J. Cell Sci. 18, 19-25(1975) 29Printed in Great Britain

THE GIEMSA-STAINING CENTROMERES OF

NIGELLA DAMASCENA

G.E.MARKSJohn Innes Institute, Colney Lane, Norwich, NR4 7 UH, England

SUMMARY

The centromere regions of each chromosome in the complement of Nigella damascena{zn = 2X = 12) stain differentially with Giemsa at interphase and throughout all the principalstages of mitosis and meiosis. Each centromere is seen to consist of a pair of sister half-centro-meres which appear as 2 differentially stained dots. The appearance and behaviour of thesedots indicates that they are kinetochores. The technique used does not stain centromeres inother plant species investigated, a fact which shows that the centromeres of Nigella are in someway different. The implications of this observation in relation to centromere polymorphismare discussed.

INTRODUCTION

Chromosome preparations of Nigella damascena (zn = zx = 1 2 ) stained withGiemsa show differential staining at the centromeres of all the chromosomes in thecomplement. This phenomenon has been studied at all the principal stages of mitosisand the results are presented in this paper.

METHODS

Root tips and anthers were collected from plants of Nigella damascena grown from commercialseed of unknown origin. Giemsa preparations were made as follows.

Root-tips

1. Fresh or pretreated (005 % aqueous colchicine for 4 h at room temperature) root tips werefixed in 3:1 ethanol/acctic acid overnight.

2. Root tips were then transferred to 45 % acetic acid (v/v) at 60 °C, incubated for 20 min,and then transferred to distilled water at room temperature. This treatment softened the roottips, allowing satisfactory squashing and also proved to be the most critical and essential stepin obtaining good differentiation with Giemsa.

3. Squash preparations were made in 45 % acetic acid at room temperature on clean slideswithout heating. There was no subsequent loss of cells if the squashing and spreading weresatisfactory; this was checked under phase-contrast. Coverslips were removed using the dry-ice method and the slides air-dried on a warm hotplate.

4. Slides were immersed in a fresh saturated solution of barium hydroxide at room tempera-ture for 5-10 min, then rinsed in distilled water and washed in tap water for 1 h.

5. The slides were then incubated in twice strength SSC (salt-sodium citrate) at 60 °C for 1 h.6. After rinsing in distilled water the slides were put into 2% Giemsa (G. T. Gurr's R66

improved stock diluted x 50 with M/15 SOrensen's phosphate buffer, pH 69). Staining waschecked after 5 min and thereafter at intervals until the preparation was optimally stained,which usually occurred within 1 h.

20 G. E. Marks

7. Slides were then rinsed rapidly in distilled water, dried on a hotplate for 12 h, rinsed inEuparal Essence and mounted in Euparal.

Pollen mother cells

1. Anthers were fixed in Pienar's fixative (6:3:2, v/v methanol:chloroform:propionic acid)for 24 h and stored for 1 week in 90% v/v ethanol at +4°C. Storage in ethanol preventsdisintegration of pollen mother cells during squashing.

2. Squash preparations were made in a drop of 45 % acetic acid and observed under phase-contrast. Slides containing suitable division stages were heated very slightly after gently pressingthe coverslip. No adhesive was necessary because it was found that if squashing and spreadingwere satisfactory there was negligible loss of cells.

3. Coverslips were removed using dry ice and the slides air-dried on a warm hotplate.4. Slides were then incubated in 45 % acetic acid at 60 °C for 20 min and washed for 15 min

in tap-water, finally rinsing in distilled water. Thereafter slides were treated as from stage 4given above for root tip preparations.

Preparations of root tips and pollen mother cells were also made using conventional stains(carmine, Feulgen, orcein, toluidine blue) for comparative purposes.

RESULTS

The chromosome complement of Nigella damascena consists of 5 pairs of metacen-tric chromosomes and one pair of acrocentric chromosomes. With conventionalchromosome stains the centromeres appear as the usual featureless unstained gaps.However with Giemsa staining this gap contains one pair of darkly stained dots which,together with one pair of telomeres, are the only regions throughout the chromo-some complement which stain differentially with Giemsa (Fig. 1). At interphase,Giemsa staining shows chromocentres (Fig. 2). In a sample of 100 cells the meannumber found was 10-18 ± 0-18. At all the principal stages of mitosis the centromereis differentially stained. At prophase (Fig. 3), prometaphase (Fig. 4) and metaphase(Fig. 5), there is a pair of dots at each centromere, whereas at anaphase each centro-mere shows a single dot (Fig. 6).

