J. Cell Set. 36, 281-299 (1977) 281

Printed in Great Britain

VISUALIZATION OF INTERPHASE

CHROMOSOMES

STEPHEN M. STACK,• DAVID B. BROWN,* ANDWILLIAM C. DEWEYfDepartment of Botany and Plant Biology* and Department of Radiology and RadiationBiologyf, Colorado State University, Fort Collins, Colorado 80523, U.S.A.

SUMMARYUsing a modified Giemsa-banding technique we have observed what appear to be chromo-

somes during interphase in nuclei from Allium cepa root tips and Chinese hamster cells (CHOline). During telophase through Gx chromosomes progressively uncoil and decondense.During S chromosomes are comparatively decondensed, but some segments have structuresimilar to chromosomes in. GL and G,. During Gt the chromosomes progressively recondenseand coil in apparent preparation for prophase. Although specific structural modifications ofchromosomes occur in Glt S, and Gx nuclei, chromosomes appear never to decondense to thepoint that they lose their 3-dimensional integrity, but remain in distinct domains throughoutinterphase.

INTRODUCTION

Chromosomes typically are visible during cell division in eukaryotes, but duringtelophase chromosomes usually become indistinct in a process referred to as de-condensation (swelling) and/or uncoiling (despiralization or unravelling) (Brown &Bertke, 1974). When chromosomes have disappeared in the interphase nucleus,usually there is no clear indication of chromosome boundaries or even at what levelthe structural integrity of chromosomes is maintained to allow them to reappear insubsequent divisions. Current models for the structure of interphase nuclei assumean unravelling of chromosomes into chromonemata that mingle in the nucleoplasm inthe form of fibres 10-30 nm in diameter (Comings, 1968; DuPraw, 1970; Brasch &Setterfield, 1974). The structural integrity of interphase chromosomes is thought todepend on numerous attachments of chromosomal fibres to the nuclear envelope.These models are based largely on electron microscopy of sectioned and whole-mounted interphase nuclei in which usually only a tangle of chromonemata can beobserved, with no sign of individual chromosomes (Wischnitzer, 1973).

In apparent contradiction to this model for interphase chromosomes, we haveobserved what seem to be distinct interphase chromosomes throughout the cell cycle ofAllium cepa and Chinese hamster (CHO line) nuclei that have been treated with a modifi-cation of the chromosome banding technique of Drets & Shaw (1971). Our observationsare generally in agreement with many older light-microscopic descriptions of interphasenuclei and chromosomes, and we interpret these observations as being compatible withand complementary to descriptions of interphase nuclei based on electron microscopy.

282 S. M. Stack, D. B. Brown and W. C. Dewey

MATERIALS AND METHODSAllium cepa bulbs were suspended in aerated tap water at room temperature until roots sprouted.

The bulbs were then transferred to damp vermiculite until the roots grew to a length of 2-5 cm.At this time the roots were either cut from the bulbs or allowed to grow one hour longer indistilled water containing 10 /iCi/ml tritiated thymidine (New England Nuclear, 20 Ci/mmol)before being cut from the bulbs. Harvested roots were immediately fixed for 1 h in acetic-ethanol (1:3). Using 1 or 2 root tips in a drop of 45 % acetic acid on a slide, meristematic cellswere picked out with dissecting needles, and the multicellular fragments were removed. Theseessentially single cell suspensions were squashed and the coverslips removed by the dry-icemethod. After the slides were air-dried briefly, they were stored in a CaClj desiccator foi4-5 h. Dried slides were then treated according to one of the schedules (I-V) in Table 1.Although many variations of schedules I I-V were tried and some were reasonably effective inshowing structure in interphase nuclei, the exact schedules in treatments I I-V were generallysuperior.

Chinese hamster cells (CHO line) were maintained in logarithmic growth in McCoy's5A medium containing 15 % foetal calf serum. Cells were synchronized in mitosis by shakingoff mitotic cells, after which separate flasks were harvested by txypsinization at o, 1, 3, 6, 10,and 12 h of incubation (Dewey, Noel & Dettor, 1972). Immediately before trypsinization, thecells were pulse labelled with 5 /tCi/ml tritiated thymidine for 15 min. Trypsinized cells werefixed for 5 min in 1:3 acetic ethanol and then squashed immediately in 50% acetic acid.Coverslips were removed by the dry-ice method, and the squashes were air dried for 1-9 daysin open slide boxes. Dried slides were then treated according to schedules I, II, or IV in Table 1.At o h the cells were 95 % mitotic. After 1 h, none of 200 nuclei scored were labelled, indicatingnuclei were exclusively in Gx. After 3 h only 7 % of the nuclei were labelled, so most cells werein late G1 while a few cells had passed into S. After 6 h 72 % of the nuclei were labelled, so bythis time most cells were in early S phase with the remainder in late Gx. After 10 h 96 % of thenuclei were labelled, indicating most cells were in middle to late 5. After 12 h 41 % of thenuclei were labelled, and mitotic cells were frequently observed. Apparently by this timesynchrony had been largely lost, since many cells had passed into and through G, while theremainder were in late S (Dewey et al. 1972).

For autoradiography, dried slides were dipped in Kodak NTB2 liquid emulsion at 41 °C,air dried overnight in a light-proof box with air circulating through it, and stored 4-6 days ina sealed light-proof box at 4 °C. The slides were developed 2 min in Kodak D19 developer at20 CC, rinsed in distilled water, and fixed 3 min in Kodak (acid) fixer. After washing 5-10 minin distilled water, the slides were air dried before further processing.

