Journal of Cell Science 103, 839-846 (1992) 839 Printed in Great Britain © The Company of Biologists Limited 1992

The relative positions of condensed chromosomes are maintained between divisions in the epidermis of ethlius

GWENDOLYN JEUN and MICHAEL LOCKE*

Department of Zoology, University of Western Ontario, London, Ontario, Canada N6A 5B7 *Author for correspondence

Summary

The larval epidermis of (, chromosomes are not all alike, suggesting that chromo- Hesperiidae) is a syncytium of doublets where sibling somes are repositioned at mitosis. Separation distances cells are twins that remain connected by residual mid- between chromosomes remain the same between but not bodies between mitoses. Twins resemble one another through cell divisions, suggesting that determinants for more than their other neighbours in such structural fea- nuclear structure are conserved through interphase and tures as the shape and number of nucleolar particles relaxed at mitosis. Although the condensed chromo- and the number of actin bundles. We have now found somes of sibling nuclei resemble one another in their that they also resemble one another in the position of separation, they differ in their orientation, as would be the condensed chromosomes that occur in female cells. expected if whole nuclei rotate in the plane of the epithe- Female lepidopteran cells contain one or more particles lium. of condensed chromatin, depending on their ploidy. In the epidermis, nuclei with two condensed chromosomes are found in pairs and are separated by the same dis- Key words: chromosome position, nuclear structure, pattern tances. However, clones of cells with multiple condensed pairing, condensed chromosomes.

Introduction same between but not through cell divisions, suggesting that determinants for nuclear structure are conserved through Two orders, Lepidoptera and Trichoptera, have het- interphase and relaxed at mitosis. erogametic females with ZW sex chromosomes (Feltwell, 1982; Robinson, 1971). Female insect cells have a con- densed chromosome presumed by Moore (1966) to be the Materials and methods Z chromosome. The nuclei of all cells of female Calpodes ethlius (Lepidoptera, Hesperiidae) contain such particles of Experimental condensed heterochromatin, which mark the position of the A colony of Calpodes ethlius (Lepidoptera, Hesperiidae) was kept inactive chromosome within the nucleus (Locke, 1990). In in a greenhouse where the larvae were reared on lily plants. epidermal cells they are roughly spherical, about 1.0 mm in The caterpillars were easily sexed because the ovaries and testes diameter. Polyploid cells of other tissues contain single pro- are visible through the transparent integument. The timing of portionately larger particles, reaching 10 mm in diameter in larval development was standardized by rearing them in an incu- oenocytes (Locke, 1969). In larval epidermal cells poly- bator at 22°C on a 12:12 h, day:night, cycle. The 5th stadium (the ploidy is reflected in the number rather than the size of the time from the 4th - 5th ecdysis until the 5th - pupal ecdysis) took particles (Locke, 1988). The point of interest for the present 8 days (Locke, 1970). Epidermal cells divide 36 h before ecdysis study is that when there are two of these particles, there are and again 90 h into the 5th stadium. Microscope observations were also two in the nucleus of an adjacent epidermal cell. made on the epidermis of 5th stage female larvae in the period between divisions. In the 5th stage larval epidermis all divisions are lateral. Sibling cells retain their midbody between mitoses that may Preparation of the epidermis be several days apart, creating an epithelium of Siamese twin cells one cell thick. Twins resemble one another more Fifth stage caterpillars are up to 7.0 cm long and 1.5 cm wide. The integument (cuticle + the single layer of epidermal cells) was than their other neighbours in such structural features as the kept flat but with its natural proportions, by coating caterpillars shape and number of nucleolar particles (Locke and Leung, with 5% aqueous gelatin, drying, and attaching them to a micro- 1985) and the number of actin bundles (Delhanty et al., scope slide with “krazy glue”. The glue adhered more firmly, but 1991). In this paper we describe how the distances sepa- not permanently, to the cuticle after coating with gelatin. The rating condensed chromosomes in twinned cells remain the glued larvae were then separated from the slide and fixed by infla- 840 G. Jeun and M. Locke tion with 5% formaldehyde. The glued integument was cut from Statistical analysis the larva and fixation continued for 15 min. The integument was Two separate one-way analysis of variance (ANOVA) tests were permeabilized for 10 min in 5% formaldehyde containing 1% used to determine whether (1) the angle and (2) the condensed Triton X-100. The glue mold, muscle and fat body were removed, chromosome separation distances were significantly different leaving cuticle with epidermal cells preserved in one plane but between the 18 pairs of measurements in 18 twinned cells. otherwise in their natural positions.

