Direct visualization of intranuclear lampbrush chromosome gene domains using videomicroscopy

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DIRECT

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VISUALIZATION

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Vol. 72, No. 9, September

OF INTRANUCLEAR LAMPBRUSH DOMAINS USING VIDEOMICROSCOPY

1988

737

CHROMOSOME GENE

B&be1 Helmut Michael F. Trendelenburg, Meissner, Tr&ter, Institute of Experimental Sigrid Berger" and Herbert Spring. Research Centre, D-6900 Heidelberg, Pathology, German Cancer *Max Planck Institute for Cell Biology, Rosenhof, D-6802, FRG., Landenburg, FRG.

chromatin arrangement Insight into the exceedingly complex within the interphase nucleus had - in the past - predominantly obtained using electron methods (for been microscopic (EM) review of the early literature, see DuPraw, that 1970). Since progress in subcellular fractionation and the increasing time, availability of defined molecular has led to a major probes improvement In understanding of the basic organizational the principles of active eukaryotic gene structure (for review, see Mathis et al., 1980; Gasser and Laemmli, 1987) and also in regard to the specific role of nuclear proteins in the three-dimensional arrangement of domains in the active gene interphase nucleus (for review, Hubert see Blobel, 1985; and Bourgeois, 1986; Gasser and Laemmli, 1987). However, due to the small dimensions of somatic interphase nuclei and also due to the high structural complexity of chromatin arrangement, interphase gene domains of interest could in almost all cases -. not be visualized directly. an impressive range of indirect Thus, experimental approaches has to be designed in order to test Rabl's initial proposal for arrangement of within an ordered chromosome domains the interphase nucleus. studies Such include (i) analyses of chromosomal rearrangements and theoretical considerations (ii) (Vogel and Schroder, 1974), of modified the use Giemsa-banding technique to obtain information on chromosome domains within squashed interphase nuclei (Stack et al., 1977), (iii) an EM analysis using rapidly Triton-buffer extracted whole mount preparations of fibroblasts and epithelial cells (Penman et al., 1983), (iv) the application of selective laser uv-microirradiation of small interphase chromatin areas and evaluation of corresponding chromosome segments during subsequent mitosis (Cremer et a1.,1984), and (v) an analysis of chromosome orientation during early Drosophila embryogenesis (syncytical blastoderm stage) using light microscopic optical through interphase nuclei containing sections prematurely condensed chromosomes after oxygen deprivation treatment. observation Direct of intranuclear gene 03OS-1651188/090737-27/$03,00/O

predominantly structures is

by using possible in

light microscopy a few, specially

@ 1988 Academic

Press Ltd

-

738

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suited such as cell the types insects (Chironomus, Drosophila) predominantly in amphibian species

Vol. 12, No. 9, September

large nuclei and in some (see below).

of some oocyte

1988

dipteran nuclei,

Detailed protocols have been worked out in order to obtain the large polytene chromosomes from dipteran salivary gland cells in an 'native' condition in almost order to allow precise localization of cloned gene probes and chromosomal proteins reviews, see Hill and Watt, (for 1978; Hill et al., 1987). In predominantly addition to these light microscopic investigations, this cell type was also extensively used for EM investigations on the fine structure of the most prominent gene domain of Chironomus polytene the Balbiani ring chromosomes, Stereo-EM of 0.1 pm sections was used to obtain (BR) genes. information of the spatial arrangement of chromatin axes of transcriptionally active individual, BR genes (Olins et al., More recently, the of events with 1980). complete sequence the biogenesis of the BR ribonucleoprotein regard to (MP) elaborated by the use of EM-computed transcripts could be the specific ultrastructural changes occurring tomography, e.g. transcript RNP fibril on the primary nascent up to the of the premessenger RNP particle through the nuclear transport pore complex of the salivary gland cell nucleus (for recent Skoglund et al., remains review, see 1986). It to be salivary emphasized that the unique features of cytogenetic allowed a precise deciphering gland polytene chromosomes have arrangement of chromosomes in of the three-dimensional these nuclei using computer based digital Drosophila salivary gland microscopy (Mathog et al., 1984; light imaging fluorescence 1987a,b; for methodological review, see Hochstrasser and Sedat, Arndt-Jovin et al., 1985). the other As to the present state of the structural analysis of system for interphase chromosome organization, important model chromosomes, had largely been lampbrush research e.g. the chromosomes of amphibians (see below lampbrush concentrated on Drosophila on lampbrush chromosomes of for details) and Hennig, 1985, Hess, 1981; spermatocytes (for reviews, see also Callan, 1986). 1987; c,f, structural analysis of amphibian lampbrush With regard to the which allow refined techniques had been developed, chromosomes, of unfixed chromosomes in a gentle way, e.g. by preparation the chromosomes can Thus, exposure to slightly dilute salt media. analysed as and be sedimented from ruptured oocyte nuclei 1966; reviews, see Gall, structures flat (for extended, 1983; Trendelenburg, Macgregor, Sommerville, 1977; 1980; Among the most Trendelenburgh et al., 1986). 1986; Callan, those from the newt are thoroughly studied chromosomes there in chromosomes are shown Table 1, these Triturus. As

