Genomic in situ hybridization to sectioned nuclei shows chromosome

domains in grass hybrids

A. R. LEITCH1'*, W. MOSGOLLER2, T. SCHWARZACHER1, M. D. BENNETT3

and J. S. HESLOP-HARRISON1

1 Cambridge laboratory], Institute of Plant Science Research, Trumpington, Cambridge CB2 2JB, UK2lnstitut fur Histologie und Embryologie, Sclnvarsspanierstr. 17, 1090 Wien, Austria3Jodrell laboratory, Royal Botanic Gardens, Keiv, Richmond, Surrey T\V9 3DS, UK

* Author for correspondencefFrom March 1990: Cambridge Laboratory, IPSR, Colney Lane, Norwich NR4 7UH, UK

Summary

In situ hybridization using biotinylated totalgenomic DNA and avidin detection systems -wasadapted for examination of thin-sectioned plantmaterial in the light and electron microscopes. Roottip material was preserved prior to sectioning, sothat the in vivo disposition of the chromatin wasmaintained. Use of total genomic DNA from Secaleafricanum as a probe enabled the chromatin fromthe two parental genomes in the grass hybridHordeum chilense x S. africanum to be dis-tinguished. The biotinylated probe preferentiallylabelled the chromosomes of S. africanum origin.DNA-DNA hybrids were visualized at the light-microscope level by Texas Red fluorescence and at

the electron-microscope level by the enzymic pre-cipitation of DAB (diaminobenzidine) or by colloi-dal gold particles. The use of thin sections allowedthe location of probe hybridization to be estab-lished unequivocally in both metaphase and inter-phase nuclei. Analysis of interphase nuclei showedthat chromatin originating from the two parentalgenomes did not intermix but occupied distinctdomains.

Key words: in situ hybridization, sections, chromosomedomains, chromosome disposition, nuclear organization,electron microscopy, wide hybrid, Hordeum, Secale.

Introduction

The three-dimensional organization of the cell nucleus isimportant because of the direct relationship between thepositions of chromosomes and their mechanical behav-iour, such as occurs during pairing and elimination.Nuclear organization must be investigated to explore itsrelationships with gene location or expression (Vogel andKriiger, 1983; Heslop-Harrison and Bennett, 1984; Bor-den and Manuelidis, 1988).

The analysis of chromosome position requires both theidentity and location of chromosomes to be known.Spread preparations where chromosomes can be ident-ified, have been used to analyse chromosome position(e.g. see Wollenberg et al. 1982; Avivi et al. 1982).However, the preparation procedure reduces the threedimensions of the original nucleus to the two dimensionsof the spread. The distortion is variable and has anunknown effect on chromosome disposition (Heslop-Harrison et al. 1985).

An alternative approach involves the reconstruction ofcells from serial sections. At mitotic metaphase andJournal of Cell Science 95, 335-341 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

prophase, volume measurements of chromosome arms inphysical sections of some grass species permitted chromo-some identification (Heslop-Harrison and Bennett, 1983,1984), and in human reconstructions a particularchromosome structure allowed identification of chromo-some 9 (Heslop-Harrison et al. 1989). Oud et al. (1989)reconstructed anaphase cells from confocal optical sec-tions and could identify the three chromosome pairs inCrepis capillaris. At interphase, polytene chromosomeshave been identified by their length and banding patternsin computer-enhanced optical sections (Agard and Sedat,1983). Using the reconstruction technique alone, analysisof chromosome position and identity is restricted toorganisms where the chromosome types vary in mor-phology, and in cell types in which either divisions occuror unusual (e.g. polytene) chromosomes are included.

In situ hybridization of labelled DNA probe tochromosomal DNA permits the identification ofgenomes, chromosomes and chromosome segments inspread preparations at all stages of the cell cycle by lightmicroscopy (e.g. see Lichter et al. 1988; Schwarzacher-Robinson et al. 1988). To find the disposition of ident-

335

ified chromosome or DNA sequences in situ hybridiz-ation must be used in combination with optically orphysically sectioned material. Optical sectioning ofwhole-mount material following in situ hybridization ofbiotinylated probes has given useful data about chroma-tin disposition (Manuelidis, 1984; Borden and Manueli-dis, 1988). Physically sectioned material has been exam-ined by light microscopy after hybridization withradioisotope-labelled probes (e.g. see Rae and Franke,1972; Neer et al. 1977; Hafen et al. 1983) and biotinyl-ated probes (e.g. see Brigati et al. 1983; Walt et al. 1989;Liesi et al. 1986). However, the resolution of lightmicroscopy is not generally high enough to allow axes ofin situ labelled interphase chromosomes to be followedaccurately. Using ultrathin-sectioned material examinedin the electron microscope, interphase chromatin axescan be followed in some species (Heslop-Harrison et al.1988) but in situ hybridization is necessary to identify theaxes.