Staining epidermal nuclei Results Sheets of integument were incubated in 10 ml RNase (ICN Bio- -2 chemicals) solution (1 mg RNase/ml 5´ 10 M phosphate buffer, Nuclei in female Calpodes contain condensed chromosomes pH 7.4) at 34-37°C overnight, washed in distilled water, cut into 1 cm2 segments and stained for 15 min in 1 ml of a solution con- Fields of epidermal cells from female larvae stained for taining 20 mg of propidium iodide. Excess stain was removed by DNA with propidium iodide showed that all nuclei con- three 5 min washes in distilled water. The integument was tained small dense particles. The particles were about 1 mm mounted in a 1:9 (v/v) phosphate buffer (pH 7.4):glycerol solu- in diameter, roughly spherical and more brightly fluores- tion, with the epidermis facing a “0” coverslip. cent than the rest of the nucleus, suggesting that their DNA is more densely packed than the surrounding heterochro- Microscopy matin (Fig. 1). The particles are presumed to be condensed Epidermal cells were examined with a Bio-Rad MRC600 con- chromosomes. focal laser-scanning microscope, using a Nikon Planapo ´ 60 oil Most epidermal nuclei were the same size and had single immersion objective (NA = 1.4) on a Nikon Diaphot-TMD particles, but some larger cells had two or more. Nuclei inverted microscope. The microscope was equipped with an argon with two (Fig. 1A,B) or three particles (Fig. 1C,D) always ion laser and a filter, excitation wavelength = 514 nm. occurred in pairs. The pairs are presumed to be sibling cells because multiple particles never occurred singly and the Selection criteria for nuclei pairs were always surrounded by cells with single particles. Large flat fields of epidermal cells were scanned using conven- tional epi-fluorescence to find paired nuclei, each containing two The separation of paired condensed chromosomes condensed chromosome particles. Clusters with more than two The positions of condensed chromosomes were determined nuclei having double particles were not used, since selection of in 18 pairs of nuclei. Nuclei were reconstructed in 3D from pairs then became subjective. stacks of 0.1 mm thick optical sections. Vertical profiles showed that the nuclei were usually ovals compressed in Optical sectioning the plane of the epithelium (Fig. 2A). The horizontal (xy Selected pairs of nuclei were optically sectioned (0.1 mm/section) distances) and the vertical (z distances) were measured as from apical to basal surfaces at zoom 4, 6 or 8 (Fig. 1A). Image in Fig. 2B. The real separation of the chromosomes, averaging of each section reduced background noise. A z-series (xy2+z2)g, was then calculated. The separation between par- consisted of 40-70 sections. An extended focus image was formed ticles in unpaired nuclei varied from 1 to 14 mm (Fig. 2D), by stacking all the optical sections. The same field was later opti- confirming that the condensed chromosomes do not have a cally sectioned at a lower zoom (and at 0.2 or 0.3 mm/section) to constant position in the nucleus (Fig. 2C). However, the show the relationship between pairs of nuclei and the surround- ing cells. separation of double chromosomes was similar in pairs of nuclei (ANOVA, P<0.0005), from which we conclude that sibling cells resemble one another more than they resem- 3D measurement of particle separation ble other cells nearby. The xy (or 2D) separation between particles was measured directly from the extended focus image. The z separation was measured by counting the number of sections between the maximal particle Double chromosomes have different orientations in paired diameter and multiplying by the stepping motor increments (i.e. nuclei 6 sections ´ 0.1 mm per section = 0.6 mm). The possible error Separation is a measure of the way that the condensed chro- described by Leung and Jeun (1992) in determining the centre of mosomes are arranged within each nucleus. The direction spherical objects was insignificant because of the small size of the condensed chromosome (less than or equal to 1 mm in diameter). The 3D separation distance was calculated using the xy and z dis- tances in the Pythagorean theorem (Fig. 2B). Only the 3D results Fig. 1. Nuclei in epidermal cells from female Calpodes larvae are presented because there was no significant difference between contain particles of condensed chromatin. Propidium iodide the 3D and 2D measurements (paired sample t-test, P>0.05), prob- staining of the epidermis for DNA shows most nuclei with single ably because the nuclei were flattened ovals rather than spheres. particles, but a few are polyploid and have two or more. Nuclei with more than one particle always occur in pairs. (A) Most cells have single particles, but one pair enlarged in (B) has two in each Measurement of the angle formed by the line joining nucleus (\). (D) The pair of cells enlarged in (C) has 3 particles and the mitotic plane chromosomes (/). Pairs of nuclei are presumed to be in sibling Straight lines were drawn on an extended focus image to connect cells because they are surrounded by fields of smaller nuclei all the double particles within each nucleus. The angles made by these having single condensed chromosomes. Confocal summing of lines with the estimated previous plane of division were measured optical sections taken from cells about 4 days after the last (Fig. 3A). mitosis. Bars, 5 mm. Condensed chromosome arrangement 841 842 G. Jeun and M. Locke