Cell Biology

characterized structures domains.

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by which

a

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particularly represent

Vol. 12, No. 9, September

the

size large transcriptionally

of

1988

lateral active

loop gene

the In addition, these chromosomes are further characterized by presence of conspicuous, large 'landmark' loop structures which precise identification had been shown to be very useful for the of individual chromosomes (for review, see Callan, 1986). that structural Despite accumulated amount of information, comparatively little is known about the specific function, e.g. for gene content, most of the individual loops, with few but notable exceptions, in particular, the containing loop pairs histone genes the in NotoohthalmU (Triturug) viridescens (for Gall, recent results, see Bromley and 1987; for review see 1977, Sommerville, 1981; 1980, 1986; Callan, Macgregor, Davidson, difficulties in 1986; 1986). The obvious gene localization to defined loop structures are thought to be largely due to the high DNA complexity of the large amphibian Triturus, Necturus genomes, e.g. (Table l), organisms with large loop structures. It thus appeared to be of interest to also on concentrate chromosome analysis of species with much smaller genomes, such as the frog Xenopus (Table 1). However, if one considers the average short length of sized loop structures in Xenopus, one realizes that a detailed analysis of loop structures using conventional phase contrast microscopy of unfixed chromosomes is very difficult, if not impossible. This also for the chromosome analysis of other organisms with holds small genomes (Table 1.) Before the advent of video (see enhanced light microscopy semithin sections high light below) the analysis of at microscopic magnification combined with EM of subsequent ultrathin sections represented one of the only means to tackle this problem (Spring et a1.,1975; Spring and Frank@, 1981; Scheer, 1987; c,f, also Callan, 1986; Trendelenburg et al., 1986, for recent review) . Only recenlty, scanning EM had been used to visualize lampbrush loop organization in the salamander Pleurodeles (Angelier et al., 1984; Bonnanfant-Jais et al., 1985). One of the most interesting findings of studies these was documentation that very small loops could be clearly visualized using SEM, but could not be detected on the same chromosome with conventional phase contrast light microscopy. A further new finding was the observation that some of the small exhibited partially coiled when loops superstructures SEM, indicative analysed by for an occurrence of higher order chromatin packing of at least some of the lateral loops. In previous investigations on the ultrastructure of Acheta rDNA genes the existence of a higher order organization of this type of transcriptionally active rDNA chromatin could be visualized, rDNA e.g. when the chromatin was gently dispersed from its

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ACTIVE GENE CHROMATIN 5’ +

9 kb Kleinschmrdt type EM spread of protein - free DNA

a

5’ +

b

+ 3’

2.8 Drn

+ 3’

Miller type EM spread of active chromatin

0.6 Drn Light microscopic observation of the in sttu chromatin organlsation C

Fig. 1. Limitations for visualization of gene the direct Gene size. domains: illustration of 1. a gene sire of 9 kb In the schematic Fig. Kleinschmidt double stranded DNA is given as example for the DNA spreading technique (a). If a gene of this size is madimally transcribed by RNA polymerases and spread from Miller chromatin spreading nuclear contents according to the technique, the length of the spread gene segment is shown (b). segment as In contrast to the length of the extended gene visualized in a Miller type KM spread preparation, the in vivo microscopy configuration of active genes as identified by light order non-spread chromatin, is often a complex, higher of structure (c) . compiled Data Miller, 1981; Trendelenburg 1987).

from (Franke Trendelenburg, 1986; et al.,

et al., 1979; Mathis et al., 1980; 1985; Trotter et al., 1983; Trendelenburg and Pucion-Dutilleul,

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741

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-----------_ -------_ -/---0 a

b

Fig.2. domains:

Limitations Position

of

for genes

the within

direct the

visualization intact nucleus.