Giemsa staining was performed by flooding the slides with a 1:9 mixture of ice-cold 620Harleco stock Giemsa stain and 0-12 M, pH 68 potassium phosphate buffer. After staining20 min, the slides were rinsed in distilled water and air dried.

Although nuclei treated for autoradiography (see above) can show structured interphasechromatin reasonably well, the clearest demonstrations are shown by nuclei that have not beentreated for autoradiography. To combine a good demonstration of interphase structure withtritiated thymidine labelling, schedule IV in Table 1 involves first treating nuclei to produceinterphase structure followed by photography, destaining, treating for autoradiography, andrestaining. Although this procedure sometimes worked reasonably well, there was always aquestion, particularly in A. cepa, whether the Giemsa stain had been sufficiently washed outbefore the treatment for autoradiography. If any Giemsa stain remains, it somehow exposesthe emulsion regardless of the presence or absence of tritium (see Acknowledgements). Forthis reason, this method was used only to a limited extent for A. cepa, but it was used extensivelyfor CHO cells where it seemed easier to wash out the Giemsa stain adequately.

All light microscopy and photomicrography were performed on a Leitz Orthoplan microscopefitted with a 4* x 5* format automatic camera using Kodak Ektapan film, or a 35-mm camerausing Kodak Panatomic X film.

Interphase stages (G1( S and G,) in A. cepa nuclei were determined by pulse labelling withtritiated thymidine and from differences in nuclear diameters. Nuclear diameters were assumedto be positively correlated with nuclear volumes that generally have been shown to be pro-

Visualization of interphase chromosomes 283

portional to DNA content in both plants (Sunderland & McLeish, 1961) and animals (Sawicki,Rowinski & Swenson, 1974). On this basis the smallest nuclei were considered to be in Gltintermediate size nuclei were considered to be in S, and the largest nuclei were considered tobe in Gt. Although the correlation of DNA content with nuclear volume seems generally re-liable for rapidly dividing cells, slowly dividing and non-mitotic cells are often exceptional

Table 1. Flaw chart outlining various treatment schedules (I—V) of dried squashes onslides. All schedules start at the top of the chart with dried slides and end at the bottomof a column indicated by a Roman numeral for the particular schedule

dried

stain in 2 % aceto-orcein for io min

4wash off coverslipand staining solutionwith ioo % ethanol and

air dryi

1 1treat for auto- mount inradiography Euparal(see Materials Iand methods) 4

1 photograph (see{ Materials and

mount in methods)Euparal 1

I 1wash off cover-slip and Eupaialwith ioo% ethanoland air dry

4treat air-driedslides accordingto schedule three

II

slidesi

submerge in a solution that is002 N NaOH and22 °C for io s

3 rapid washes in

3 rapid washes in

0-114 M NaCl at

470 % ethanol

495 % ethanol

1

submerge in 2 x SSC (Drets & Shaw,1971) at 60 °C for

1.Giemsa stain(see Materialsand methods)

1

1 4photo- photo-graph graphIII /

/remove coverslip,Euparal, andGiemsa stain withmethanol andethanol washesand air dry

4treat for auto-radiography

14Giemsa stain

1

4mount in EuparalIV

1 min1

Iflood with ice-cold O-I2 M, pH68 potassium phosphatehuflrer flnrl Allow tostand for 10 min

11rinse in distilledwater and air dry

4treat for auto-radiography

Giemsa stain4

mount inEuparal

V

(Macleod & MacLachlan, 1974; Leuchtenberger & Schrader, 1951). To avoid the problem ofcontaminating rapidly dividing meristematic cells with the slowly or non-di aiding cells of theroot cap, apical meristem, and zone of elongation, these parts of the root tip were largely cutaway and only a section approximately 2 mm long containing the primary meristems was used toprepare squashes. The single cells dissected from this section were assumed to be cycling

284 S. M. Stack, D. B. Brown and W. C. Dewey

rapidly and to show the correlation between nuclear volume and DNA content (Sunderland &McLeish, 1961). CHO cells were harvested only from non-confluent cultures in their log phaseof growth to assure rapidly cycling cells.

Because diameter is also correlated positively with the amount of pressure applied duringsquashing, it is necessary to look at cells that have been comparably squashed for accuracy inestimating interphase stages by nuclear diameters. Although this problem initially seems almostinsurmountable because of the variation in pressure applied from one thumb squash to another,the solution appears to be intrinsic to the method of visualizing structured interphase chromatin.In a thumb squash on an 18- or 22-mm square coverslip, there is an area of high pressure inthe centre that integrates with comparatively low pressure at the periphery where the thumbis not in contact. The central nuclei are usually over-squashed, in the sense that nuclear dia-meters are very large and no internal structure is visible. However, more peripherally there isa ring of generally intermediate-size nuclei that surround the central group, and it is here thatnuclei with structured chromatin can be observed. More peripherally, the nuclei are generallysmall, dark-staining, and without apparent internal structure. Presumably in this most peripheralzone the pressure was insufficient to flatten nuclei to the extent necessary to reveal internalstructure. All measurements and photography were concentrated in the intermediate zonewhere the pressure was most appropriate. Using this population of cells that varied to someextent from slide to slide in its precise distance from the centre of the squash, a good correlationbetween slides was observed, as diameters of hundreds of nuclei were related to classificationsin Gu S, and Gz.