Radioactive probes have been used (e.g. see Steinerteial. 1976; Geuskens and May, 1974) but probe localizationis imprecise because of the high background and offsetsignal detection. Non-radioactive probe hybridizationwith more precise localization has been used by Hutchin-son et al. (1982) and Narayanswami and Hamkalo (1986)on whole-mount chromosome preparations wherechromosome position could not be analysed. Binder et al.(1986) detected in situ hybridization of biotinylatedmitochondrial nucleic acid to thin sections of Drosophilaovaries and Wachtler et al. (1990) investigated tran-scribed rDNA sequences in sectioned lymphocytes.Manuelidis (1984) showed that the location of satelliteDNA sequences in the mouse cerebellum was cell-typespecific by hybridizing and detecting biotinylated probeto thick vibratome sections, which were re-embedded andultrathin sectioned for electron microscopy.

In the present paper, we report the results of usingDNA-DNA in situ hybridization of biotinylated probeswith diverse detection systems on thin-sectioned plantmaterial for both light and electron microscopy. The useof thin sections allows the location of probe hybridizationto be established unequivocally in both metaphase andinterphase nuclei, and detection of probes in the electronmicroscope allows the resolution of the technique to besubstantially increased. The system enabled us to estab-lish the identity and analyse the positions of genomes orchromosomes that would otherwise be unidentifiable atboth interphase and metaphase.

Materials and methods

Plant materialHybrids from the grass species Hordeum chilense Roem. andSchult. and Secale africanum Stapf. (2n=2x=14) were ob-tained by cross pollination and subsequent embryo culture(Jensen, 1975). The parents, H. chilense (2«=2x=14) IPSRaccession 401 000 1 (formerly Line 1), 5. africanum (2n=2x=14) IPSR accession 301 000 1 (formerly R102) and thehybrids have been growing in the glasshouse and have beenpropagated vegetatively for several years. The hybrids grewvigorously and were karyotypically stable. They had previously

been described at mitotic metaphase in three-dimensionalreconstructions by Schwarzacher-Robinson et al. (1987) and atall cell cycle stages by in situ localization of parental genomes incell spreads (Schwarzacher et al. 1989). Root tips used in theexperiments were obtained from plants transferred for 3-10days into hydroponics.

DNA probesTotal genomic DNA was isolated from leaves of S. africanumfollowing a DNA isolation procedure adapted from Maniatis etal. (1982). The isolated DNA was mechanically sheared andlabelled with biotin-14-dATP by nick translation (BRL BioNickLabeling System). After labelling the DNA was approximately150-250 bp (base-pairs) long.

The probe was prepared by mixing biotinylated DNA to afinal concentration of 5/igml~ in a solution of 50% (v/v)formamide, 10% (w/v) dextran sulphate, 0 .1% (w/v) SDS(sodium dodecyl sulphate), and 2XSSC (0 .3 M NaCl, 0 . 0 3 Msodium citrate) immediately prior to denaturation.

Cell spread preparationsCell spreads were obtained using the methods described bySchwarzacher et al. (1989).

Preparation of sectionsRoot tips were fixed for 1 h at about 20 °C in freshly prepared1 % (v/v) glutaraldehyde (diluted from Taab vacuum-distilled10 ml sealed ampoules) and 0.25% (v/v) saturated aqueouspicric acid solution in phosphate buffer (0 .05 M Na2HPO4,0.05 M KH2PO4). They were then dehydrated through a gradedethanol series, embedded in LR White (medium) resin andpolymerized at 65°C for 16 h. Ultrathin (0.1 jim, gold) and thin(0.25 jUm, blue) sections were cut with a Reichert Ultracut forelectron microscopy and light microscopy, respectively. Allsections were expanded in the knife trough containing 1 % (v/v)benzyl alcohol in water. Ultrathin sections were picked up on200 gold mesh grids and thin sections were transferred to glassslides coated with poly-L-lysine solution (Sigma). Slides wereincubated overnight at 60°C prior to in situ hybridization.

PretreatmentImmediately prior to in situ hybridization, slides with cellspreads were refixed in 3 parts methanol to 1 part acetic acid,washed in methanol, then ethanol, and air dried. They werethen incubated in 100 fig ml"1 DNase-free RNase in 2xSSC at37°C for 1 h, washed twice in 2XSSC for 10min at roomtemperature, dehydrated through a graded ethanol series andair dried.