The rapid identification of a gene unifixed intact, nucleus is illustrated microscopic 'optical' sections. In microscopic image of the structures of the nuclear membrane in medium of and subsequent gentle sedimentation of an appropriate centrifugation chamber text; c.f. also Trendelenburg, 1983; Trendelenburg et al., 1986).

compact discussion, 1986).

intranuclear see Troster

organization et al.,

of

gene

of

interest within an in using light (a) general, a much better is obtained after opening isotonic ionic strength nuclear chromatin within (b) for details see Troster et al., 1985;

(Figs. 1 and 2; 1985; Trendelenburg

for

further et al.,

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The above results were only possible by a systematic adaptation of video enhanced light microscopy (Fig. 3., for details, see Allen and Allen, 1983; Allen, 1985; Ino&, 1986; c.f. also Rebhun, to the special requirements for visualization of 1986) unfixed, unstained, hydrated chromatin (see e.g. Trendelenburg et al., 1986; Montag et al., 1988).

h

offse:

I,,

gain J

hn/,

backgrou,nd hn, digital enhancement J

major steps in generation of Fig. 3. Scheme of the operational videomicroscopic images. The first steps (upper optimizing the videocamera setting by consists of panel) and 'offset' and r gain' functions. Electronic storage real the background image is then followed by a subtraction of time operation (lower panel). final, digital contrast enhancement and Allen (1983), Allen (1985), Inoue details, Allen For see et al. (1986). (1986) I Trendelenburg

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743

observing chromatin domains within Due to the possibility of nuclei, using nuclear isolation conditions isolated oocyte which had been shown to prevent deterioration of chomatin structures during the time needed for image recording (Zentgraf et al., 1979; Schultz et al., 1981; c.f. also Gurdon and Wakefield, 1986), it was possible to obtain images of the intranuclear structure of lampbrush chromosomes and marker loop structures in isolated nuclei of Xenopus laevis and using also, related nuclear isolation conditions, in nuclei of Acetabularia and Acheta (for details, see below). Further studies of this type will allow a detailed comparison of chromosome and loop compaction when visualized in situ, with the dimensions of the identical chromosomes following isolation and sedimentation. We believe that this type of analysis may contribute to the further elucidation of the in viva structure of the presently intensively discussed loop domain model for transcriptionally active genes (for details, see Ptashne, 1986; Gasser This is of and Laemmli, 1987) . particular if we take into interest, account that a direct intranuclear visualization of specific gene domains in interphase nuclei of difficult, somatic cells is exceedingly even when very sophisticated methods can be applied (for discussion, see McDowall et al., 1986; Ringertz et al., 1986).

es

In

wrmrv

nuclei

of

Acetabularia

wdlterranea Visualization, precise counting and a possible identification of individual lampbrush chromosomes of Acetabularia primary nuclei is particularly difficult using the conventional chromosome preparation techniques. Among the reasons are (i) the relatively small diameter of appropriate primary nuclei of, on average, 80-120 )xn, (ii) the small size of the individual lampbrush chromosomes (iii), and the intranuclear location of the chromosomes in close vicinity to the large, dense nucleolar units (Fig. 4). As outlined in previous studies (Spring et al., 1974, 1975, 1978) it was found to be exceedingly difficult to obtain complete unstretched and/or unfragmented lampbrush chromosomes using conventional the chromosome preparation methods. it appeared to be of major interest, to use situation, In this of the A situ visualization for videomicroscopic methods within intact, isolated primary the chromosomes lampbrush own including our investigations, From numerous nucleus. A. mediterranea primary nuclei known that was experience, it isolation appropriate are particulary stable, when isolated in Brachet, see Schweiger and Berger, 1979; medium (for review,

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Survey contrast light micrograph of a semithin 4. phase and through a glutaraldehyde-fixed section )yn) Note the Epon-embedded rhizoid of Acetabularia gediterranea. nucleus (N). In the primary peculiar structure of the giant section one clearly recognizes the outline of the shown, median centrally nucleolar located almost nuclear envelope, the lampbrush them, individual adjacent to structures, and, not are loop structures Lateral chromosomes (arrows). appropriately detectable (arrowheads). also Franke et a1.(1974), Spring et see text; c.f. For details 15 um. Bar indicates al. (1975, 1978), Scheer et al. (1979). Fig.