For electron microscopy, root tips of A. cepa were fixed in phosphate-buffered glutaraldehydeand osmium tetroxide and embedded in Epon-Araldite (see Packard & Stack, 1976, for details).Embedded root tips were both thick (i-/im) and thin (80-nm) sectioned on an LKB 4801Aultramicrotome. Thin sections were stained with uranyl acetate and lead citrate and photo-graphed in an AEI EM6b electron microscope. Thick sections were mounted on glass slides,stained with 1 % methylene blue in 012 M dibasic potassium phosphate, and photographed lightmicroscopically. CHO cells were fixed in Karnovsky's fixative and embedded in Epon. Thinsections were post-stained with phosphotungstic acid and photographed in a Philips 200electron microscope.

RESULTS

When A. cepa root tip cells or Chinese hamster (CHO line) cells in interphase weretreated by one of schedules II-V in Table 1 (called sodium hydroxide treatmentssubsequently), chromatin could be seen condensed into distinct, separate masses thatoften resembled chromosomes.

To determine whether chromatin of A. cepa differs in appearance during the variousstages of interphase, nuclei were identified as being in Glt S, or G2 by tritiated thymi-dine pulse labelling and by comparisons of the diameters of the squashed nuclei (seeMaterials and methods). Using these techniques we have arranged the unsynchronized,sodium-hydroxide-treated nuclei from A. cepa root tips in a sequence that representsa light-microscopic interpretation of the changes that chromatin undergoes throughthe mitotic cycle. A familiar point at which to begin such a sequence is metaphase.At this time the condensed chromosomes are scattered over the metaphase plate,indicating A. cepa does not have a hollow spindle (Fig. 1). Often it can be seen thatthe chromosomes have heterochromatic telomeres. In anaphase the chromosomes takeon a pole-to-pole orientation with the heterochromatic telomeres trailing to the insideof each group of separating sister chromosomes (Fig. 2). During telophase the nuclearenvelope forms around the chromosomes as they maintain their relic anaphase

Visualization of interphase chromosomes 285

orientation, and the chromosomes begin to swell (Fig. 3).Gx follows telophase. Because the cells were not synchronized, nuclei could not be

classified as being in early, middle or late Gx by this means. However, assuming aprogressive decondensation of chromatin during Gx as is generally reported (Taylor,1963; Lafontaine & Lord, 1974; de la Torre, Sacristan-Garate & Navarrete, 1975), onecan arrange Gx nuclei in a series that appears to show progressive decondensation ofchromosomes that retain their relic anaphase-telophase orientation (Heitz, 1932;Stack & Clarke, 1974; Fussell, 1975) (Figs. 4-9). Also, the chromosome-like structuresoften appear to have heterochromatic telomeres (Figs. 4, 6, 9) like chromosomes fromprophase through anaphase (Figs. 1, 2, 20, 21).

Nuclei in early S phase resemble those interpreted to be late Gx nuclei (Figs. 10—11).As the cell proceeds through S phase the chromatin becomes increasingly diffuse, butthere are usually some clear divisions between chromatin masses (Figs. 12, 13). Ifthe cells are squashed intensely, long strands of finely dispersed chromatin are evident(Fig. 14). These strands probably correspond to chromonemata of linearly intactchromosomes whose 3-dimensional structure has been destroyed (Rohme, 1975). Latein S phase, structure again becomes more obvious as nuclei begin to resemble G2

nuclei (Fig. 15).G2 nuclei have highly structured chromatin that often resembles long chromosomes.

Although cells cannot be classified as early, middle, or late G2 on the basis of synchrony,G2 nuclei can be arranged in an order that appears to show the progressive condensa-tion of chromosomes for prophase (Figs. 16-19). This order is supported by theobservation that large, lightly labelled nuclei (Fig. 15) that are interpreted to be inearly G2 or late in S phase have less distinct chromosomes than large, unlabellednuclei (Figs. 16-18) that are interpreted to be in middle to late G2 (see Materials andmethods). Except for occasional heterochromatic telomeres (Fig. 17), G2 chromosomesdo not appear to be double, but since this technique does not show sister chromatidsat prophase or metaphase (Figs. 1,21), this is not surprising. The condensing chromo-somes in late G2 or early prophase vary in diameter apparently because condensationor coiling does not proceed at the same rate even within individual chromosomes(Figs. 19, 20). Finally in middle to late prophase, all of the chromosomes appear tohave the same diameter and retain their relic anaphase-telophase orientation(Fig. 21).

Aceto-orcein-stained nuclei were prepared (Table 1, schedule I) to'compare theirmore familiar appearance with sodium hydroxide-treated nuclei. Small unlabelledtelophase nuclei (Fig. 22) typically show chromosome structure that continuesthrough early Gx (Fig. 23), but structure seems largely to be lost by late Gx (Fig. 23).During S the nuclei increase in size but have little recognizable structure (Figs. 24,25). During G2 some thin strands and clumps of chromatin are visible, but these aredifficult to interpret as chromosomes (Figs. 26, 27). By early prophase distinctthickened strands are visible in relic anaphase-telophase orientation (Fig. 28). Ascondensation continues toward middle prophase the strands retain their orientationand become thinner, with more even diameters (Fig. 29). From these observations itappears that chromosomes look similar after aceto-orcein staining and sodium

19 CEL 26

286 S. M. Stack, D. B. Brown and W. C. Dewey

16

Visualisation of interphase chromosomes 287

hydroxide treatment from early prophase through early G1( but during most of inter-phase aceto-orcein staining provides little evidence for the persistence of chromosomestructure.