Grids with ultrathin sections and slides with thin sectionswere incubated in RNase and washed as above. They were thentreated with 5(Ugml~' proteinase K (BDH) in 20 mMTris-HCl, pH7.4, 2mM CaClz buffer for 10 min at 37°C andthe reaction was stopped by washing the sections in the bufferwith 50 mM MgCl2. After washing, the sections were refixed infreshly depolymerized 4 % (w/v) paraformaldehyde for 5 min atroom temperature and washed in 2XSSC for 5 min.

Chromosome and probe denaturationSlides of cell spreads and thin sections were denatured in 70 %(v/v) deionized formamide in 2xSSC for 2 min at 68-72°C.They were then dehydrated in a graded ethanol series at 4°Cand air dried. The probe DNA was denatured at 70°C for5-30 min, loaded onto the slide preparations and sealed under acoverslip with rubber solution. The ultrathin sections were pre-incubated in 50% (v/v) formamide in 2xSSC for 10 min atroom temperature and denatured by placing the grids for 10 min

336 A. R. Leitch et al.

Fig. 1. Root tip cells from the hybrid H. chilense X 5. africanum after genomic in situ hybridization using biotinylated totalgenomic DNA from S. africanum as a probe. A,B. Metaphase spread showing that only chromosomes of S. africanum originare probed; C-E and F -H showing three consecutive metaphase sections; I -K and L-N showing three consecutive sectionsof interphase cells (a,c,d) and a prophase cell (b). The sites of hybridization of the genomic probe are detected by Texas Redfluorescence (B, C-E, L-N) , and simultaneous DAPI staining shows DNA (A, F-H, I -K) . The parental origin of chromatinat interphase can be determined; it is not intermixed but occurs in domains. Arrows: a chromosome arm that does not subtendthe full thickness of the section in D and G. DAPI staining intensity is reduced between F and G but Texas Red fluorescenceremains constant between C and D. X1500.

under a coverslip in a drop of denatured probe mixture in ahumid chamber at 90°C.

DNA-DNA hybridization and post-hybridizationwashingIn all cases hybridization was carried out overnight at 37°C.Afterwards, the cell spreads and sections were washed in2XSSC at 40°C for 5 min, and then given a stringent wash forlOmin in 50% (v/v) deionized formamide in 2XSSC at35-42°C. Following the stringent wash the slides or grids werewashed twice for 5 min each in 2xSSC at 42°C and then atroom temperature.

Detection of hybridization to cell spreads and tosections on slidesThe detection method was the same for spread material and forthin-section material and followed that of Pinkel et al. (1986).The slides were transferred to BT buffer ( 0 . 1 M NaHCO3,0.05% (v/v) Tween 20, pH8) for 10 min, treated with 5%(w/v) BSA (bovine serum albumin) in BT buffer for 5 minfollowed by incubation with l - lO^gml" 1 avidin conjugatedwith Texas Red (Vector Laboratories, maximum excitation595-604nm, green; maximum emission 606-615 nm, red) inBT-BSA buffer for l h at 37°C. After incubation with thelabelled avidin the preparations were washed twice in BT bufferat 37°Cfor 10min.

The signal was amplified after a 5 min block with 5 % (v/v)normal goat serum (Vector Laboratories) in BT buffer byincubation with 25^gml~ biotinylated anti-avidin-D (VectorLaboratories) in BT-goat serum buffer for 1 h at 37 °C. Afterwashing in BT buffer, the preparations were re-treated with thelabelled avidin as above.

All slides were treated with l -2 jUgmr ' DAPI (4',6-di-amidino-2-phenylindole) in Mcllvaine's citrate buffer (0.01 Mcitric acid, 0 .08M Na2HPO4, pH7), which fluoresces blue onexcitation with ultraviolet (u.v.) light (340-380 nm). The slideswere mounted in BT buffer mixed with an equal volume ofantifade solution (10% (w/v) p-phenylenediamine dihydro-chloride in PBS (phosphate-buffered saline: 0 .12M NaCl,2.7 mM KClin phosphate buffer, pH7.4), mixed 1:9 (v/v) withglycerol). The slides were examined with a Zeiss epifluor-escence microscope with suitable filter sets (02 and 12).Photographs were taken with Fujicolor 400 or Kodak Ektar 1000colour print film.

Detection of hybridization to ultrathin sections on gridsTwo detection methods were used to locate probe signal onultrathin sections: 20 nm streptavidin-gold (Biocell) andstreptavidin-HRPO (horseradish peroxidase; Vector Labora-tories). Streptavidin-gold detection followed the detectionmethod described for light-microscope preparations (above),but using fluoresceinated avidin, biotinylated anti-avidin D andmodifying the final step of the amplification procedure by usinga 1:10 dilution of streptavidin-gold in buffer (0 .1% (w/v)BSA, 20% (w/v) glycerol, 20 mM Tris-HCl, pH7.4, 20 mMsodium azide, 225 mM NaCl, pH8.2).

