J. Cell Sci. 14, 253-261 (1974) 253Printed in Great Britain

MOLECULAR HYBRIDIZATION OF MOUSE

SATELLITE DNA-COMPLEMENTARY RNA

IN ULTRATHIN SECTIONS PREPARED

FOR ELECTRON MICROSCOPY

J. JACOB, KATHERINE GILLIES, D. MACLEOD AND K. W. JONESDepartment of Genetics, University of Edinburgh, Edinburgh, Scotland

SUMMARY

The feasibility of in situ hybridization in tissue sections prepared for electron microscopy hasbeen examined using mouse satellite DNA-complementary RNA and mouse L cells. Theresults obtained are encouraging, although certain technical aspects require further clarifi-cation. In interphase cells, hybrid-forming sites occur in chromatin patches positioned alongthe nuclear envelope. It is also confirmed that satellite DNA occurs in nucleolus-associatedchromatin. The results suggest that satellite sequences are present in intranucleolar and peri-nucleolar chromatin. A similar distribution is indicated for ribosomal cistrons.

INTRODUCTION

The technique of in situ hybridization of nucleic acids to cytological preparationsdeveloped by John, Birnstiel & Jones (1969) and Gall & Pardue (1969) has proved tobe a powerful tool for localizing well denned stretches of DNA within the genome.Localization of particular DNA sequences in interphase nuclei and subcellular entitieswould, however, be greatly facilitated if the procedure could be extended to electron-microscope preparations. The feasibility of molecular hybridization in ultrathinsections has so far been demonstrated in only one instance (Jacob, Todd, Birnstiel &Bird, 1971) and remains to be examined in other situations. The present reportsummarizes data obtained in attempts to locate mouse satellite DNA at the ultra-structural level using satellite-complementary RNA and mouse cells.

From in situ hybridization experiments at the light-microscope level (Jones, 1970;Pardue & Gall, 1970; Jones & Robertson, 1970), it has been established that satelliteDNA is predominantly located in pericentromeric heterochromatin of all chromo-somes except the Y. Biochemical experiments (Schildkraut & Maio, 1968) and cyto-logical hybridization studies (Jones, 1970; Rae & Franke, 1972) have also locatedsatellite DNA in nucleoli. Knowledge of these facts makes the mouse cell a favourabletarget for attempting in situ hybridization in ultrathin sections with a view to evalu-ating the practicability of the approach and the specificity of the reaction.

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254 J- Jacob, K. Gillies, D. Macleod and K. W. Jones

MATERIALS AND METHODS

Preparation of target cells for electron microscopy

Mouse L cells growing on Falcon plastic dishes were washed gently with Dulbecco bufferto remove as much of the growth medium as possible. The cells were then detached with arubber policeman, transferred to a centrifuge tube and spun gently for about 5 min at 1000 g.The supernatant was then poured off and the cells were resuspended in 2-5 % glutaraldehyde(Taab Laboratories, Reading, England) in o-i M phosphate buffer, pH 7-2. After about 20 minthe fixed cells were pelleted by centrifugation, the supernatant discarded and the cells re-suspended in the buffer. After a 10-min wash the cells were spun to a firm pellet and embeddedin glycol methacrylate (Polysciences Inc., Rydal, Pa., U.S.A.). Polymerization was effected byultraviolet light at a low temperature. Sections showing gold interference colour (approx.120 nra thick) were collected on nickel or gold grids (Mason and Morton, Harrow, England)covered with Formvar and carbon.

