6-282 (1967)

AUTORADIOGRAPHIC

NUCLEOLUS

STUDY

OF RNA IN THE

OF SMITTIA JACOB

Genetics, MRC

of Edinburgh, Received

Press Inc.

Epigenetics Research Group, Edinburgh, Scotland

January

16, 1967

AN earlier

work [7] had demonstrated the occurrence of chromatin-like material containing DNA within the nucleolus of the salivary gland cells of the chironomid Smittia and the present study was carried out to determine the possible role of this intranucleolar DNA as template for the synthesis of nucleolar RNA. This question is of particular interest since evidence from previous investigations summarised elsewhere [ 1 ] do not favour attributing an active role in this process to the better defined region of the nucleolar organizer. As a result of recent investigations using biochemical techniques, the ribosomal RNA genes are now believed to be concentrated in the organizer chromatin. Apart from this and the long established fact that the organizer is the specific site of formation of the nucleolus, little is known of the functional and morphological complexities of this specialized chromosomal locus.

MATERIAL

AND

METHODS

Explants of the salivary glands from late larvae were incubated for IO to 12 min in Morgan Morton and Parker’s medium 199 with added tracer (3H-uridine, 100 PC/ml; specific activity 3.0 c/mM; Radiochemical Centre, Amersham) and fixed in cold (4”C), 6.5 per cent glutaraldehyde in phosphate buffer at pH 7.5. The glands were embedded in glycol methylate [ia] and silver sections were mounted on carbonized formvar coated nickel grids. These were then covered with a film of Ilford L4 nuclear emulsion using a loop [4]. Films showing pale gold interference colour in reflected safe light were used and test grids examined without development in the electron microscope showed the silver halide crystals to be closely and evenly distributed as a monolayer. The preparations were exposed at 4°C in plastic boxes with Drieite. Development was in Microdol X for 4 min at 18°C and fixation was in acid hardening fixer (Johnson’s FIX-SOL). The autoradiographs were stained with saturated aqueous uranyl acetate or sequentially with uranium and lead [14] and examined in an AEI. EM6 electron microscope. Exoerimenial

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RESULTS

The optical resolution and contrast of the double stained electron microscope autoradiographs are such that it is easy to distinguish and delimit the two main regions of the nucleolus. The improved quality of these preparations is partly due to the partial removal of the gelatin of the emulsion and by comparing the double stained ARGs with those stained with uranium alone, there was no suggestion of either loss or displacement of the developed silver grains. As it was essential in the present study to clearly distinguish the different regions of the nucleolus, the double stained preparations were used for analysis. After an exposure for 42 weeks, the only cellular constituent labelled was the nucleolus and even this was unlabelled in quite a number of preparations. The organizer is a compound band situated in chromosome II and extends for about 2 ,u [lo], but in sections cut perpendicular or parallel to the long axis of the chromosome no labelling was ever noted. Extensions of the organizer are known to occur in the nucleolus [7] and no labelling or pattern of labelling could be associated with such continuations of the organizer chromatin. In the large nucleolus of Smittia, different parts have been recognized [lo] and sections of these were examined. In all instanc.es, most of the silver grains were located over the amorphous looking portions described previously [lo] as the intermediate part. Representative EM-ARGs are shown in Figs. 1 to 4. Twenty-five such preparations were analysed and the combined data are presented in Table I. In each ARG, the areas of the amorphous and granular portions were determined and the overlying silver grains scored. In any one section, the areas of the two portions are usually unequal and sometimes are widely different. Taking into account this and other heterogeneity involved in the sample used for the present study, the data were subjected to a sophisticated statistical analysis using a modified form of the relative risk technique [16]. The maximum likelihood estimate of R, the ratio of grains per unit area in the amorphous portion to that in the granular portion was found to be 3.62 with approximate 95 per cent confidence limits at 2.49 and 5.25. The heterogeneity chi-square value between autoradiographs was 23.756 for 20 d.f. corresponding to P < 0.3. The results are thus consistent with the supposition that R does not vary markedly in the different preparations. About a third of the silver grains located over the granular portion (Table I) are placed within 0.25 ,u of the amorphous portion. Taking into account the effective path in methacrylate of the average beta particle from tritium [3] and other factors that influence EM-ARG resolution, it has been approximated [ 131 Experimental