Detection with streptavidin-HRPO was modified fromWachtlereJ al. (1990). Following post-hybridization washes thegrids were transferred to PBS-Tween buffer (PBS, 0.05%(v/v) Tween 20, pH7.4) for 5 min at room temperature andthen blocked with 5% (w/v) BSA in PBS-Tween buffer for5 min. This was followed by incubation with 5^gml~streptavidin-HRPO in BSA block for 1 h at 37°C in a humidchamber. Following washing in PBS-Tween for 30 min (3changes of solution) the grids were incubated for 20 min at 4°Cin 10 mM imidazole, 50 mM Tris-HCl, pH7.4, 0.05% (w/v)

diaminobenzidine (DAB) and 0.015% (v/v) hydrogen per-oxide.

All grids were washed six times for 2 min in distilled waterand air dried. Ultrathin sections were examined withoutstaining at 40-80 kV in a Philips 201 transmission electronmicroscope.

Measurement of chromatin areasMicrographs of 50 interphase nuclei were randomly selected.The areas occupied by in situ labelled chromatin and unlabelledchromatin were measured using a computerized digitizingtablet.

Results

In situ hybridization on cell spreadsFig. 1A, B shows fluorescent micrographs of a root-tipmetaphase spread of the hybrid H. chilense X 5. africa-num. All 14 chromosomes showed bright blue DAPIfluorescence after excitation with u.v. light (Fig. 1A).The parental origin of the chromosomes was not dis-tinguishable by staining intensity. The seven largerchromosomes in the nucleus of this hybrid are of5. africanum origin (Schwarzacher-Robinson et al.1987). Following in situ hybridization with biotinylatedgenomic DNA from S. africanum the hybridization siteswere detected with Texas Red-conjugated avidin undergreen light excitation (Fig. IB). The seven largerchromosomes, of S. africanum origin, fluoresced brightred (Fig. IB). The seven smaller chromosomes, ofH. chilense origin, gave only the faintest indication of anyprobe hybridization and were almost invisible.

In situ hybridization on thin sectionsFig. 1C-N shows fluorescent micrographs of genomic insitu hybridization and simultaneous DAPI staining tosections of 0.25 fim thickness. Three consecutive sectionsof a metaphase cell showed some chromosome pieces thatwere labelled strongly and fluoresced bright red, whileothers were labelled weakly and appeared dull red(Fig. 1C-E). The labelling pattern was similar on each ofthe consecutive sections. All chromatin was equallystained with DAPI (Fig. 1F-H). Differential labelling ofchromatin in nuclei (Fig. 1I-N) can also be seen atinterphase (cells a, c, d) and at prophase (cell b).

Domains in thin sectionsIn sections of interphase nuclei, DAPI stained allchromatin (Fig. 1I-K). There were large domains occu-pied by labelled, Texas Red positive chromatin and otherdomains of almost unlabelled chromatin (Fig. 1L-N).The four nuclei in Fig. 1I-N showed a weakly fluor-escing nucleolus near their centres and included oneinterphase cell (cell c) with alternating domains oflabelled and unlabelled chromatin and two interphasecells (cells a, d) with domains occurring beside eachother. In the prophase nucleus (cell b), all the chromatinis labelled except one piece, visible only with DAPIfluorescence, at the bottom of the nucleus. Similarchromosome labelling patterns were found in all nucleiexamined.

In situ hybridization on sections 337

B • •- - c*' . : - . . . ,

Fig. 2. Electron micrographs of sections of root tip cells from the hybrid H. chilense X S. africanum. Genomic in situhybridization with biotinylated total genomic DNA from 5. africanum detected with DAB allows the two parental genomes inthe hybrid plant to be identified. A. Interphase cells showing the localization of label to domains within the nucleus. X5000.B. Probe label within an interphase nucleus is restricted to some chromatin axes that occur in domains. X 10400. C. Ametaphase cell shows three chromosome pieces of//, chilense origin (light grey) and three of S. africanum origin (dark grey,arrows). X8000.

In 50 randomly selected interphase nuclei sections themean ratio of areas of nuclei, which included labelledchromatin, to those of unlabelled chromatin, was1.58±0.21:l.