(2.5

1987; 1985, Berger et al., 1987). In addition, the relatively small size of the chromosomes (c-f. Table 1) facilitate should visualization of the whole chromosomes, using optical sectioning videomicroscopy (Fig. 2). A typical optical section is shown in Fig. 5. One complete chromosome is shown to be located in a 15 F wide zone between a large nucleolus and the nuclear membrane. In the central part of chromosome, the the typical lampbrush chromosome organization can be clearly recognized, consisting of the chromosome axis the and lateral loop structures. Although a typical chromomere structure of chromosome the axis is much clearly visible less than in amphibian lampbrush chromosomes, a more compact chromatin

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structure fluorescence

of of

the the

Reports,

Vol. 12, No. 9, September

chromosome axis same specimen.

can

(Fig.

be inferred 5b)

from

(pg)

% non repetitive DNA sequences/ genome

mean size of lampbrush loops oyn)

Acetabularia mediterranea

0.92

n.d.

2-5

Achetea domesticus

2.3

n.d.

n.d.

Xenopus laevis

3.1

55

3-8

Triturus cristatus

23

Species

Necturus

C value

-90

<45

(10

745

1988

the

DNA

30-50

7100

Table 1. Genome characteristics in relation to mean lampbrush organisms. chromosome sizes of (n.d. = not loop selected Bier, determined, data compiled from 1970; Sonunerville, 1979; Scheer 1977; Spring et al., 1978; Scheer et al., and Sommerville, 1982; Trsster et al., 1985)

available information on With regard to the presently still the only well Acetabularia lampbrush chromosomes documented case for the plant kingdom (for recent review, see Callan, 1986) the videomicroscopic analysis presented here may be of particular value to contribute to the following intriguing topics: (i) A precise determination of the number of lampbrush chromosomes individual primary contained in an nucleus, (ii) the possible visualization of a direct structural interrelationship of particular lampbrush chromosomes with the large nucleolar units (for discussion, see e.g. Spring et al,, (iii) to possible 1978), and document changes in the organization of individual chromosomes during the long differentiation period of the primary nucleus concomitant with the vegetative phase of the Acetabularial life cycle (Franke et al., 1974, Brachet, 1985; 1987, Berger et al., 1987).

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Fig. 5. Videomicroscopy of lampbrush chromosome structures within an isolated, intact Acetabularia mediterranea primary nucleus. A large complete chromosome and a short segment of an adjacent chromosome is shown in (a). Note the structural arrangement of the central chromosome axis and the lateral loop structures as visualized in interference contrast videomicroscopy. For comparison, the same specimen is shown in fluorescence videomicroscopy in (b). The path of the central chromosome axis is recognizable due to DNA-specific DAPI fluorescence. Bar indicates 5 w.

Cell Biology

cT Acheta

.

International

Reports,

.

oocvte

Vol. 12, No. 9, September

r

-

1988

R'