Nuclei of synchronized CHO cells were identified as being Gx, S or G2 according to(1) the incubation time, (2) whether they were labelled with tritiated thymidine, and(3) by comparing nuclear diameters. In the following description of changes in thestructure of sodium hydroxide-treated (schedule IV in Table 1) nuclei during thecell cycle, all of these means of determining stage were in general agreement.

In the o-h sample, most nuclei were in metaphase (Fig. 30). The chromosomes arearranged at the periphery of the metaphase plate, which indicates the presence of a' hollow spindle' that is characteristic of many animals (DuPraw, 1970). This arrange-ment is carried through anaphase-telophase, with the chromosome arms radiatingfrom an apparently empty polar area (Fig. 31). In the o-h sample, there were also afew telophase nuclei that showed what appeared to be progressive stages in the un-coiling and swelling of chromosomes (Figs. 32, 33).

Figs. 1-29. Chromosomes and nuclei horn A. cepa root tips, x 1200. Figs. 1-21 repre-sent sodium hydroxide-treated preparations. The clearest of these were not treatedfor autoradiography (see Materials and methods). The Roman numerals in parenthesesthat precede the descriptions of each figure indicate which of the schedules in Table 1was used to make the preparation. Schedule V involves autoradiography whileschedule III does not. Fig. 1: (III) Polar view of a metaphase chromosome spread.Note the heterochromatic telomeres. Chromosomes are scattered over the metaphaseplate indicating the absence of a hollow spindle. Fig. 2: (III) Lateral view of lateanaphase or early telophase. Again note the heterochromatic telomeres and theorientation of chromosomes. Fig. 3: (V) Later al view of telophase showing continuedorientation of chromosomes. Fig. 4: (III) Polar view of an early G^ or late telophasenucleus. Observe the radiating chromosomes with heterochromatic telomeres (arrows).Fig. 5 : (V) Polar view of a more advanced Gx nucleus with radiating chromosomes thatare swollen compared to Fig. 4. Fig. 6: (III) Polar view of a Gl nucleus that is quitesimilar to the nucleus in Fig. 5 except that the radiating chromosomes are clearer andshow heterochromatic telomeres (arrows). Fig. 7: (V) Lateral view of Gx nucleus withswollen chromosomes visible on one side (large arrow). Fig. 8: (III) Lateral view ofGi nucleus that is very similar to the nucleus in Fig. 7 except the oriented chromo-somes are much clearer. Fig. 9: (III) Lateral view of a late Gj 01 early S-phase nucleuswith oriented chromosomes and heterochromatic telomeres. Chromosomes generallyare becoming less distinct. Fig. 10: (V) Lightly labelled early S-phase nucleus in whichchromosomes are less clear, but divisions still separate chromatin domains that probablyrepresent chromosomes. Fig. 11: (III) Early S-phase nucleus comparable to thenucleus in Fig. 10. Heterochromatic telomeres continue to be visible on somechromosomes. Fig. 12: (V) Labelled mid- to late S-phase nucleus still showing someseparate chromatin domains, but in general such nuclei have more diffuse chromatinthan Gx nuclei. Fig. 13: (III) Mid- to late 5-phase nucleus comparable to the nucleuain Fig. 12, but chromatin domains are more distinct. Fig. 14: (IV) Labelled 5-phasenucleus that was over-squashed to reveal a complex network of threads that probablyrepresent chromonemata that were forced out of their domains. Autoradiographicemulsion was applied after this photograph was taken. Fig. 15: (V) Lightly labelledlate S or early Gt nucleus with more distinct chromatin domains. Fig. 16: (V) EarlyG, nucleus with distinct domains of chromatin.

rg-2

288 S. M. Stack, D. B. Brown and W. C. Dewey

29

Visualization of interphase chromosomes 289

Cells in Gx were studied in the 1- and 3-h samples. In early G± nuclei, swelling(uncoiling) of the chromosomes can be observed to occur asynchronously or unevenlyalong the length of individual chromosomes (Fig. 34). In late Gx nuclei more finelydivided chromatin masses, some of which still seem recognizable as chromosomes, canbe observed (Fig. 35).

In the 6-h sample, early 5 phase nuclei had both chromosomes in the process ofuncoiling and finely divided chromatin that may represent uncoiled chromosomes(Figs. 36-39). Other nuclei that were interpreted as being later in S phase showed acombination of finely divided and clumped chromatin (Figs. 40, 41).

In the 10-h sample, lightly labelled nuclei were considered to be very late in S phaseor early in G2. These nuclei showed considerable internal structure that often re-sembled what could be interpreted as condensing chromosomes (Figs. 42, 43).

Finally, in the 12-h sample, chromosome condensation was underway. Chromo-somes appeared to condense (perhaps coil) asynchronously along their length (Figs. 44,45), and this condensation continued until the chromosomes became very thin byearly prophase (Fig. 46). As prophase continued, chromosomes became thicker andshorter (Fig. 47) until they reached their metaphase length (Fig. 30).