In situ hybridization on ultrathin sectionsStructural preservation following fixation, sectioning andin situ hybridization was good. The hybridization sites ofthe biotinylated probe were detected by the precipitationof electron-dense DAB (Fig. 2) or by gold particles(Fig. 3). The DAB label was restricted within nuclei tosome chromosome or chromatin axes and non-specificbackground label was low (Fig. 2). The metaphase cell in

Fig. 2C shows three labelled (arrows) and three un-labelled chromosome pieces. The labelled chromatin inlongitudinal section (Fig. 2C) shows varying density ofDAB deposit on each chromatid, with the smallest clearlyresolvable dots being 100 nm in diameter. Within inter-phase cells labelled chromatin domains could be seen(Fig. 2A,B).

The detection of the biotinylated probe with 20 nmgold particles allowed quantification of the signal(Table 1). In the two consecutive metaphase cell sectionsin Fig. 3B,C four chromosome pieces show a highdensity of gold particles (21-30 gold particles |Um~2) andtwo show a low density (6—7 gold particles fim~z), both

338 A. R. Leitch et al.

V

B

f \ \

3A

Fig. 3. Electron micrographs of sections of root tip cells from the hybrid H. chilense X S. africanum. Genomic in situhybridization with biotinylated total genomic DNA from 5. africanum detected with avidin-conjugated 20 nm gold particles.A. Interphase cell showing domains of high and low gold density (chromatin of low gold density, arrowheads). X15 400.B,C. Two consecutive sections through a metaphase cell showing four chromosome pieces of S. africanum origin (i, ii, iii, v)with high gold density on both sections and two chromosome pieces of H. chilense origin (iv, vi, arrows) with low gold densityon both sections. X8000. The density of gold particles per ^m is given in Table 1.

Table 1. The density of20nm gold particles on cellcomponents following genomic in situ hybridization toelectron microscope thin sections (average values from

Fig. 3B.C)

Cell component

iiiiiiivVviCytoplasmCell wallIntercellular spaceVacuoles

Gold particles(number ;im~ )

2626306

21710a0

Identity ofchromosome piece

SecaleSecaleSecaleHordeumSecaleHordeum

-

r -

—

being higher than background levels (0-3 gold particlesfxm~ ). Interphase cells showed differential chromatinlabelling, with domains of high and low density of goldparticles (Fig. 3A).

Discussion

Genomic in situ hybridizationMetaphase spreads from root-tip cells of H. chilense XS. africanum (e.g. Fig. 1A,B) demonstrated that theseven larger chromosomes originating from S. africanumhybridized with the biotinylated 5. africanum genomic

DNA probe. Following probe detection with Texas Red-conjugated avidin the seven larger chromosomes in thehybrid metaphase cell fluoresced bright red (Fig. IB).The smaller chromosomes originating from H. chilenseshowed only weak hybridization signal. Similar differen-tial probe hybridization was shown by Schwarzacher etal. (1989).

In the sectioned nuclei (Fig. 1I-N) strongly andweakly labelled nuclear domains were visible. The ratioof areas of strongly labelled domains to weakly labelleddomains was 1.58±0.21:l. The DNA content of aprophase (4C) nucleus of 5. africanum is 29.7 pg, andthat of//, chilense is 21.8 pg (Bennett and Smith, 1976),giving an expected ratio of 1.36:1 for the proportions ofthe hybrid nucleus filled by each genome. The Jack-knifeprocedure (Miller, 1986) showed the two ratios were notsignificantly different (49 degrees of freedom P>0.05).This is consistent with the labelled chromatin originatingfrom S. africanum, as expected from the cell-spread data(Fig. 1A,B). The areas of chromatin with weak hybridiz-ation, visible only as red specks, represented chromatinoriginating from H. chilense. The differentiation of theparental origin of the chromatin is entirely due todifferential hybridization of the probe. DAPI fluor-escence demonstrates that the two genomes have similarDNA staining properties in both cell spreads and sections(Fig. 1A.F-H.I-K).

Chromosome domainsThere was no evidence that the chromosomes of the two

In situ hybridization on sections 339

parental sets were intermixed in the sectioned interphasenuclei. This result confirmed the impression gained fromspread preparations of interphase nuclei that chromo-somes are arranged in domains (Schwarzacher et al.1989). In spreads, the three-dimensional positions of thechromatin is distorted, and the extent of intermixing ofunlabelled chromatin with labelled chromatin could notbe determined. Sectioned material allows quantificationof probed areas and the localization of chromatin pos-ition. The accessibility of probe and reagents to chroma-tin is unaffected by residual cytoplasm and wall debris,and the whole surface of the section is uniformly access-ible to probe and detection reagents. Section labellingmay have been mainly on the section surface, since DAPIstaining showed intensity variations where chromosomesdid not extend through the full section thickness, whilethere was little variation in the intensity of Texas Redfluorescence (e.g. Fig. 1D,G, arrows). Material for sec-tioning was fixed with high-grade glutaraldehyde, amethod that does not materially change the relativeposition of the chromatin, unlike fixatives such asmethanol-acetic acid that are used for spreading (Skaerand Whytock, 1976). Thus, the domains are true rep-resentations of in vivo interphase chromosome dis-position.