Fig. 2. The separation of condensed chromosomes measured on 3D reconstructions of nuclei. Nuclei similar to those in Fig. 1B were reconstructed from stacks of 0.1 mm thick optical sections and the vertical (z distance) separation of particles was measured. (A) Lateral view of a nucleus through the condensed chromosomes to show their vertical (z distance) separation and the shape B of the nucleus. z Separation was found by collecting 0.1 mm thick optical sections of nuclei similar to those in Fig. 1B. The sections were viewed sequentially in order to measure the distance between maximum chromosome diameters. Bar, 5 mm. (B) The surface view of this nucleus allowed measurement of the separation in the plane of the surface (the xy distance). xy Separation was measured as in B from an extended focus image. The 3D separation, (xy2+z2)g, of the particles could then be calculated. (C) Although separation between unpaired nuclei varied widely, the separation of double particles was similar in pairs of nuclei (ANOVA, P<0.0005). (D) The 3D distances separating condensed chromatin particles vary widely between nuclei. of the separation is a measure of nuclear orientation in rela- separations might remain constant. We therefore measured tion to structures outside the nucleus. If chromosome ori- the angles subtended by the lines joining chromosomes and entation is conserved in the whole nucleus between mitoses, the plane between paired cells. as with the separation distances, then we might expect that The angles subtended by the lines joining double parti- the orientation of the double chromosome pairs would cles and the plane separating paired cells were measured as remain as mirror-images. With other nuclear components in Fig. 3A. Angles measured in 18 nuclear pairs similar to such as nucleolar particles, for example, orientation as well Fig. 3B were found not to be mirror imaged (ANOVA, as separation is mirror-imaged in recently divided cells P>0.05). The orientations of sibling nuclei to one another (Locke and Leung, 1985). On the other hand, if nuclear are therefore not constant, even though they may be paired components can rotate, then the orientation of the hete- with respect to chromosome separation. We conclude that rochromatin might differ between pairs, even though their the components that conserve the separation distance Condensed chromosome arrangement 843

Table 1. Pairs of condensed female chromosomes in clones of four or more nuclei are not all separated by the same distances Chromosome separation, 3D measurement (mm)

Clone 1 Clone 2 Clone 3 Clone 4 2.5 5.2 4.4 5.2 4.4 6.1 2.5 8.9 11.8 3.3 8.9 7.3 12.4 4.0 6.6 2.2 3.6 3.9 5.1 4.6 8.0 8.4 2.8 2.5

The separations of chromosomes in nuclei like those shown in Fig. 4 were measured. Although the nuclei could be subjectively grouped in pairs according to chromosome separation, they were not all alike.

over the whole range recorded in Fig. 2D. These observa- tions suggest that whatever may determine the arrangement of chromosomes between mitoses, it is not conserved through mitoses.