f

chroxnosomeg

The existence of a typical lampbrush chromosome organization on Acheta oocyte nuclei had originally been propsed by Kunz (1969) largely based on Feulgen squash chromosome preparations of the pachytene and very early diplotene oogenetic stages. In the meantime, several detailed structural investigations had been carried out on mid-diplotene (for oocytes references, see Troster et al., 1985) which contain oocyte nuclei of 80-100 um in diameter (largest size during Acheta oogenesis). This stage of oogenesis is characterized by a maximal further transcription rate of both, amplified and ribosomal chromosomal RNA genes (Spring et al., 1974; Franke et al., 1979; Troster et al., 1985; Davidson, 1986), it thus corresponds closely to the diplotene stage of amphibian oocytes, where lampbrush chromosomes the are typical nuclear constituent (see introduction). However, if with procedures identical the typical lampbrush chromosome preparation method 1979; (Scheer et al., Trendelenburg, 1983; Callan, 1986) are applied to Acheta oocyte nuclei following isolation from mid-diplotene oocytes, from chromatin structures can be sedimented nuclear contents, but the characteristic structure of thickened chromatin axes and protruding lateral loop profiles could not be seen in such preparations (Troster et atl., 1985; Trendelenburg et al., This is line 1986). essentially in with Kunz' observations (1969) for the transition from the late pachytene stage TO diplotene stage of oogenesis, 'where a major despiralization of lampbrush chromosomes which takes place finally renders the chromosome structure non-detectable using Feulgen squash preparation or conventional phase contrast rof mocroscopy unfixed chromosomes' (translated from Kunz, 1969) The typical structural aspect of a median section through a mid-diplotene Acheta oocyte nucleus is shown in Fig. 6. The conspicuous most chromatin element is the very dense extra-chromosomal micronucleoli which are characteristically distributed within one half of the oocyte nucleus (lower left in Fig. 6). In the other half of the nucleus, small areas, which only are slightly denser than surrounding the nucleoplasm, can be seen. Analysis of consecutive ultrathin sections using electron microscopy reveals that zones these consist of typical ribo-nucleoprotein (RNP) structures and are thus indicative for the transcriptionally active, chromosomal loci. However, it is gene clear from the specimen shown in Fig. 6 that no small axial chromosome segments can be identified close to these RNP complexes. the chromosome Thus, organization at this stage of oogenesia is different to that of

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Fig. 6. Phase contrast light micrograph of an Epon section through an early vitellogenic oocyte of Acheta domesticus. In this semithin secion (2.5 )un) the arrangement of the major nuclear chromatin components is clearly detectable. Micronucleoli, which contain extrachromosoma, amplified circular Acheta rDNA units are seen in the lower left of the large oocyte nucleus. Much less dense chromatin areas of almost identical are size located in the upper part of the nucleus. These structures likely to are represent RNP transcript complexes associated with the chromosomes of the oocyte nucleus. For details, see text; c.f. also Troster et al. Trendelellburg et al. (1986). Bar indicates 15 pm.. (1985), typical lampbrush chromosomes. For comparison, are easily identified in similar semithin Acetabularia primary nuclei (Fig. 4) or through Xenopus oocytes (Fig. 8a). Recently, only clear conspicuous

progress had marker structure chromosomal

such structures sections through nuclei of small

been made in identifying probably the of Acheta chromosomes, oocyte the nucleolar organizer (NOR) complexes

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1988

(Fig. 7; c.f. Troster et al., 1985; Trendelenburg et al., 1986). with a mean diameter of 6 um were These structures first identified using DIC 'opital sections' (Fig. 2) through isolated oocyte nuclei (Fig. 7a) and then further analyzed following sedimentation of from isolated nuclear NOR's When contents. isolated in medium of isotonic ionic salt strength, NOR complexes retain most of their inu structural characteristics (compare Fig. 7a with Fig. 7b). In addition it thin sectioned NOR's using could be deduced from results of well as conventional transmission electron microscopy (TEM) as from micrographs of 5 um sections using scanning transmission NOR's represent electron microscopy that Acheta (STEM) particularly for the detailed analysis of favourable material ribosomal active the in situ organization of transcriptionally RNA genes (Troster et al., 1985; Trendelenburg et al., 1986). types of By contrast to almost all thoroughly studied different review see Macgregor, 1982; nucleoli or NOR structures (for contain closely Stahl, 1982; Hadjiolov, 1985) the Acheta NOR's packed active pre-rRNA transcription units without most of the components and skeletal nucleolar pars granulosa typical absence of these nucleolar nucleolar proteins. Due to the unravel rapidly upon constituents, the compact NOR structures 7b with isolation buffer (compare Fig. exposure to dilute Fig.7~) to large subunits. Following prolonged exposure to low salt medium, e.g. using conditions similar Miller-type to the chromatin spread preparation (Miller and Beatty, 1969; Miller, 1981; recent for review see Trendelenburg Puvion-Dutilleul, and 1987) the compact NOR submits dissociate into fine rDNA chromatin strands of 0.6-0.8 pm in diameter. This is indicative of a characteristic higher order structure of the actively transcribing Acheta rDNA genes, since this type of gene shown was to have a mean length of 5.5 um when visualized according to the Miller-type chromatin spreading technique (for discussion, see Troster et al., 1985; Trendelenburg et al., 1986). lear