Preparation of satellite-complementary RNA

Satellite DNA was isolated from caesium chloride gradients of mouse liver DNA preparedaccording to the method of Marmur (1961). The crude satellite fraction was then purifiedthrough 2 further CsCl gradients and finally analysed in the Beckman Model E analyticalultracentrifuge, where it was observed to form a single symmetrical band at 1-691 gem"3.Tritium-labelled, purified satellite DNA obtained by such a procedure has been checked forsite specificity against metaphase chromosomes in in situ hybridization experiments at thelight-microscope level. Ten microgrammes of pure satellite DNA were transcribed into comple-mentary RNA using Escherichia coli RNA polymerase prepared according to the method ofChamberlain & Berg (1962). Approximately equimolar (0-4 HIM) amounts of each of thetritium-labelled nucleoside triphosphates, ATP (22-7 Ci/mM), UTP (14-0 Ci/mM), CTP(208 Ci/mM) and GTP (9-9 Ci/mM) in solution (Radiochemical Centre, Amersham, England)were lyophilized together with the DNA in water in a 10-ml conical centrifuge tube. To thelyophilizate was added 0-3 ml of o-i M Tris-HCl buffer (pH 7-9 at 30 °C) containing i-6 mMspermidine, 1 fi\ mercaptoethanol ( i m l = i-n68g, Sigma), 1-5 mM MnCl,, 10 mM MgCl2and s units of RNA polymerase. The reaction was carried out for 90 min at 30 °C. Afterincubation, 5 mM MgClj and 40 /tg/ml RNase-free DNase (Worthington) were added anddigestion was carried out for 10 min at 37 °C; 500 fig non-radioactive E. coli RNA were addedas carrier and the mixture made 0-2% in sodium lauryl sulphate and o-i M in NaCl. After2 min at 37 °C, the mixture was shaken with an equal volume of water-saturated phenol for10 min at room temperature. After 10 min centrifugation at 12100 g in a SS-34 rotor of aSorvall RC2B centrifuge, the upper aqueous layer was removed, set aside, and the phenol oncemore extracted with an equal volume of water. The 2 aqueous fractions were combined andapplied to a 1-7x15 cm column of Sephadex G 50 equilibrated with o-i SSC (SSC = 0-15 MNaCl, 0-015 M trisodium citrate, pH 7-2). The column was developed with o-i SSC and o-5-mlfractions were collected. The radioactive excluded fractions containing carrier and c-RNA werepooled, lyophilized and then adjusted to 4 x SSC for use. The final specific activity obtainedwas estimated to be approximately 1-2 x io8 dpm/fig. The possibility that polymerized nucleo-tides were the product of polynucleotide phosphorylase contamination in RNA polymerase wasexcluded since enzyme controls exhibited lack of incorporation into TCA-insoluble materialon the omission of DNA. The product of primed reactions was RNase-sensitive and chromosom-ally site-specific.

Preparation of Xenopus ribosomal RNA

The sH-labelled 28s ribosomal RNA used in one set of experiments reported here wassynthesized in vivo using cultures of a Xenopus kidney cell line as previously described (Jacobet al. 1971). The specific activity of the sample kindly supplied by Dr Birnstiel was 1-5 x 10'dpmjfig.

In situ hybridization in EM preparations 255

In situ hybridization and autoradiographic localization of labelled molecules

The procedure for hybridization was essentially aa described before (Jacob et al. 1971) inexperiments with ribosomal RNA. Ultrathin sections were taken through 0-2 % solution ofbovine pancreatic RNase (Koch-Light Laboratories) for periods ranging from 30 to 60 min at37 °C, 001 % protease (from Streptomyces griseus, repurified type VI, Sigma) for 30-45 minat 37 °C, and 01 M NaOH for 1 or 2 h at room temperature, before they were floated on'H-c-RNA (33 x 104 or 9-3 x 10* dpm//il) for about 5 h at 70 °C. After this, the sections weretreated with 0-05 % RNase for 15-30 min at 37 °C, stained with uranyl acetate in ethanol andcovered with a layer of carbon. The preparations were finally coated with a monolayer of pre-gelled Ilford L-4 nuclear emulsion by the method of Caro & Van Tubergen (1962), which hasseveral advantages over other coating methods (Jacob, 1971).