Fig. 3. Pairs of double chromosomes do not have consistent orientations to one another. (A) The angles made by the lines Discussion subtended by chromosomes and the previous plane of division were measured. (B) Measurements were made on an extended The arrangement of condensed chromosomes in sibling focus image. The orientation was not mirror-imaged in nuclear nuclei allows five conclusions: (1) after mitoses, sibling pairs (ANOVA, P>0.05). Bar,10 mm. cells are alike in the position of condensed chromosomes. Patterns must therefore be replicated. (2) Chromosome position is conserved through interphase. Pattern is there- fore not only replicated, but inherited. (3) Patterns do between chromosomes during interphase are free to change not survive through mitosis. The inheritance is transient. their orientation in the plane of the epithelium (Fig. 6, (4) There is no constant position of chromosomes in fields below). of epidermal cell nuclei. The repositioning of chromosomes during the cell cycle allows separation to vary, causing dif- Pairs of chromosomes in clusters are not all separated by ferences from cell to cell across the epithelium. (5) Nuclei the same distance may rotate in the plane of the epithelium, changing the ori- Double chromosomes usually occurred in pairs (as in Fig. entation between nuclei but not the separation of chromo- 1A,B) but occasionally they were in fours (Fig. 4A) or even somes within nuclei. The conclusions are summarized in larger clusters (Fig. 4B). The low frequency of double chro- Figs 5 and 6. mosomes in pairs makes it unlikely that the clusters are chance associations of pairs. The clusters were all enclosed within fields of nuclei having single particles. Since the Pattern replication clusters were usually in fours, we presume that they are Since the early work of Heitz (1957), numerous studies clones derived by division of pairs. Particles in these clus- have shown similarities between recently divided cells ters of four did not all have the same separation. The sep- (Jordan et al., 1982; Locke and Leung, 1985; Rawlins and aration was in two pairs as in Fig. 4A. Such double pairs Shaw, 1990; Rawlins et al., 1991; Mathog and Sedat, 1989), were excluded from the calculations to show pairing, giving evidence from a variety of cell types that similar because it would have required the subjective selection of spatial information determining the distribution of compo- pairs from fours. Although the separations were clearly nents is passed to daughter cells. In Calpodes epidermal grouped in twos, each pair differed from the other pair, pre- nuclei, the inheritance of the spatial arrangement of con- sumably because they were separated in lineage by an addi- densed chromosomes can be recognized even several days tional mitosis (Table 1). In larger clones (Fig. 4B), pairs after division, perhaps because of the conservation of mid- could be matched subjectively but the separations extended bodies. 844 G. Jeun and M. Locke

Fig. 4. Pairs of double chromosomes that are in clones are not all separated by the same distance. (A) Nuclei with double chromosomes occasionally occur in fours that are presumed to be the daughters of sibling pairs. The chromosomes of one pair are separated by 3.3 and 4.0 mm, and the other pair by 5.2 and 6.1 mm. Bar, 10 mm. (B) Larger clones can be grouped subjectively into pairs with separations that may extend over the whole range. Bar, 25 mm. See Table 1.

