Genetica 78: 51-55, 1989 © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands 51

The R-banding pattern of the chinese hamster Don cell line

A. Martinez l, J. J. Pasantes 1, A. Gonz~ilez 1, V. Goyanes 2 & J. M6ndez 1 1Departamento de Gendtica, Colegio Universitario de La Corufia, Spain 2Departamento de Gen~tica, Hospital "Teresa Herrera" 15006 La Corufia, Spain

Received 15.5.1986 Accepted in revised form 3.10.1986

Abstract

Chinese hamster cells (Don line) were treated in vivo with 5-BrdU and 33258-Hoechst fluorochrome for obtain- ing the partial inhibition of condensation that causes the R-banding pattern. Untreated chromosomes were stained by a standard G-banding method. Statistical measurements show significant differences in the band numbers between the two treatments. The Don cell line in the authors' laboratory presents some karyotypical differences from Don cell lines studied by other authors.

Introduction

During the last few years chromosomal analysis was improved by the introduction of several detailed banding methods applied to prophasic chromo- somes, obtained from partially synchronized cell cultures (Yunis, 1976; Viegas-Pequignot & Dutril- laux, 1978; Yunis, 1981). This has allowed to increase the number of G-bands of the human karyotype from 338 bands (Paris Conference, 1971) to 850 bands now observed when high-resolution banding methods are applied to cultures treated with acridine orange before trypsine Giemsa banding (Matsubara & Nakagome, 1983). This method is based on a par- tial inhibition of condensation caused by acridine orange during prophase (Nakagome & Matsubara, 1981).

On the basis of the finding that condensation- decondensation cycles relationed with the phos- phorylation and de-acetylation of histones produce changes in chromosomal morphology during the mitotic cycle (Marcus et al., 1979), Goyanes and M6ndez (1981) developed a high resolution R- banding method by cell treatment with 5- bromodeoxyuridine (5-BrdU) and 33258-Hoechst.

33258-Hoechst is a fluorescent benzimidazol compound which shows a specific binding to A - T rich regions in DNA (Kucherlapati et al., 1975; Go- yanes & M6ndez, 1981) and produces a bright fluorescence in these areas (Rocchi et al., 1976); Hay- man & Sharp, 1981). It also stains centromeric regions of mouse chromosomes, and inhibits the condensation of these areas (Rocchi et al., 1976). The addition of 33258-Hoechst fluorochrome to a cell culture interferes partially with chromosomal condensation during G 2 phase and early mitosis (prophase and metaphase), and inhibits normal metaphase condensation. This phenomenon produces the appearance of metaphasic chromo- somes which are longer than chromosomes isolated from untreated cultures (Marcus et al., 1979). Inspite of this effect, 33258-Hoechst does not interfere with cell division (Marcus et al., 1979).

The thymine analogue 5-BrdU acts on heter- ochromatic regions (late replicating regions), delay- ing their condensation (Zakharov & Egolina, 1972) and producing inequalities in sister-chromatid con- densation. Acting during two continous cell cycles, it produces chromosomes with one chromatid slight- ly longer than its sister (Goyanes & M~ndez, 1981).

52

Material and methods Results and discussion

Chinese hamster (Cricetulus griseus) Don cells were cultured in RPMI 1640 medium supplemented with 15% fetal calf serum, antibiotics and L-glutamine.

Exponentially growing cultures in monolayer at 80-90% confluence were grown in flasks of 25 cc. Metaphase blocking was performed with colchicine for 2 h. Metaphase cells were detached from the monolayer by horizontal shaking of the culture flasks. After hypotonical treatment with KC1 (0.56%), at 37 °C for 5 min, cells were fixed three times in methanol-acetic (3"1), dropped on slides and air-dried.

R-bands: treatment by BrdU (20 #g/ml) and 33258-Hoechst (40/~g/ml) was performed for 12 h. After this treatment the cultures were finished with colchicine as described above and stained with 4% Giemsa at pH 6.8 for 5 min.

G-bands: 48 h after harvesting the metaphases were G-banded by treatment with 0.05 % trypsin for 7 s, incubated in 5% fetal calf serum at pH 6.88 for 10 s and washed in phosphate buffer at pH 6.88. Metaphases were stained by 4% Giemsa at pH 6.88 for 5 min.

After treatment with BrdU and 33258-Hoechst, chromosomal pairs have been obtained that clearly differ in length in each of the 60 Chinese hamster Don metaphases studied. Identical results have been obtained in chromosomes of 58 metaphases treated with the G-banding method. As pointed out by Hsu and Zenzes (1964), size variability is higher for big chromosomes than for small ones. Differences in the number of bands per chromosome are not statisti- cally significant in R-banded chromosomes or in G- banded chromosomes.

As previously reported, the mean number of bands per chromosome significantly differs between chromosomes where condensation has been inhibit- ed (R-banding) (Fig. 1) and untreated chromosomes (G-banding) (Fig. 2). Taking this fact into account, the higher number of bands in chromosomes treated with BrdU and 33258-Hoechst acting during one cell cycle may be interpreted as a result of the condensa- tion inhibitory effect produced by these chemicals (Goyanes & M6ndez, 1981).