OBSERVATIONS

Following in situ hybridization of mouse satellite DNA-complementary RNA onto ultrathin sections of mouse cells, autoradiographic preparations can be obtainedwith silver grains restricted almost entirely to nuclear regions. Some technicalproblems have, however, been encountered. One of these is variability in labellingintensity between closely placed sections. Factors such as the unavoidable variation inthickness of sections cut with an ultramicrotome (Williams, 1969) and the variablelocation of the target material in relation to the cut surface (Bernhard & Tournier,1962) may account for this. Another problem was the presence of background grains,which other workers have also reported in hybridization studies (Amaldi & Buon-giorno-Nardelli, 1971; Gall, Cohen & Polan, 1971) in conventional cytologicalpreparations. When hybridization was carried out with labelled Xenopus ribosomalRNA synthesized in vivo, background was usually low or negligible. In experimentswith mouse RNA transcribed in vitro, the background tended to be higher andvariable. This is probably caused by the presence in c-RNA of RNase-resistantduplexes which may stick to sections and especially to the supporting Formvar-carbon membrane of grids. Data presented in this report are from grids or areas ofgrids with low background; the background ranged from 1 grain per 72 to 270 /tm2

in the cytoplasm of the cells analysed. It may be possible to avoid this problemaltogether by the use of c-RNA transcribed from isolated strands of satellite DNA orby the use of H or L strands of DNA labelled in vivo prior to extraction (Jones, 1970).

The preparations shown (Figs. 1-4) are representative of interphase cells obtainedfrom several experiments. In Fig. 1, fairly large patches or blocks of chromatin areassociated with the nuclear envelope, while more diffuse chromatin is distributedthroughout the nucleus. In the nucleus in Fig. 2, most of the chromatin, including thenucleolus-associated chromatin, is in compact form and appears more electron-densethan in the previous instance. A diffeient organization of chromatin is evident inanother interphase nucleus (Fig. 3), where chromatin patches of intermediate size anddensity are dispersed amongst large amounts of diffuse chromatin. The silver grainspresent in Fig. 1 can be ascribed to radioactivity present in the chromatin patchesassociated with the nuclear envelope. Hybrid-forming sites are apparently moredispersed in the nucleus shown in Fig. 3. Most of the grains in Fig. 2 are localized

256 J. Jacob, K. Gillies, D. Macleod and K. W. Jones

over the chromatin associated with nucleoli, suggesting that this chromatin is rich insatellite DNA sequences. The distribution of satellite DNA in the nucleolus-associ-ated chromatin is better illustrated in Fig. 4. Many grains in this instance are localizedover the chromatin surrounding the nucleoli. Stretches of chromatin can also be seenextending into the interior of the nucleoli and some of these are also clearly labelled.The autoradiographic resolution obtainable in electron-microscope preparationspermits such an interpretation and it would be difficult to attribute the grains inquestion to radioactive sources in perinucleolar chromatin.

It has been demonstrated earlier (Brown, Weber & Sinclair, 1967; Pardue, Gerbi,Eckhardt & Gall, 1970) that ribosomal RNA from one eukaryote can hybridize withribosomal DNA from another eukaryote. An in situ hybridization experiment wastherefore performed on electron-microscope sections of mouse cells using 3H-labelled, Xenopus ribosomal RNA. The outcome of this was interesting, in that thedistribution of silver grains on nucleoli (Figs. 5, 6) is similar to that obtained afterhybridizing satellite-complementary RNA to sections of mouse cells.

DISCUSSION

The electron-microscope hybridization experiments with satellite-complementaryRNA show that in some of the interphase cells, hybrid-forming sites are located inchromatin patches apposed to the nuclear envelope, as was pointed out in the pre-liminary report (Jacob, 1972). A recent light-microscope study of mouse liver andtestis cells (Rae & Franke, 1972) also demonstrated that hybridization occurs withheterochromatin blocks associated with nuclear envelope. In view of the well estab-lished location of satellite DNA in centromeric regions of metaphase chromosomes(Pardue & Gall, 1970; Jones, 1970), the simplest explanation of the present obser-vation would be that, at some particular stage of interphase, the centromeric hetero-chromatin comes to be positioned predominantly along the nuclear envelope. Thisevent may be related to the phenomenon of chromosome condensation to the envelopeduring early prophase of mitosis as reported in several electron-microscope investi-gations (Porter & Machado, i960; Robbins & Gonatas, 1964; Comings & Okada,1970). Alternatively, such a location of satellite DNA-containing chromatin may beindicative of the period of replication of the DNA. It is known (cf. Flamm, 1972) thatmost of the satellite DNA in mouse replicates in the third quarter of 5-phase. Butwhat is highly controversial is whether replication or initiation of replication ofnuclear DNA in eukaryotes occurs in association with the nuclear membrane or awayfrom it (Comings & Kakefuda, 1968; Mizuno, Stoops & Peiffer, 1971; Fakan, Turner,Pagano & Hancock, 1972; O'Brien, Sanyal & Stanton, 1972; Ockey, 1972; Huberman,Tsai & Deich, 1973; Wise & Prescott, 1973).