Aceto-orcein-stained CHO nuclei were prepared to compare their structure tosodium-hydroxide-treated nuclei. After aceto-orcein staining, G± and G2 nucleicontained generally diffuse chromatin with some clumping evident (Figs. 48, 50).S phase nuclei consistently contained more diffuse chromatin (Fig. 49).

Since aceto-orcein staining did not seem to represent a treatment that wouldnecessarily interfere with subsequent sodium hydroxide treatment to show structuredinterphase chromosomes, we thought it might be possible to make a direct comparison

Fig. 17: (III) Two early G% nuclei similar to the nucleus in Fig. 16, but the chro-matin domains have more the appearance of separate chromosomes with hetero-chromatic telomeres, one of which appears double (arrow). Fig. 18: (V) Lateral viewof a Ga nucleus in which there is a suggestion of oriented chromosomes. Fig. 19: (III)Lateral view of a late Gt or early prophase nucleus with oriented chromosomes thathave rather uneven diameters and suggestions of coiling. Fig. 20: (III) Lateral view ofan early prophase nucleus with oriented chromosomes that have uneven diameters andsuggestions of coiling. Many of the telomeres are heterochromatic. Fig. 21: (III) Lateprophase nucleus with oriented chromosomes that have comparatively even diametersand heterochromatic telomeres. Figs. 22—29. Nuclei that have been aceto-orceinstained according to schedule I in Table 1. Fig. 22: Telophase nuclei in which chromo-somes are still visible to some extent. Fig. 23: On the left is a late telophase or early Gjnucleus in which chromosomes remain visible. On the right is a late Gx nucleus in whichchromosomes are not visible in the comparatively diffuse chromatin. Fig. 24: Labelledearly to middle S phase nucleus in which chromosomes are not visible in the diffusechromatin. Fig. 25: Labelled late S phase nucleus in which chromosomes are notvisible in the diffuse chromatin. Fig. 26: Early G, nucleus in which chromatin appearsin clumps and thin strands. Fig. 27: Late G% nucleus in which chromatin seems to be inmore distinct strands than in Fig. 26. Fig. 28: Early prophase nucleus in which thechromatin is in the form of thick chromosomes with irregular diameters, and thechromosomes have retained their relic anaphase-telophase orientation. Fig. 29:Middle prophase in which the chromosomes are long and thin with more regulardiameters than in Fig. 28. Again the chromosomes can be seen to have retained theirrelic anaphase-telophase orientation.

290 S. M. Stack, D. B. Brown and W. C. Dewey

47

48 49 50

Visualization ofinterphase chromosomes 291

of the same nuclei after aceto-orcein staining and after the sodium hydroxide treat-ment (schedule II in Table 1). Although boundaries between clumps of interphasechromatin looked fuzzy after this treatment of A. cepa nuclei, the procedure workedreasonably well for CHO cells. A comparison of Figs. 51 and 53 with Figs. 52 and 54,respectively, indicates the sodium hydroxide treatment causes some swelling ofchromatin and the appearance of distinct chromatin domains that may correspond tointerphase chromosomes.

As a test of the hypothesis that the 10-30-nm chromosomal fibres must havenumerous attachment sites to the nuclear envelope in order for chromosomes toreorganize at prophase (Comings, 1968, and see Discussion), A. cepa and CHO cellswere examined in prophase by both light and electron microscopy. As the hypothesispredicts, prophase chromosomes generally are condensed against the nuclear envelopein CHO cells (Fig. 55). However, in conflict with the hypothesis, A, cepa prophasechromosomes condense throughout the nucleoplasm (Figs. 56-58). Apparently inA. cepa nuclear envelope attachment sites are not necessarily needed for the reorgan-ization of chromosomes during prophase.

Figs. 30—50. Chromosomes and nuclei from Chinese hamster cells (CHO line). Thenuclei in all figures except 47 were obtained from synchronized cultures. Thesampling time is given in parentheses at the beginning of the description of each figure,x 1500. Figs. 30-47 illustrate sodium hydroxide-treated nuclei (schedule IV in Table 1).Fig. 30 (oh) : Polar view of a metaphase chromosome spread. The arrangementof chromosomes is characteristic of a hollow spindle. Fig. 31 (oh): Polar view of earlytelophase chromosomes that have the arrangement characteristic of a hollow spindle.Fig. 32 (oh) : Middle telophase in which the chromosomes begin to swell.Fig- 33 (oh) : Late telophase or early Gx nucleus in which the chromosomes continueto swell and uncoil (arrow). Fig. 34 (1 h): Middle Gj nucleus in which the chromo-somes have swollen unevenly along their lengths (arrow). Fig. 35 (3 h): Late G1 nucleusin which the chromosomes are less distinct than in Fig. 34. Fig. 36 (6 h): Early S-phasenucleus in which much of the chromatin is rather diffuse, but a few chromosomesare still in the process of uncoiling (arrows). Fig. 37: Autoradiograph of the nucleusin Fig. 36. Fig. 38 (6 h): Early 5-phase nucleus in which the chromosomes are evenmore loosely coiled (arrow), but many distinct clumps of chromatin remain. Fig. 39:Autoradiograph of the nucleus in Fig. 38. Fig. 40 (6 h): Nucleus in middle S phase inwhich part of the chromatin is finely divided while the rest is still clumped. Fig. 41 :Autoradiograph of the nucleus in Fig. 40. Fig. 42 (1 o h): Late S-phase nucleus in whichthick chromatin strands begin to reappear. Fig. 43. Autoradiograph of the nucleus inFig. 42. Fig. 44 (12 h): Lateral view of a Ga nucleus in which thick chromosome-likestructures are visible in their relic anaphase-telophase orientation (arrows). Fig. 45(12 h): Mid- to late G, nucleus in which chromosomes have contracted into distinctthickened strands with uneven diameters. Apparently contraction like swelling (seeFig. 34) does not occur at the same rate along the length of chromosomes. Fig. 46(12 h): Early prophase nucleus in which some of the chromosomes have contracted tovery thin strands, but many thick regions remain. Fig. 47 (non-synchronized): Pro-phase nucleus in which the chromosomes are thin and have comparatively evendiameters. Figs. 48-50: Aceto-orcein-stained nuclei photographed prior to theapplication of autoradiographic emulsion. These nuclei have a fine network ofchromatin fibres with clumping more evident during Gx and G, compared to S phase.Fig. 48 (1 h): Gj nucleus. Fig. 49 (10 h): Middle (left) and late (right) S phase nuclei.Fig. 50 (12 h): G2 nucleus.