Lichter et al. (1988) used chromosome-specific probes,rather than genome-specific probes, to show that indi-vidual human chromosomes occupy domains withininterphase nuclei. From the evidence of Lichter et al.(1988), it is likely that the individual chromosomeswithin each genome also lie in domains. Chromosomedomains have also been shown by genomic in situhybridization to whole mounts of human X hamsterhybrid nuclei that include only a few human chromo-somes (Manuelidis, 1985; Schardin et al. 1985).

In the electron micrographs (Figs 2 and 3), DAB orgold particles are seen in domains, and overlie certainchromosomes or chromosome axes. Because of the simi-larity of the method to that used on sections examined inthe light microscope (differing principally in the finaldetection system), we conclude that the DAB or goldparticles identify the sites of probe hybridization thatdistinguish the parental genomes. The demarcation ofthe domains, and the lack of intermixing of chromatinaxes from the two parental genomes even at their edges, isevident in the ultrathin sections.

Detection systemsIn the light microscope, the Texas Red detection systemrevealed the labelled chromosomes with high contrast inboth sectioned and spread preparations (Fig. 1). Fluor-escein can also be used as the detection system (Pinkel etal. 1986; Schwarzacher et al. 1989) but the contrastbetween fluorescein-labelled and unlabelled chromatinwas lower. The ability to counterstain fluorescein-labelled nuclei with a DNA-specific fluorochrome that isexcited at the same wavelength is valuable but notpossible with Texas Red fluorescence. Autofluorescenceof the sections (LR White medium resin) was much lowerat the Texas Red excitation wavelength than at thefluorescein excitation wavelength (results not shown).

In the electron microscope, hybridized biotin-labelledDNA was detected by the enzymic precipitation of DAB(Fig. 2) and by avidin-linked gold (Fig. 3). The DABgives a strong signal, and is particularly clear at lowmagnification (<x5000; Fig. 2A). At higher magnifi-cations (Fig. 2B) it is apparent that chromatin detail ispartly obscured by the DAB. The density of DAB is notuniform over the whole area of labelled chromatin(Fig. 2C); this might reflect differences in DNA packing,sequence composition or differential denaturation. Goldlabelling is less easy to visualize at low magnifications, butultrastructure is not obscured and the number of goldparticles can be quantified (Table 1). Quantification ofgold particle density over a long series of consecutivesections would permit accurate comparisons of the rela-tive abundance and intrachromosomal distribution ofDNA sequences complementary to the probe in the twogenomes. Some of the background detected by goldlabelling may have arisen from incomplete RNase treat-ment.

High-resolution in situ hybridizationThe high-resolution in situ hybridization shown here(Figs 2, 3) and developments of it will become increas-ingly important for the physical mapping of DNAsequences. It may be used to identify the locations ofsingle genes, gene clusters and repetitive sequences bothspatially within the nucleus and along chromosome arms.Various methods have the potential to increase theresolution of in situ mapping of DNA sequences usinglight or electron microscopy (transmission and scanning).Both spread and sectioned interphase and metaphase cellsmay be used. Interphase nuclei have particular advan-tages. (1) They are present in all tissues, so differentiatedcells can be studied. (2) Closely linked probe hybridiz-ation sites may be further apart than at metaphase as thechromatin is decondensed and hence can be mapped.(3) In sectioned chromatin, denaturation, reagent ac-cessibility and the lack of interference from cytoplasmmay make in situ hybridization more reproducible andsensitive.

Interphase reconstructionsFinch et al. (1981) showed that metaphase chromosomesof one parent were peripheral in the hybrid H. vulgareXS. africanum and it was noted that the chromosomes thatwere more peripheral dominated the plant phenotype(Bennett, 1984). In mammals, Manuelidis (1984) andManuelidis and Borden (1988) found that chromosomeposition was cell-type specific during differentiation inmouse and human cerebellum. The high-resolution gen-omic in situ hybridization shown here, and extensions ofthe method, have the potential to examine order in anycell at interphase and can investigate whether patternsvary with cell type or during development. Borden andManuelidis (1988) provided the first direct evidence thatchromosome position and cell behaviour were correlatedby showing that the position of the X chromosomedomains in normal and epileptic cortex cells were differ-ent. The reconstruction of whole cells following in situhybridization to localize whole genomes, chromosomes,

340 A. R. Leitch et al.

chromosome segments or genes will help to elucidate anyrelationship between nuclear architecture and gene ex-pression or chromosome behaviour.