At all stages of meiosis the centromere is likewise differentially stained with Giemsa.It is clearly seen at pachytene (Fig. 7) and its doubleness is evident at metaphase I(Fig. 8), anaphase I (Fig. 9) and metaphase/anaphase II (Figs. 10, 11). It appears asa single dot at anaphase II (Fig. 12).

DISCUSSIONThe term centromere is used here simply to describe that region of the chromosome

concerned with chromosome attachment and movement on the spindle. Essentiallythe results show that at the centromere of each chromosome in Nigella there is a Giemsa-staining body bridging the space across each chromatid arm. Chromosomes thereforehave a pair of dots and chromatids a single one. At metaphase of mitosis the pairs ofdots are clearly orientated parallel to the spindle axis in an amphitelic manner (Fig. 5),whereas at metaphase I of meiosis the pairs are co-oriented syntelically (Fig. 8).Such orientation in mitosis and meiosis is what we expect of centromeres from thestudy of chromosome orientation and movement in Tipula oleracea (Bauer, Dietz &Robbeln, 1961). At anaphase, both in mitosis and meiosis, it is the stained dots whichlead the way to the spindle poles (Figs. 6, 9).

Nigella centromeres 21

From the foregoing evidence there seems little doubt that what stains differentiallywith Giemsa is some structure within the confines of the centromere. Lima-de-Faria(1956) has described structures at the centromeres of chromosomes in many species,including both animals and plants, and has postulated a generalized structure for thecentromere. One part of this structure contained chromomeres which were thoughtto be concerned with spindle attachment, that is, they were 'kinetochores'. Stack (1974)considers the centromeric dots which he describes in Allium, Ornithogalum, Rhoeo andTradescantia chromosomes to be kinetochores and claims that the Giemsa techniqueemployed is specific for demonstrating them. Likewise, similar pairs of dots at thecentromeres of human metaphase chromosomes have been demonstrated by Eiberg(1974) who considers these also to be the result of using a specific Giemsa method.Evans & Ross (1974) equated these pairs of dots in human chromosome centromereswith the pairs of unstained spheres often seen in the centromeres of human, mouse andother mammalian preparations pretreated with colcemid and stained conventionallywith orcein or Giemsa. They also believe that the pair of raised prominences seenat the centromere regions in optically shadowed unstained human chromosomespretreated for either C or G banding to be equivalent to the dots. Since it is knownfrom ultrastructural studies that the regions occupied by the raised prominencescontain paired disk-shaped kinetochores (Jokelainen, 1967) it is suggested by Evans &Ross (1974) that the structures seen with light microscopy are indeed the kinetochores.If their conclusion is correct and it applies equally to the paired dots in Nigella chromo-somes we can expect these also to have demonstrable kinetochores under the electronmicroscope. Investigations of this possibility are now in progress.

Centrometric chromomeres are known in some cases to contain DNA (Lima-de-Faria 1950, 1956). The Nigella centromere, however, does not stain with Feulgen,neither does it show allocycly with respect to Giemsa staining. Such allocycly is,however, evident in the Giemsa-stained centromeres described by Stack (1974), for inthe examples given the centromeres are visible only during division and they do notproduce chromocentres at interphase.

The Giemsa method described here is not specific for staining centromeres becauseit fails to stain them in other species. In fact it stains telomeres only in Clematismontana, pericentric heterochromatin in Rhoeo discolor and it differentiates intercalarybands only in the chromosomes of various Anemone species (Marks & Schweizer, 1974,and G. E. Marks, unpublished). If the technique has any specificity, therefore, itmust be for a particular chromosome component which is common to all those chromo-some regions which it stains. Telomeres sometimes show neo-centric activity as inSecale and Zea (Prakken & Muntzing, 1942; Rhoades, 1952), and they may take overthe role of the centromere, as in scorpion chromosomes (Piza, 1974). However, theseindications of functional similarities between centromeres and telomeres are notparalleled by the abilities of these regions to stain differentially with Giemsa, becausethe pair of differentially stained telomeres in Nigella do not show neo-centric activity(Fig. 6).

Because centromeres from different species react differently to the same techniquewe must conclude that centromeres are not necessarily similar even in the chromo-

22 G. E. Marks

somes of species within related genera (e.g. Anemone, Clematis and Nigella). If weaccept the concept of centromere polymorphism then caution is needed in formulatinggeneralizations about centromere structure from scattered observations made ona range of organisms.