S. M, Stack, D. B. Brown and W. C. Dewey

58

Figs. 51-54. Nuclei from non-synchronized CHO cells prepared according toschedule II in Table 1. Autoradiography was not performed on these nuclei, x 1200.Fig. 51: Aceto-orcein-stained telophase (lower arrow) and Gx nuclei (upper arrows).In the Gj nuclei the chromarin is in a fine reticulum while in the telophase nucleus thinchromosomes are visible. Fig. 52: The same nuclei illustrated in Fig. 51 after sodiumhydroxide treatment. Apparently the chromatin domains in the G, nuclei and chromo-somes in the telophase nucleus swelled after the sodium hydroxide treatment. Fig. 53 :Aceto-orcein-stained S phase (left) and late G2 or early prophase (right) nuclei. Thechromatin is in a fine reticulum in the S-phase nucleus, but thin chromosomes arevisible in the late G, or early prophase nucleus. Fig. 54: The same nuclei illustratedin Fig. S3 after sodium hydroxide treatment. Some clumping of chromatin is visiblein the 5-phase nucleus and distinct strands are visible in the late G, or early prophasenucleus. Again it is apparent that the sodium hydroxide treatment induced someswelling of chromarin. Fig. 55: Electron micrograph of a CHO cell in prophaseshowing that chromosomes condense primarily along the nuclear envelope, x 5000.Fig. 56: Electron micrograph of A. cepa root rip cell in prophase showing condensationof chromosomes throughout the nucleoplasm. x 7000. Figs. 57, 58: Light micrographsof sectioned A. cepa root rip cells in prophase showing condensation of chromosomesthroughout the nucleoplasm. x 1200.

Visualization of interphase chromosomes 293

DISCUSSION

Concern about the physical state of chromosomes during interphase can be tracedback to the old arguments over whether chromosomes even exist as separate entitiesduring interphase. Although subsequently the persistence of chromosomes as unitshas been demonstrated adequately by both genetic and morphological evidence(Boveri, 1909; Wilson, 1928), the physical state and boundaries of chromosomesduring interphase remain unclear.

The most common means of visually examining chromosomes and nuclei at presentinvolve light microscopy of squash preparations and electron microscopy of thinsections and whole mounts. Feulgen, aceto-orcein, and aceto-carmine-stained squashesgenerally do not reveal structures in interphase nuclei of higher plants and animalsthat are recogni2able as chromosomes. Thin sections and whole mounts of interphaseand non-mitotic nuclei examined in the electron microscope show a tangle of 10-30-nmfibres attached at numerous sites to the nuclear envelope (Feldherr, 1972). Lafontaine& Lord (1974) recorded changes in the pattern of clumped chromatin in A. porrumroot tip nuclei through interphase, but it is difficult to relate these changes to thestructure of individual chromosomes. In A. cepa and CHO cells chromatin fibresbecome increasingly dispersed from Gx through S, and at least in A. cepa thechromatin fibres condense somewhat during G2 (Dewey et al. 1972; de la Torre et al.1975). In all, the results from electron microscopy of interphase nuclei have beenscanty and difficult to interpret, but in a recent review Wischnitzer (1973) comes tothe positive conclusion that the results of electron microscopy as early as the 1950s' ...established that discrete interphase chromosomes are absent'. In agreement withthis interpretation, recent models for the structure of chromosomes during interphaseassume a general uncoiling of the 10-30-nm fibres of which chromosomes appear toconsist (DuPraw, 1970) so that the main pattern of chromosome 3-dimensionalstructure is maintained only by numerous attachments of the fibres to localized regionsof the nuclear envelope (Comings, 1968; DuPraw, 1970; Brasch & Setterfield, 1974).As the chromosomes begin to condense during prophase the fibres are somehowretrieved from intermingling in the nucleoplasm to reform chromosomes against thenuclear envelope (Comings & Okada, 1970a, b).

In apparent conflict with this interpretation of the structure of chromosomes duringinterphase, there is an extensive literature based mainly on light microscopy of livingcells and cells that have been fixed and sectioned that suggests chromosomes do notlose their 3-dimensional structure during interphase.