Our observations with the electron microscope on the localization of label overnucleoli are consistent with the fact known for some time (Schildkraut & Maio, 1968;Mattoccia & Comings, 1971), that DNA sedimenting with nucleoli is considerablyenriched in satellite sequences compared with DNA from whole nuclei. That peri-nudeolar chromatin is rich in satellite DNA was recently shown by the Hght-micro-

In situ hybridization in EM preparations 257

scope hybridization experiments of Rae & Franke (1972). With the increased opticaland autoradiographic resolution provided by the present electron-microscopeprepaiations, hybrid-forming sites are also demonstrable in the chromatin stripswhich penetrate interphase nucleoli.

In mouse cells hybridized with Xenopus ribosomal RNA, the indications are thatthe ribosomal cistrons are contained in chromatin within and around nucleoli. Sucha distribution of ribosomal cistrons was also indicated in Xenopus cells (Jacob et al.1971). It is now generally accepted that ribosomal DNA is localized in nucleolusorganizer segments of chromosomes, but one of the still-unanswered questions is theprecise location and extent of the organizer in interphase cell nucleoli (Lafontaine &Lord, 1973). From the above-mentioned observations relating to this question, it canbe expected that analysis of hybrid-forming sites at the ultrastructural level will proveuseful for further clarification of this and other similar problems in molecular cytology.

As pointed out earlier in this report, our hybridization experiments suggest thatribosomal DNA as well as satellite DNA in mouse cells have a similar location inrelation to nucleoli. The nucleolus organizers of mouse chromosomes are situated nearthe centromeric heterochiomatin (Levan, Hsu & Stich, 1962) which contains thelight satellite DNA (Jones, 1970; Pardue & Gall, 1970), but electron-microscopehybridization experiments indicate that ribosomal cistrons and satellite sequences areprobably more intimately linked than hitherto assumed. Any speculation on thepossible significance of a close spatial association between ribosomal DNA and otherhighly repetitive DNA sequences must await further study and confirmation in manymore organisms.

The technique of in situ hybridization in ultrathin sections is in its infancy and thefirst results obtained are encouraging. More work will, however, be needed before thepotential usefulness of this procedure can be evaluated fully. A parallel approach thatalso requires to be explored further is that of examining hybrid-forming sites inelectron-microscope autoradiographic preparations of cells after carrying out in situhybridization in them by conventional procedure. The first step in this direction hasalready been taken by Rae & Franke (1972). Development of these techniques mayprove useful in our attempts to gain further insight into the molecular organization ofthe genome.

This work was aided by grants from the Medical Research Council and the World HealthOrganization.

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260 J. Jacob, K. Gillies, D. Macleod and K. W. Jones

Figs. 1-4. Autoradiographs of ultrathin sections of mouse L cells following in situhybridization using 'H-RNA complementary to mouse satellite DNA. The bars indi-cate 1 /tm. In Fig. 1 the silver grains (arrows) are located exclusively on chromatinpatches associated with the nuclear envelope. In Fig. 2 most of the grains (arrows)overlie the nucleolus-associated chromatin. Label is obviously more dispersed inthe interphase nucleus shown in Fig. 3: many grains overlie small chromatin patchesin the central region of the nucleus in addition to those over the nucleolus-associatedchromatin. The distribution of grains over nucleolus-associated chromatin is shownespecially well in Fig. 4: both intranucleolar chromatin (we) and perinucleolarchromatin (pne) are labelled, n, nucleolus.

In situ hybridization in EM preparations 261

Figs. 5, 6. Mouse L cells hybridized with 3H-28s ribosomal RNA of Xenopus.Practically all the silver grains (arrows) in Fig. 5 are over nucleolar chromatin. Intra-nucleolar labelling is indicated in Fig. 6. n, nucleolus. Bars represent 1 /im.