We thank BP Venture Research Unit for support, Mrs J.A.Coates and K. Anamthawat-J6nsson for their technical assist-ance and Dr J. Brown for his statistical advice.

References

AGARD, D. A. AND SEDAT, J. W. (1983). Three-dimensionalarchitecture of a polytene nucleus. Nature, Land. 302, 676-681.

AVIVI, L., FELDMAN, M. AND BROWN, M. (1982). An orderedarrangement of chromosomes in the somatic nucleus of commonwheat, Triticum aestivum L. I. Spatial relationships betweenchromosomes of different genomes. Chromosoma 86, 17-26.

BENNETT, M. D. (1984). Nuclear architecture and its manipulation.In Gene Manipulation in Plant Improvement (ed. J. P.Gustafson), pp. 469-502. New York: Plenum Press.

BENNETT, M. D. AND SMITH, J. B. (1976). Nuclear DNA amounts inangiosperms. Phil. Trans. R. Soc. B 274, 227-274.

BINDER, M., TOURMENTE, S., ROTH, J., RENAUD, M. AND GEHRING,W. J. (1986). In situ hybridization at the electron microscopelevel: Localization of transcripts on ultrathin sections of LowicrylK4M-embedded tissue using biotinylated probes and protein A-gold complexes. J. Cell Biol. 102, 1646-1653.

BORDEN, J. AND MANUELIDIS, L. (1988). Movements of the Xchromosome in epilepsy. Science 242, 1687-1691.

BRIGATI, D. J., MYERSON, D., LEARY, J. J., SPALHOLZ, B., TRAVIS,S. Z., FONG, C. K. Y., HSIUNG, G. D. AND WARD, D. C. (1983).Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes.Virology 126, 32-50.

FINCH, R. A., SMITH, J. B. AND BENNETT, M. D. (1981). Hordeumand Secale mitotic genomes lie apart in a hybrid. J. Cell Sci. 52,391-403.

GEUSKENS, M. AND MAY, E. (1974). Ultrastructural localization ofSV40 viral DNA in cells, during lytic infection, by in situmolecular hybridization. Expl Cell Res. 87, 175-185.

HAFEN, E., LEVIN, M., GARBER, R. L. AND GEHRING, W. J. (1983).An improved in situ hybridization method for the detection ofcellular RNAs in Drosophila tissue sections and its application forlocalizing transcripts of the homeotic Antennapedia gene complex.EMBOJ. 2, 617-623.

HESLOP-HARRISON, J. S. AND BENNETT, M. D. (1983). The spatialorder of chromosomes in root-tip metaphases of Aegilopsumbellulata. Proc. R. Soc. Land. B 218, 225-239.

HESLOP-HARRISON, J. S. AND BENNETT, M. D. (1984). The positionof centromeres on the somatic metaphase plate of grasses. J. CellSci. 64, 163-177.

HESLOP-HARRISON, J. S., CHAPMAN, V. AND BENNETT, M. D.(1985). Heteromorphic bivalent association at meiosis in breadwheat. Heredity 55, 93-103.

HESLOP-HARRISON, J. S., HUELSKAMP, M., WENDROTH, S.,ATKINSON, M. D., LEITCH, A. R. AND BENNETT, M. D. (1988).Chromatin and centromeric structures in interphase nuclei. In KeviChromosome Conference III (ed. P. E. Brandham), pp. 209-217.London: HMSO.

HESLOP-HARRISON, J. S., LEITCH, A. R., SCHWARZACHER, T.,SMITH, J. B., ATKINSON, M. D. AND BENNETT, M. D. (1989). Thevolumes and morphology of human chromosomes in mitoticreconstructions. Hum. Genet. 84, 27-34.

HUTCHINSON, N. J., LANGER-SAFER, P. R., WARD, D. C. ANDHAMKALO, B. A. (1982). In situ hybridization at the electronmicroscope level: Hybrid detection by autoradiography andcolloidal gold. J. Cell Biol. 95, 609-618.

JENSEN, C. J. (1975). Barley monoploids and double monoploids:Techniques and experience. In Barley Genetics III (ed. H. Gaul),pp. 316-345. Miinchen: Thiemig.

LICHTER, P., CREMER, T., BORDEN, J., MANUELIDIS, L. AND WARD,D. C. (1988). Delineation of individual human chromosomes inmetaphase and interphase cells by in situ suppression hybridizationusing recombinant DNA libraries. Hum. Genet. 80, 224-234.

LlESI, P . , JULIEN, J. P . , VlUA, P. , GROSVELD, F . AND RECHARDT,L. (1986). Specific detection of neuronal cell bodies: In situhybridization with a biotin-labeled neurofilament cDNA probe. J.Histochem. Cytochem. 34, 923-926.