The chromosomes of some algae and protozoa remain distinctly visible throughinterphase (DuPraw, 1970; Grell, 1973). However, because of the long phylogeneticseparation of these organisms from higher plants and animals and their possessionof certain other differences in mitosis that are considered anomalous or primitive,these cases are not ordinarily used as evidence for the condition of interphase chromo-somes in higher organisms. But there are special cases among higher plants and animalsin which chromosomes do remain visibly distinct during interphase.

Chromosomes are said to be visible in the spermatozoa of 2 nematodes (Meves,

294 s- M- Stack> D- B- Brown and W. C. Dewey

1915; Mulsow, 1912). Large polytene chromosomes of both animals and plants remaindistinct through endomitotic interphases (Brown & Bertke, 1974). Sex chromatin isa well known example of the visibility of one heterochromatic X chromosome throughinterphase in somatic cells of mammals (Barr & Bertram, 1949; Davidson & Smith,1954), and constitutively heterochromatic parts of chromosomes generally are reportedto be visible through interphase in both higher plants and animals (Yunis & Yasmineh,1972; Stack & Clarke, 1973). Finally, in some instances chromosomes may formseparate 'vesicles' during telophase, and these 'chromosomal vesicles' may remaindistinct but associated through interphase. Usually this has been reported in rapidlydividing cells such as occur in cleaving animal embryos and spermatogonia (Wilson,1928), but 'chromosomal vesicles' have also been reported to constitute interphasenuclei in normal and malignant fibroblasts of the rat and mouse (Lewis, 1948), non-mitotic cells of frogs and rats (Kater, 1927,1928), and Phaseolus (bean) root tips (Kater,1926). In these cases the authors usually assume that each chromosome is surroundedby its own membranous envelope. This may be the case for some cleavage and possiblyspermatogonial cells, but it seems unlikely for the other examples (Ris & Mirsky,1949). Still, regardless of the authors' explanations of how the chromosomes remainvisible, they claim to be able to observe chromosomes as individual units throughinterphase.

Several different experimental treatments of living but primarily non-mitotic cellsof a variety of plants and animals have been reported to cause chromosome-likestructures to appear in nuclei. Effective treatments for specific cell types includepricking nuclei with sharp instruments (Chambers, 1924; Ris & Mirsky, 1949),treating cells with most of the common fixatives that contain acetic acid (Chambers,1924; Ris & Mirsky, 1949), and exposing cells to solutions that vary in dissolved salts(Anderson & Wilbur, 1952), pH (Philpot & Stanier, 1956), and tonicity (Kuwada &Nakamura, 1941; Chambers & Black, 1941; Ris & Mirsky, 1949). These observationswere consistently interpreted to indicate that intact nucleoplasm consists of a gel ofhydrated and intimately associated chromosomes that can be dehydrated by variousmeans to reveal structures that are similar to prophase chromosomes. It has also beensuggested that such chromatin condensations may be comparable to the condensationof chromatin during mitotic prophase (Chambers, 1924; Philpot & Stanier, 1956).However, since most of these studies were on non-mitotic cells, the nuclei were prob-ably structurally comparable to Gt interphase nuclei (Brown & Bertke, 1974), whichordinarily do not condense into prophase chromosomes. One can still interpret thiswork to indicate that chromosomes of some non-mitotic nuclei and possibly Gx nucleiremain 3-dimensionally intact but swollen.

Support for this interpretation of chromosomes in interphase and non-mitotic cellscomes from a variety of other sources. Chambers & Black (1941) described thechromatin in the normal non-mitotic nuclei of living onion bulb scales andTradescantialeaf hairs as resembling, ' , . . . brain coral, with sinuous winding strands without be-ginning or end and filling the entire nucleus except for one or two rounded nucleoli'.Interphase and non-mitotic chromosomes have been isolated from the nuclei ofa variety of vertebrate tissues (Claude & Potter, 1943; Mirsky & Ris, 1947, 1951;

Visualization of interphase chromosomes 295

Yasuzumi, 1951; Van Winkle, Renoll, Garvey & Prebus, 1952), leukaemic humancells (Polli, 1953), and insect muscle and follicle cells (Pfeiffer, 1950). In these casesunfixed nuclei were broken, and ehromatin in the form of recognizable chromosomeswas isolated. The chromosomes consisted of one or two chromatids that were longerthan normal metaphase chromosomes, and these chromosomes could be induced toswell or contract depending on the medium in which they were suspended (Mirsky &Osawa, 1961). Finally, our own observations of sodium-hydroxide-treated G± nucleialso indicate the nucleoplasm consists largely of elongate, swollen chromosomes.