I wish to acknowledge the helpful discussions of the work presented in this paper withProfessors D. R. Davies and L. F. La Cour, F.R.S., and Dr D. Schweizer.

REFERENCES

BAUER, H., DIETZ, R., & ROBBELN, C. (1961). Die Spermatocytenteilungen der Tipuliden.I l l Mitteilung. Das Bewegungsverhalten der Chromosomen in Translokationsheterozygatenvon Tipula oleracea. Chromosoma 12, 116-189.

EIBERG, H. (1974). New selective Giemsa techniques for human chromosomes, Cd staining.Nature, Lond. 248, 55.

EVANS, H. J. & Ross, A. (1974). Spotted centromeres in human chromosomes. Nature, Lond.249, 861.

JOKELAINEN, P. T. (1967). The ultrastructure and spatial organization of the metaphase kine-tochore in mitotic rat cells. J . Ultrastruct. Res. 19, 18-44.

LIMA-DE-FAHIA, A. (1950). The Feulgen test applied to centromeric chromomeres. Hereditas36, 60-74.

LIMA-DE-FARIA, A. (1956). The role of the kinetochore in chromosome organisation. Hereditas42, 85-160.

MARKS, G. E. & SCHWEIZER, D. (1974). Giemsa banding: karyotype differences in some speciesof Anemone and in Hepatica nobilis. Chromosoma 44, 405-416.

PIZA, S. DE T. (1974). Scorpion chromosomes with centromeres at both ends. In ChromosomesToday, vol. 4 (ed. J. Wahtman & K. Lewis), p. 431. New York and Jerusalem: John Wiley& Sons Ltd. and Israel University Press.

PRAKKEN, R. & MUNTZING, A. (1942). A meiotic peculiarity in rye, simulating a terminalcentromere. Hereditas 28, 441-482.

RHOADES, M. M. (1952). Preferential segregation in maize. In Heterosii, pp. 66-80. Ames:Iowa State College Press.

STACK, S. M. (1974). Differential Giemsa staining of kinetochores and nucleolus organizerheterochromatin in mitotic chromosomes of higher plants. Chromosoma 47, 361-378.

(Received 13 November 1974)

Figs. 1-6. Microphotographs of mitosis in Nigella damascena. Fig. 1 is from a colchi-cine-treated root tip. Figs. 2-6 are from untreated material. All preparations areGiemsa stained, x 1600.

Fig. 1. Colchicine metaphase showing full complement of 5 pairs of metacentricand 1 pair of acrocentric chromosomes (ac). All chromosomes have stained centromeresand one pair of metacentrics show faintly stained telomeres (arrows).

Fig. 2. Interphase nucleus showing 12 distinct and 4 indistinct chromocentres. Theformer most probably correspond to the centromeres and the latter probably represent2 pairs of sister telomeres.

Fig. 3. Prophase. Centromeres consist of a pair of stained dots (sister half-centro-meres).

Fig. 4. Prometaphase. Repulsion between sister half-centromeres is evident in mostchromosomes.

Fig. 5. Metaphase. Amphitelic orientation of sister half-centromeres is clear in allchromosomes.

Fig. 6. Anaphase. Half centromeres lead the way on the spindle and are clusteredtowards the poles. The stained telomeres (arrows) do not show neo-centric behaviour.

Nigella centromeres

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24 G. E. Marks

Figs. 7-12. Microphotographs of meiosis in Nigella damascena. Giemsa. X 1600.Fig. 7. Pachytene. Pairing is complete and all centromere regions and one telomere

region are differentially stained. The stained telomere is strictly terminal (arrow) whereasthe centromere of the acrocentric bivalent is subterminal (ac).

Fig. 8. Metaphase I. Six bivalents all showing distinct differentially stained centro-meres. Some of the centromeres are clearly double structures in which the syntelicorientation of the sister half-centromeres is evident.

Fig. 9. Anaphase I. The doubleness of each centromere is evident in most chromo-somes.

Fig. 10. Metaphase II. Polar view showing the centromeres located at theperiphery of the spindle.

Fig. 11. Metaphase/anaphase II. Slightly oblique polar view showing the amphitelicorientation of sister half-centromeres.

Fig. 12. Anaphase II. Spread out pair of groups showing a single half-centromerelocated to one side of each chromatid.

Ntgella centromeres

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