The G-banding pattern obtained in this work is in agreement with those obtained by Kato and Yosida

Figs. 1-2. Cricetulus griseus, metaphase from Don cell line. (1, left) R-banding; - (2, right) G-banding.

53

3 33

3 2

_ _ 3 ] . . . .

~:Z 2 2 . 5

_ _ 2 2 . ~ _

~:~ 1 2 . 2

2 2 2 . 4

~:~

p 2 3t

2

P ,

m

| -

3 4

I

6 7

5

8 9 10

q

Y

Fig. 3. Cricetulus griseus, Don cell line, R-banded idiogram.

54

2 ~

__ +C___

i+3

1 4

1 l

t £

1

__ !~___

- - 3 1 - - -

3 2

ml

I I

P 2 2

2 24

2

- - 3 1 - - - P 2 22

2

3

p-~- ~l--- ~ ' I:'--- P';]

4

p, I ~3 P' ' + ,!! --i~--- q . . . . . . q 2 ~ 2

5 6

p ' IP ~ , +-++'--- +~

8 9

P I

q - - + ~ . . . .

2 2 3 2 4

7

q 1

!0

P . . . . . . •

3

X Y

Fig. 4. Cricetulus griseus, Don cell line, G-banded idiogram.

(1972), Deaven and Petersen (1973) and Campbell and Worton (1977), but the cell line of our laboratory shows an extra-band in one of the homologous chro- mosomes of pair one (lp+), as reported by Chen (1985). In addition, this cell line shows two different characteristics in relation to those studied by other authors. First, one of the chromosomes of pair five shows two more telomeric bands than its homologue (Sq+). Second, the Y chromosome is small and metacentric and has suffered a deletion in the telo- meric region of its long arm (Yg-).

R- and G-banding schemes (Figs. 3 and 4) have been drawn in reference to the mean number of bands observed in the cell populations studied. Bands are named according to Ray and Mohandas' nomenclature (1976).

References

Campbell, C. E. & Worton, R. G., 1977. Chromosome replication patterns in a established cell line (C. H. O.). Cytogenet. Cell Genet. 19: 303-319.

Chen, T. R., 1983. The Chinese hamster Don cell line differs from normal diploidy by one chromosome band. Cytogenet. Cell Genet. 39: 57-63.

Deaven, L. L. & Petersen, D. E, 1973. The chromosomes of CHO, an aneuploid Chinese hamster cell line: G-bands, C-bands and autoradiographic analyses. Chromosoma 41: 129-144.

Goyanes, V. J. & Mendez, J., 1981. Karyotyping chromosomes by electron microscopy. Condensation-Inhibition of G-bands in human and Chinese hamster chromosomes by a BrdU-Hoechst 33258 treatment. Cancer Genet. Cytogenet. 39: 45-51.

Hayman, D. & Sharp, P., 1981. Hoechst 33258 induced uncon-

55

densed sites in marsupial chromosomes. Chromosoma 83: 249 - 262.

Hsu, T. C. & Zenzes, M. T., 1964. Mammalian chromosomes 'in vitro' XVII. Idiogram of the Chinese hamster. J. natn. Cancer Institute 32, 4: 857-869.

Kato, H. & Yosida, T. H., 1972. Banding patterns of Chinese ham- ster chromosomes revealed by a new techniques. Chromosoma 36: 272-280.

Kucherlapati, R. S., Hilwig, A., Gropp, A. & Ruddle, E H., 1975. Mammalian chromosome identification in interspecific hybrid cells using 'Hoechst 33258'. Humangenetik 27: 9-14.

Marcus, M., Nielsen, R., Goitein, R. & Gropp, A., 1979. Pattern of condensation of mouse and Chinese hamster chromosomes in G-2 and mitosis of 33258-Hoechst-treated cells. Expl Cell Res. 122: 191-201.

Matsubara, T. & Nakagome, Y., 1981. High resolution banding by treating cells with acridine orange before fixation. Cytoge- net. Cell Genet. 35: 148-151.

Nakagome, Y. & Matsubara, T., 1981. High resolution banding: a very simple procedure. Ann. Rev. Nat. Inst. Genet. 31: 103 - 104.

Ray, M. & Mohandas, T., 1976. Proposed banding nomenclature for the Chinese hamster chromosomes (Cricetulus griseus). Cytogenet. Cell Genet. 16: 83-91.

Rocchi, A., Prantera, G., Pimpinelli, S. & Di Gastro, M., 1976. Effect of Hoechst 33258 on Chinese hamster chromosomes. Chromosoma 56: 41-46.

Viegas-P6quignot, E. & Dutrillaux, B., 1978. Une m~thode simple pour obtenir des prophases et des prom&aphases. Ann. Genet. 21: 122-125.

Yunis, J.J., 1976. High resolution of human chromosomes. Science 191: 1268-1270.

Yunis, J. J., 1981. Mid-prophase human chromosomes. The attain- ment of 2000 bands. Human Genet. 56: 293-298.

Zakharov, A. F. & Egolina, N. A., 1972. Differential spiralization along mammalian mitotic chromosomes. Chromosoma 38: 341- 365.