MANIATIS, T., FRITSCH, E. F. AND SAMBROOK, J. (1982). MolecularCloning. A Laboratory Manual. Cold Spring Harbor LaboratoryPress, NY, USA.

MANUELIDIS, L. (1984). Different central nervous system cell typesdisplay distinct and nonrandom arrangements of satellite DNAsequences. Proc. natn. Acad. Sci. U.S.A. 81, 3123-3127.

MANUELIDIS, L. (1985). Individual interphase chromosome domainsrevealed by in situ hybridization. Hum. Genet. 71, 288-293.

MANUELIDIS, L. AND BORDEN, J. (1988). Reproducible

compartmentalization of individual chromosome domains in humanCNS cells revealed by in situ hybridization and three-dimensionalreconstruction. Chromosoma 96, 397-410.

MILLER, R. G. (1986). Beyond ANOVA, Basics of Applied Statistics.New York: J. Wiley and Sons.

NARAYANSWAMI, S. AND HAMKALO, B. A. (1986). Electronmicroscope in situ hybridization using biotinylated probes. Focus8, 3-6.

NEER, A., BARAN, N. AND MANOR, H. (1977). In situ hybridizationanalysis of polyoma DNA replication in an inducible line ofpolyoma-transformed cells. Cell 11, 65-71.

OUD, J. L., MANS, A., BRACKENHOFF, G. J., VAN DER VOORT, H.T. M., VAN SPRONSEN, E. A. AND NANNINGA, N. (1989). Three-dimensional chromosome arrangement of Crepis capillaris inmitotic prophase and anaphase as studied by confocal scanninglaser microscopy. J. Cell Sci. 92, 329-339.

PINKEL, D., STRAUME, T. AND GRAY, J. W. (1986). Cytogeneticanalysis using quantitative, high-sensitivity, fluorescencehybridization. Proc. natn. Acad. Sci. U.S.A. 83, 2934-2938.

RAE, P. M. M. AND FRANKE, W. W. (1972). The interphasedistribution of satellite DNA-containing heterochromatin in mousenuclei. Chromosoma 39, 443-456.

SCHARDIN, M., CREMER, T., HAGER, H. D. AND LANG, M. (1985).Specific staining of human chromosomes in Chinese hamsterXmanhybrid cell lines demonstrates interphase chromosome territories.Hum. Genet. 71, 281-287.

SCHWARZACHER, T., LEITCH, A. R., BENNETT, M. D. AND HESLOP-HARRISON, J. S. (1989). In situ localization of parental genomes ina wide hybrid. Ann. Bot. 63, 315-324.

SCHWARZACHER-ROBINSON, T . , CRAM, L . S. , MEYNE, J . AND

MOYZIS, R. K. (1988). Characterization of human heterochromatinby in situ hybridization with satellite DNA clones. Cytogenet. CellGenet. 47, 192-196.

SCHWARZACHER-ROBINSON, T . , FlNCH, R. A., SMITH, J. B. AND

BENNETT, M. D. (1987). Genotypic control of centromere positionsof parental genomes in HordeumXSecale hybrid metaphases. jf.Cell Sci. 87, 291-304.

SKAER, R. J. AND WHYTOCK, S. (1976). The fixation of nuclei andchromosomes. J. Cell Sci. 20, 221-231.

STEINERT, G., THOMAS, C. AND BRACHET, J. (1976). Localization byin situ hybridization of amplified ribosomal DNA duringXenopuslaevis oocyte maturation (a light and electron microscopy study).Proc. natn. Acad. Sci. U.S.A. 73, 833-836.

VOGEL, F. AND KRUGER, J. (1983). Is there a general relationshipbetween estimated chromosome distances in interphase andlocation of genes with related functions? Hum. Genet. 63, 362-368.

WACHTLER, F., MOSGOLLER, W. AND SCHWARZACHER, H. G. (1990).Electron microscopic in situ hybridization and autoradiography:localization and transcription of rDNA in human lymphocytenucleoli. Expl Cell Res. (in press).

WALT, H., EMMERICH, P., CREMER, T., HOFMAN, M. C. ANDBANNWART, F. (1989). Supernumerary chromosome 1 ininterphase nuclei of atypical germ cells in paraffin-embeddedhuman seminiferous tubules. Lab. Invest, (in press).

WOLLENBERG, C , KEIFABER, M. P. AND ZANG, K. D. (1982).Quantitative studies on the arrangement of human metaphasechromosomes VIII. Localization of homologous chromosomes.Hum. Genet. 60, 239-248.

(Received 30 October 1989-Accepted 8 December 1989)

In situ hybridization on sections 341