More recently, complementary evidence concerning the state of chromosomesthroughout interphase has come from experiments on fusion of mammalian non-mitotic or interphase cells to metaphase cells utilizing Sendai virus. Such fusion oftenresults in 'premature chromosome condensation (PCC)' in interphase nuclei (seeSperling & Rao, 1974, for a review). In as little as 5-10 min after fusion the nuclearenvelopes of interphase cells break down, and the ehromatin in G1 and G2 nucleicondenses to form recognizable chromosomes with one or two elongate chromatids,respectively. S phase ehromatin does not condense appreciably, but apparently spillsout of nuclei as very long intact strands that probably correspond to individualchromosomes (Rohme, 1975; Sperling & Rao, 1974). Fusion of metaphase cells tonon-mitotic cells such as lymphocytes, spermatozoa, and erythrocytes results inGj-type PCC (Johnson, Rao & Hughes, 1970). Similarly, G^type PCC is observedin brain nuclei injected into Rana pipiens primary oocytes at the time of germinalvesicle breakdown (Ziegler & Masui, 1973). The appearance of interphase chromo-somes after sodium hydroxide treatment is similar to prematurely condensed chromo-somes with the exceptions that prematurely condensed chromosomes are betterseparated from one another and can be seen to consist of 2 chromatids at G2- These2 differences are expected, since an intact nuclear envelope holds sodium hydroxide-treated interphase chromosomes together and, for reasons that are not clear, thesodium hydroxide technique does not allow the visualization of sister chromatidseven at prophase or metaphase (Figs, i, 20, 21, 30, 46, 47).

If one assumes that all of these techniques for observing interphase chromosomesare probably revealing comparable structures in interphase nuclei, an importantquestion is how the sodium hydroxide technique allows visualization of interphasechromosomes, particularly when it does not employ pretreatment or even observationof living nuclei as most of the other methods do. Although we have only begun toexamine this question experimentally, we suspect the successive treatments causeshrinkage and swelling of ehromatin that results in its being visibly separated intodistinct domains or clumps that correspond to individual chromosomes. The use ofacetic acid in fixation has been reported to cause condensation of ehromatin duringinterphase (Ris & Mirsky, 1949). Following squashing and air drying, the slides areimmersed in a dilute solution of sodium hydroxide that probably causes the ehromatinto swell (Drets & Shaw, 1971; Comings, Avelino, Okada & Wyandt, 1973; and seeFigs. 51-54). The slides are quickly washed in 70 and 95 % ethanol which shoulddehydrate the ehromatin and cause it to contract strongly (Willey, 1971). Finallythe brief incubation in 2 x SSC may allow a partial swelling and restoration of

296 S. M. Stack, D. B. Brown and W. C. Dewey

chromosome structure after the extremes of swelling and contraction produced bythe previous steps.

The preceding descriptions of metaphase nuclei also suggest an explanation for thefailure to observe interphase chromosomes as individual units by electron microscopy.Unlike the sodium hydroxide technique, fixation for electron microscopy is usuallya careful attempt to alter the structure of cellular components as little as possible. Ifone assumes the nucleus is composed essentially of swollen chromosomes betweenwhich there is ordinarily no space other than that occupied by nucleoli, the 10-30-nmchromosomal fibres may intermingle to some extent on the borders of chromosomes.This would blur the boundaries of chromosomes at the levels of both light and electronmicroscopy. Since most of the chromatin of the nucleus has the same density,refractive index, and staining properties, it would be surprising if individual chromo-somes could be seen without somehow separating them from one another.

With reference to present models for interphase chromosome structure, it seemsclear that complete decondensation of chromosomes does not occur in the intactinterphase nucleus, but rather the chromosomes swell while otherwise maintainingtheir relic anaphase-telophase arrangement. Furthermore, attachment of chromosomefibres to the nuclear envelope probably is not the sole basis of maintaining the structureof chromosomes through interphase, because many of the chromosomes of A. cepacondense at prophase without apparent association with the nuclear envelope. Thisobservation may be related to the model for nuclear-envelope-dependent chromosomecondensation (Comings & Okada, 1970a, b) by the recent suggestion of Comings &Okada (1976) that chromosomes condense in association with a protein networkcalled the 'nuclear matrix' (Berezney & Coffey, 1976), that includes not only thenuclear-envelope-pore complex but a fibrous network throughout the nucleoplasm.The precise relationship between this fibrous network and individual chromosomesis not yet clear.

Apart from its apparent capability of allowing some morphological study of inter-phase chromosomes, the sodium hydroxide technique may find further use in allowingreasonable estimates of whether interphase nuclei are in Gv S or G2 without resortingto tritiated thymidine labelling or microspectrophotometry. At present, apart from thefluorescent technique of Moser, Muller & Robins (1975), there is no comparablyaccurate and generally applicable technique based on staining (Alvarez & VaJladares,1972; Dewse, 1974; Nescovic, 1968) or morphological differences (Nagl, 1970;Lafontaine & Lord, 1974; Das & Alfert, 1968; de la Torre et al. 1975) to identify thestages of interphase.

This research was supported in part by grants to S.M.S. from the Donald F. Jones Fund ofResearch Corporation, and from the Faculty Improvement and Biomedical Support Committeesat Colorado State University and, to W.CD. from NIH Grant CA 08618.

We thank Dr T. C. Hsu and Dr Frances Arrighi in the Cell Biology Section of the Universityof Texas M. D. Anderson Hospital and Tumor Institute at Houston, Texas, for warning usof the problem in attempting autoradiography of Giemsa stained material, Dr Larry Hopwoodffor assistance in autoradiography, Dr Marny Barrauf and Kathy Packard* for electron micro-graphs of CHO and A. cepa nuclei in prophase, and Dr David E. Comings in the Department

Visualization of interphase chromosomes 297

of Medical Genetics, City of Hope National Medical Center, Duarte, California, U.S.A., forcritically reading the manuscript.

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(Received 23 August 1976 - Revised 7 January 1977)