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that the source of radioactivity producing a silver grain may be 0.3 ,u away from the centre of the grain developed from L4 emulsion. In the present EM-ARGs, most of the label is in the amorphous portion and it is probable that a third of the grains overlying the granular portion may also be due to the label incorporated in the amorphous portion. If this is so, about 90 per cent TABLE I. Areas of the two morphologically distinct portions and their respectioe grain counts. Amorphous

portion

Granular

of the nucleolus

portion

Total no. grains

Total area (p*)

Grains 100 p2

Total no. grains

Total area (fi2)

196

939.48

21.0

33

546.20

Grains 100 f.l’2 6.0

of all developed grains would be due to label in the amorphous portion. However, the alternate possibility that the grains in question over the granular portion are actually due to decaying atoms in this portion itself cannot be eliminated. The background in the autoradiographs used for this study was estimated to be about 1 grain per 200 ,u2. DISCUSSION

in selectively labelling RNA has been well The specificity of 3H-uridine established by earlier work on the salivary gland cells of Smittia. In the present work it is seen that following a pulse labelling lasting 10 to 12 min and a 42 week exposure, the chromosomes, nucleolar organizer and nuclear sap are unlabelled. In those sections where the nucleolus is labelled, the bulk (W-90 per cent) of the newly synthesized RNA is localized over a morphologically well defined portion-the amorphous intermediate portion, which forms most of the inner region of the nucleolus [lo]. This is also the region to which small dispersed patches of intranucleolar DNA were found [7] to be restricted. The small (50 to 170 mp in average diameter) patches mentioned above could not be made out in the EM-ARGs since the conditions which revealed Fig. I.-A section of the nucleolus at the region chromosome. Some extensions of the organizer there are no associated silver grains (see also portions are easily made out and practically all

of the organizer cut parallel to the long axis of the chromatin into the nucleolus are seen (arrows) but Fig. 4). The amorphous (AP) and granular (GP) the grains lie over the former. x 9000.

Fig. 2.-Also EM-ARG of a section passing through the organizer; note nucleolar material (NM) at the organizer. Most of the grains are located over the inner amorphous portion. x 12,000. Experimental

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their presence are not all reproducible in the present preparations; further their small size makes them liable to be easily masked by the large silver grains developed from the L4 emulsion. However, the distribution of the newly synthesized RNA is what would be expected if the dispersed patches of intranucleolar DNA are active in their synthesis. The limitations of the technique and those imposed by the material make it seem unlikely that a closer link between this DNA and the RNA synthesized can be demonstrated. A small proportion of the labelled RNA is found in the outer granular portion of the nucleolus. From light microscope studies, a shift of labelled RNA has been known to occur from the inner to the outer regions (cf. [15]) and a morphological basis for such a transfer is apparent from a recent study [8] of nucleolar ultrastructure. With a shorter pulse labelling than that in the present study, it may be possible to effect a more precise localization of labelled RNA to the inner region, but this must be weighed against the very low sensitivity of the EM-ARGs of thin sections. In this context, it may be pointed out that in this study while the period of incubation in tracer was half that used in an earlier one [9], the exposure required was about 5 times longer. There have so far been, apart from an earlier one in Smittia [9], only two investigations at the ultrastructural level where the synthetic activity of the organizer has been looked into. Using EM-ARG, the synthesis of nucleolar RNA in monkey kidney cells was shown to occur [B] in association with both the peripherally situated nucleolus associated chromatin and the intranucleolar chromatin, while in amphilian embryonic cells, it was concluded [ll ] that the intranucleolar DNA and not the associated chromatin participates in this process. The present work on Smittia also point to the intranucleolar DNA dispersed in the inner region of the nucleolus as the primer for the synthesis of nucleolar RNA. During the last 3 or 4 years various investigators (cited in [7]) have shown using the electron microscope, the presence within the nucleolus of chromatin material that was not evident by light microscope cytochemical methods. With increasing evidence to this effect, it may be necessary to regard the submicroscopic intranucleolar DNA as an internal organizer and thus distinguish it from the more obvious (external) organizer of the usual cytological preparaFig. 3.-EM-ARG