The inheritance of chromosome position and its not larger groups of nuclei (Locke and Leung, 1985). conservation through interphase Release from the old nuclear skeleton and assembly on the Similarities of condensed chromosome separation and mitotic spindle, followed by release from the spindle and nucleolar pattern persist in sibling nuclei for 5-6 days recapture by the new nuclear skeleton, might be expected between mitoses, implying that particular nuclear structures to introduce some randomness to the position of a chro- are stable during interphase. The idea that chromosomes mosome. Relative chromosome position is reordered during maintain their position during interphase agrees with studies the transition from prophase to metaphase in Crepis capil - on Drosophila embryo cells, where the relative positions of laris, where all chromosome pairs are associated with their decondensation sites at the beginning of interphase are the homologues during prophase, but by metaphase the associ- same as early condensation sites at the next prophase ation only persists between nucleolar organizer chromo- (Hiraoka et al., 1989). Chromosomes in interphase nuclei somes (Oud et al., 1989). A relationship between chromo- are not like bowls of spaghetti, as they are depicted in even somes and cytoplasmic microtubules precedes the the most up-to-date textbooks (Alberts et al., 1989, pp. 762 metaphase rearrangement in mouse 3T3 fibroblasts. and 767; Darnell et al., 1990, p. 155). Either as a result of Prometaphase chromosome configuration translates inter- their association with the nucleoskeleton or because of close phase spatial order into order at the metaphase plate (Chaly and Brown, 1988). Once on the metaphase plate even quite packing, interphase chromosomes occupy discrete domains large movements do not affect the relative positions of chro- that are not intermixed with other chromosomes (Heslop- mosomes. These relative positions might be expected to be Harrison and Bennett, 1990). transferred to the new interphase nuclear skeleton. The redetermination of chromosome separation distance at mitosis The variability of chromosome position in fields of nuclei Clones are not formed from similar cells, suggesting that Because the interphase patterns shared by sibling cells do chromosomes are repositioned between interphases. Nucle- not survive through the next mitosis, genetic clones may olar patterns also resemble one another in sibling pairs but consist of cells with differently patterned nuclei. Even Condensed chromosome arrangement 845

tribution of condensed chromosomes in Calpodes. Although these examples show that chromosomes can differ in posi- tion in otherwise identical cells, other nuclear components may have tissue-specific positional determinants or domains that are similar in a general way from cell to cell. Three overlapping kinds of domain have been described in inter- phase nuclei: chromosomal, nucleolar and heterochromatin (Hilliker and Appels, 1989). For example, centromeres and telomeres lie at opposite poles in Drosophila blastoderm nuclei (Hiraoka et al., 1990); the nucleolar organizer regions on chromosomes 4 and 7 in Pisum sativum occupy special sites (Wolff and Quednau, 1988); human chromo- somes are confined to domains in human-hamster hybrid cell lines (van Dekken et al., 1989); centromeres may be organizing centres for cell type-specific interphase chro- Fig. 5. The separation of condensed female sex chromosomes is mosome patterns (Manuelidis and Borden, 1988). Even conserved during interphase but not between mitoses. Barr bodies, which have variable positions, may have pre- Chromosomal rearrangement probably occurs when they are ferred domains near the nuclear envelope (Belmont et al., released from the nuclear matrix in the transition from prophase to metaphase. After separation to the poles and decondensation, 1986). Variability in position does not imply that there is chromosomes become fixed in a new location in the nuclear no determinant for that position, only that such a determi- matrix where they remain until the next division. Although the nant may be differently placed in otherwise similar nuclei. separation of the chromosomes remains constant during interphase Attention was drawn to this transient inheritance of patterns the entire nucleus may rotate to change the orientation of the in otherwise variable sibling cells by calling it somatic chromosomes. inheritance (Locke, 1990). Nuclear rotation with conservation of separation clones of four cells are not all alike in the positions of their The idea that nuclei may rotate in the plane of the surface sex chromosomes. Our findings confirm observations show- agrees with observations on the orientation of the nuclear ing that some chromosomes have different positions in groove. The apical nuclear surface has a single deep fold, nuclei of cells that are otherwise genetically and develop- appearing in whole mounts as a clear bar with only half the mentally similar. For example, the separation of twin Barr thickness of the nucleus (Locke, 1985). The grooves have bodies in tissue cultures of XXX human fibroblasts varies all orientations in the plane of the epithelium. Rotation of from cell to cell (Belmont et al., 1986), much like the dis- linked nuclear material in the plane of the surface but not in the vertical plane is the simplest explanation for the con- servation of separation distance concurrent with changed chromosome orientation. Rotation in the plane at right angles to the surface is less probable because it would require nuclear components to bend, changing separation distances (Fig. 6). Nuclear rotation has been described in other cell types, for example in mouse neurones (DeBoni and Mintz, 1986).

We thank H. Leung and J. Sparks for technical assistance, R. Bailey and R. Green for advice on statistical analysis, and D. Carter, S. Caveney, A. Kiss, M. Sass, R. Shivers, S. Singh and W. Zeng for helpful discussions. The work was supported by NSERC grant A6607 to M.L.

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