. 0roanlzatLon

.

of

Xenooug

In contrast to the situation in Acetabularia and Acheta (see above) transcription parameters in oocytes of Xenopus laevis are particularly well understood (for recent revies, see Macgregor, 1980, 1986; Trendelenburg 1983; Hrachet, 1985; Callan, 1986; Davidson, 1986; Gurdon and Wakefield, 1986) . However, problems exist in regard to a precise correlation of transcription lampbrush parameters to chromosome loop organization (for recent review, see Scheer et al., 1979; Macgregor, 1980, Sommerville, 1981; 1986; Callan, 1986;

750

Fig. 7. chromosomal bar in (a) interference NOR complex

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micronucleoli and the Extrachromosomal Acheta organizer nucleolar region (NOR), denoted by large identified an isolated, intact oocyte nucleus with contrast videomicroscopy. In (b) the chromosomal is shown at high light magnification microscopic

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the nuclear membrane and gentle sedimentation opening of after of low salt buffer Upon addition of the rDNA gene structure. isolation medium, the NOR structure starts to isotonic to the irregularly shaped sub-units (phase dissociate into numerous, 10 um (a); 5 pm videomicroscopy, contrast [cl 1 * Bars indicate (brc) .

Davidson, 1986). Up to now, structural analysis of Xenopus lampbrush chromosomes had been concentrated mainly due to from technical difficulties on chromosomes isolated oocyte stages IV to VI, e.g. large oocytes during their final phase of 1972) . It was oogenetic growth (Dumont, found that Xenopus lampbrush chromosomes relatively small, e.g. when are (i) Triturus compared to the large lampbrush chromosomes of species (ii) and that chromosomes prepared from these stages of exhibit oogenesis do not easily identifiable landmark loop structures (c.f. also Introduction; for details, Muller, see 1974; Scheer et al., 1980, 1979; Macgregor, 1986: 1983; Callan et al, 1987). Trendelenburg, Only very limited information is available concerning the fine lampbrush structure of loops during early oogenesis of Xenopua laevis (for review, see e.g. Macgregor, 1980) . Thus, we have visualize chromosomes in oocyte nuclei attempted to lampbrush III, during early oocyte differentiation (e.g. stages II and A survey light micrograph of the Dumont, 1972). according to . . in situ organization of lampbrush chromosomes in a late stage II oocyte is shown in Fig. 8a. During this stage of oogenesis, the extrachromosomal nuclei are transcriptionally inactive and closely associated with the nuclear membrane. lampbrush chromosomes within The are arranged the nucleolus-free central area of the nucleus. When this semithin section is observed at high magnification (Fig. 8b) the typical chromosome constituents, e.g. axial chromomeres and lateral loop structures are detectable in some areas of the section. with Compared the light microscopic evaluation of 3 urn sections (Fig. 8) a substantially larger amount of information on k chromosome structure can be obtained using optical aiul sections through nuclei isolated oocyte (see Introduction). Lampbrush chromosome structures within a nucleus of a stage III are shown in oocyte Fig. 9, using DIC videomicroscopy (for details, see Introduction). The micrographs shown were taken at focus level 35 )nn inside the nucleus (Fig. 9a) and at 45 )~m (Figs. 9b,c). In the optical section shwon in Fig. 9a, parts of two closely associated lampbrush chromosomes are shown. The majority of detectable loop structures have a mean length of 2-5 um. Examples of individual large loops, e.g. with lengths

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Fig. 8. Phase contrast light micrograph of a semithin section nuclear area of a previtellogenic Xenopus through the (3 l-d laevis oocyte. The specimen shown represents a grazing section through the nuclear periphery, approximately 25 um below the nuclear envelope (a). surface Most of the nuclear area is nucleoli. occupied by extrachromosomal, amplified In the center of this area the exclusive zone of lampbrush chromosome shown at high magnification in begins. This central area is contrast videomicroscopy. Note short (b) using interference chromosome axial where typical chromomere segments the organization as well as the loop structures can be recognized. Bar indicates 15 um (a); 5 p (b).