of distal part [lOI of the nucleolus.

x 15,000.

Fig. 4.-Section cut perpendicular to the long axis of the chromosome. This is one of a number of serial sections of the nucleolus at the level of the organizer. Arrows point to chromatin material that were followed through in other sections to the organizer; no label was detected at or near these. All grains are confined to the inner amorphous portion of the nucleolus. x 6000. Experimental

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tions. This distinction also seems necessary, since the organizer complex appears to be capable of a functional divisibility as well. The nucleolar organizer is generally regarded as a polygenic heterochromatic region and this type or state of chromatin is thought of (cf. [2]) as part of a mechanism regulating gene action. Viewed in this light, the absence of nucleoli in early embryonic cells could be ascribed to a complete shut off of all genes in the organizer. That at other times, either all or few of the constituent genes may be operative in nucleolar RNA synthesis is suggested by a few investigations [5, 6,113 including the present one. The postulates proposed in this study are based on limited evidence and must remain so pending substantiation by further electron microscope studies on the organizer in a wide variety of materials. SUMMARY The result of an electron microscope autoradiographic study following a pulse labelling is consistent with the interpretation that in the salivary gland cells of late larval stage of Smiffia, the synthesis of nucleolar RNA is primed by the intranucleolar DNA dispersed in the inner part of the nucleolus. From the limited evidence so far available, it is postulated that the nucleolar organizer complex may consist of an internal and an external entity. Such a of the distinction finds support in what appears to be a functional divisibility organizer. These postulates await confirmation. The author wishes to express his thanks to Dr B. Woolf for doing the statistical analysis and to Mr M. D. McKean for his skillful technical assistance. This work was supported by the British Empire Cancer Campaign for Research and the Damon Runyon Memorial Fund for Cancer Research. REFERENCES 1. BIRNSTIEL, M. L., JACOB, J. and SIRLIN, J. L., Arch. Biol. (Lihge) 76, 565 (1965). 2. BROWN, S. W., Science 151, 471 (1966). 3. CARO, L. G., J. Cell Biot. 16, 189 (1962). 4. CARO, L. G. and VAN TUBERGEN, R. P., J. Celf Biol. 15, 173 (1962).

5. Eos&Bnq J. E., B&hem. Biophys. Res. Commun. 18, 595 (1965). 6. GRANBOULAN, N. and GRANBOULAN, P., Exptl Cell Res. 38, 604 (1965). 7. JACOB, J., Nature 211, 36 (1966). 8. In m-euaration. 9. JACOB, J: and SIRLIN, J. L., Nature 202, 622 (1964). 10. ~ J. Uttrastruct. Res. 11, 315 (1964). 11. KARASAKI, S., J. Cell Biot. 26, 957 (1965). 12. LEDUC, E., MARINOZZI, V. and BERNHARD, W., J. Roy. Microscop. Sot. 81, 119 (1963). 13. MOSES, M. J., J. Histochem. cytochem. 12, 115 (1964). 14. REYNOLDS, E. S., .7. Cell Biot. 17, 208 (1963). 15. WADDINGTON, C. H., in New Patterns in Genetics and Development, plate VII. Columbia University Press, New York and London, 1962. 16. WOOLF, B., Ann. Human Genetics 19, 251 (1955). Experimental

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