of 8-12 jr~~, are shown in Figs. 9b,c. It is will be complete information available analysis of chromosomes of isolated the Trendelenburg et oogenetic stage (see discussion.)

clear that more from concomitant identical early al., for 1986,

An example of the potential for using videomicroscopy at high microscopic light magnification further for the structural characterization of lampbrush chromosome bivalents from large shown in Xenopus oocytes is Fig. 10 (Fig. lOa, conventional contrast light phase microscopy; Fig. lob, DIC videomicroscopy). bivalent The shown belongs to the group of chromosomes, it is characterized by large two chiasmata, are fused and is thus likely to correspond to telomeres not bivalent 8 according to Muller (1974) ir bivalent II according Callan et al. (1987). Compared with the photograph of Fig. to lOa, the conspicuous large loop structures present at left the only be analyzed sufficiently using end of the bivalent can videomicroscopy (Fig. lob). Further analysis of this kind is thought to provide essential data on the location and structure small - Xenopus loop of the - comparatively marker structures. data should increase the possibilities of a precise Such identification of individual lampbrush chromosome bivalents. Concludina Lampbrush chromosomal organization reseach preferentially particularly concomitant

remark% chromosome provide an gene domains excellent model for on basic principles of system studies of transcriptionally active genes. In the past, carried on these chromosomes been had out by using chromosomes of amphibian species with large genomes Triturus 23 pg DNA) and (e.g. conspicuously large lampbrush chromosomes.

In the meantime, nuclear genomoes of amphibians

DNA analyses preferentially

had

shown used

that for

the analysis

large of

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Fig. 9. Visualization of intranuclear lampbrush chromosome structures immediately after nuclear isolation. The micrographs shown are from a nucleus of an vitellogenic early oocyte. The close lateral association of lampbrush chromosomes in situ is shown in (a). Examples of unusually large lateral loops are given in and (c). Note also the compact (b) structure of average-sized loops. Bar indicates 3 urn.

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Fig.10. and (a) lampbrush

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Comparison of interference chromosome

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755

conventional phase contrast microscopy contrast videomicroscopy (b) of a Xenopus preparation. Whereas the overall

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the bivalent shown satisfactorily organization of can be loops, analyzed in (a), details of loop structures (giant large adjacent large arrowheads) can arrows; loops, only be high magnification. identified using videomicroscopy at Note small Bar indicates 15 the size of average-sized loops. also urn (a); 5 )1"~ (b). lampbrush percentage fact This defined exceedingly

chromosome of interspersed, renders the loop strucutres difficult.

structure contain an unusually high repetitive DNA sequence elements. further molecular characterization of of such amphibian chromosomes

In the present study, evident is presented tht it also be may sufficiently possible to visualize characteristic loop structures located on lampbrush chromosomes of eukaryotes with smaller much sizes, e.g. ranging from 0.9 pg DNA in genome Acetabularia (unicellular green 2.3 pg DNA in Acheta akra), (orthopteran insect) to 3.1 pg DNA in Xenopus (frog). Visualization of sizes of loop 2-5 JIM is facilitated using video enhanced light microscopy at high magnification. This not only lampbrush chromosomes prepared by the holds for the imaging of conventional preparation technique but also for lampbrush chromosomes observed the in situ arrangement of within manually isolated intact nuclei. well as the Since the giant primary nuclei of Acetabularia as nuclei of Acheta and Xenopus are particularly suitable oocyte phenomena, the objects for studying nucleocytoplasmic transport possibility of visualizing defined intranuclear chromosome domains may finally allow the testing of essential parameters functional nuclear gene hypothesis of gating of the organization.

We thank Herbert excellent preparing

Ansgar our colleagues SteinbeiBer for helpful technical assistance and some of the thick sections.

also greatly indebted We are Microscopy, Carl Zeiss, Oberkochen, Forschungsgemeinschaft, Deutsche the TR 147/6-2) for support.

Montag Hofmann, Markus discussions, Roger Fischer help Birgit Schuhmann for

to

the Division of Germany Fed. Rep. Bonn-Bad Godesberg

and for in

Applied and to (grant

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1988

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